Chlorophyll, and what it tells us about intelligent design

For biochemists, chlorophyll biosynthesis provides a challenge to mechanistic understanding, because it involves compounds that are inherently unstable and reactive in the presence of oxygen and light, and yet it can take place in full sunlight in an atmosphere that is enriched in oxygen.

My comment: Consider that if there was no oxygen in the atmosphere prior to the supposed great oxygenation event, cyanobacteria, if they came to the surface, they would be killed by UV radiation. They would also have had to evolve from anoxygenic energy supply to oxygenic photosynthesis energy supply. But in that process, how and why would enzymes evolve, producing intermediate stage biochemical products, which, as being unstable, there were no protective mechanisms to deals with these compounds adequately?  



The paper: Cyanobacterial Evolution: Fresh Insight into Ancient Questions admits as follows:
Because of the importance of how cyanobacteria came to become masters of oxygenic photosynthesis, understanding the origins of this biological feat has perplexed scientists from different fields spanning disciplines such as biology, chemistry, geology, and paleontology. 1



Study of chlorophyll biosynthesis has also revealed several surprises to biochemists, including the existence of alternative routes or processes used to form certain intermediates in different types of organisms, variant mechanisms for forming intermediates under anaerobic vs aerobic conditions, and the existence of previously overlooked chlorophyll structural variants that may have important physiological roles.

(steps 1–6)
the initial steps  that divert general metabolic intermediates into the formation of the first cyclic tetrapyrrole, uroporphyrinogen III; 
(steps 7–9);
transformation of uroporphyrinogen III to protoporphyrin IX along the oxidative branch 
(step 10; the heme/bilin branch begins with insertion of Fe2C into protoporphyrin IX); 
the reductive branch leads from uroporphyrinogen III to siroheme, heme d1, factor F430, and corrinoids); insertion of Mg2C into protoporphyrin IX to begin the branch leading to chlorophylls 
(steps 11–14);
formation of the isocyclic ‘fifth’ ring that is present on all chlorophylls  
(step 15), 
reduction of a peripheral vinyl group to an ethyl group 
(step 16); 
followed or preceded by reduction of the macrocyclic ring system to form a chlorin, the defining oxidation state of true chlorophylls 
(step 17).
and addition of a polyisoprene alcohol to the tetrapyrrole to complete the structure of chlorophyll a






1. https://www.cell.com/current-biology/pdf/S0960-9822(14)01649-2.pdf
2. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151250#pone.0151250.ref007

Crystal structure of glutamate-1-semialdehyde-2,1-aminomutase from Arabidopsis thaliana.
https://www.ncbi.nlm.nih.gov/pubmed/27303897

The enzymes that synthesize chlorophyll



Next processing step. And that procedure repeats 17 times. In the end, there is a fully formed chlorophyll.  The pathway must go all the way through, otherwise, chlorophyll is not synthesized.
The enzymes need to be lined up like in a factory production line.  
What good would there be, if the pathway would go only up to the 15th step? none
What good would there be, if the pathway would go all the way through the 17th step? Chlorophyll would be produced, BUT:
What good for survival would there be for chlorophyll on its own, if not fully embedded in the photosynthesis process? none.
What good would there be for a light-harvesting complex without chlorophyll? none. 
What good would there be for photosynthesis without chlorophyll in place, capturing light, and transmitting it to the photosystem? none, since capturing
light is essential for the whole process.

The structures of Heme and Chlorophyll pigments are closely related, and they share the first ten biosynthesis steps. 



‘Why would evolution produce a series of enzymes that only generate useless intermediates until all of the enzymes needed for the end product have evolved?’ Therefore, the Chlorophyll biosynthesis pathway is irreducibly complex.



Promiscuous enzyme activity during evolution? Did you read this? Is that a Japanese way to say: "Let's fill the gap of evidence with an ad-hoc assertion, which sounds sciency " ?  Frankly speaking, this is a ridiculous claim and comes equally of throwing the towel, or giving up a rational discourse, and sticking to whatever comes in mind. 

This is confirmed by the fact that scientific papers have no evidence whatsoever how enzymatic biosynthesis pathways in general could have evolved. The quest becomes even more dramatic when it comes to the life-essential metabolic pathways essential for life, which had to emerge prior when life began. Science-based on methodological naturalism has only speculation and guesswork. 

This is a key problem for evolution:
Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system.
In other words: Why would natural selection select an intermediate biosynthesis product, which has by its own no use for the organism, unless that product keeps going through all necessary steps, up to the point to be ready to be assembled in a larger system?  
A minimal amount of instructional complex information is required for a gene to produce useful proteins. A minimal size of a protein is necessary for it to be functional.   Thus, before a region of DNA contains the requisite information to make useful proteins, natural selection would not select for a positive trait and play no role in guiding its evolution.

Natural selection would not select allele variants, unless a huge, and just right lateral gene transfer would take place, suddenly permitting the synthesis of all seventeen highly complex enzymes used in the chlorophyll biosynthesis
pathway

Natural selection could not operate to favour a system with anything less than all seventeen being present and functioning. What evolutionary process could possibly produce complex sophisticated enzymes that generate nothing useful until the whole process is complete? Some advocates of evolution argue that the assumed primeval organic soup had many of the simpler chemicals and that only as they were used up did it become necessary to generate the earlier enzymes in the pathway.

In The Mystery of Life’s Origin: Reassessing Current Theories, the authors set forth the good basic chemistry that demonstrates that there could never have been an organic soup, and present some of the evidence out there in the world indicating that there never was. Denton and Overman also cite a number of experts who suggest that there is no evidence for such a primitive soup but rather considerable evidence against it.

Life on Earth starts with an anaerobic metabolism that still nowadays persist in the form of bacteria living in oxygen-poor environments. In the early earth, nearly all oxygen was bound in compounds, like water and silicate rocks. But nearly 3 billion years ago the “invention” in nature of plant photosynthesis turned the anaerobic world into our present type of environment with aerobic life. It is clear that the introduction of oxygen into the anaerobic world obliged the organisms existing at that time to adapt since a lot of the by-products of oxygen metabolism are toxic compounds.




The biosynthesis pathway of Chlorophyll



Since cyanobacteria emerged first in the evolutionary timeline, their pathway on the left in which we will give a closer look. 

Steps involving more than two enzymes are shown by two arrows. 
Two separated arrows indicate that the two enzymes are distinct, and two overlapped arrows indicate that two enzymes are isoforms.  A protein isoform, or "protein variant" is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences.
Enzymes that have an essential role under aerobic conditions are shown in red. 
Enzymes that operate mainly under microoxic conditions and the transcription of the genes encoding these upregulated enzymes are shown in blue. 
Enzymes that require oxygen for catalysis are shown by “O2” in the red background, and 
enzymes that are inactivated by oxygen are shown by red “x-O2” symbols.

There are at least four enzymatic steps that require oxygen for catalysis. On the other hand, there are also oxygen-sensitive enzymes that are readily inactivated by oxygen in this biosynthetic pathway. These enzymes may be inactivated under aerobic conditions. Thus, to cope with the circumstances of various oxygen levels, cyanobacteria have an elaborate mechanism involving two enzymes that catalyze the same reaction. One enzyme functions under aerobic conditions and the other under anaerobic/microoxic conditions. ( microxic = with very small amounts of oxygen in the atmosphere  )The expression of genes encoding these enzymes is mainly controlled at the transcriptional level in response to cellular oxygen tension.

Comment:  If the atmosphere was anaerobic, without oxygen, before oxygenic photosynthesis brought the atmosphere to levels as today by the great oxygenation event, of about 20% oxygen, why would cyanobacteria evolve enzymes that only function in aerobic conditions?   Did Cyanobacteria have foresight, and know that once oxygenic photosynthesis evolved by themselves and the atmosphere would become aerobic, these enzymes that only work in aerobic conditions would be required? Evidently, that makes no sense whatsoever. And on top of that, the use of either aerobic or anaerobic enzymes is regulated by transcription factors !! How did that regulation emerge? Truth is, the existence of both enzymes, one adapted for aerobic conditions, and the other for anaerobic conditions, and both regulated to adapt the various conditions had to exist from day one. It seems that the atmosphere was always aerobic, but there are ecological niches, where oxygen is low or even non-extant, and cyanobacteria, which occupy all ecological systems on earth, were created to be able to adapt to all kind of atmospheric conditions.

There are  37 cyanobacterial representative species. The aerobic-type enzymes are ubiquitously conserved in all species. In contrast, the anaerobic/microoxic-type enzymes are missing in about half of these species, and some anaerobic enzymes, in only two species.

The first stage 

The first stage in tetrapyrrole synthesis is the synthesis of 5-aminoaevulinic acid ALA via two possible routes: 

(1) condensation of succinyl CoA and glycine (C4 pathway) using  ALA synthase, or 

(2) decarboxylation of glutamate (C5 pathway) via three different enzymes:

glutamyl-tRNA synthetase to charge a tRNA with glutamate, 
glutamyl-tRNA reductase to reduce glutamyl-tRNA to glutamate-1-semialdehyde (GSA), and 
GSA aminotransferase to catalyse a transamination reaction to produce ALA.



5-aminolevulinic acid  (ALA) can be considered to be the first universal, committed tetrapyrrole precursor.which is made either via the C4 or Shemin pathway or by the C5 Beale pathway

Route 1: the C4 or Shemin pathway 



Remarkably, this pathway shares the same first ten steps with the pathway of heme biosynthesis.

The Shemin pathway is used by eukaryotes that do not contain plastids (e.g., animals, yeasts, fungi) and members of the subgroup of purple bacteria, to form ALA. 

ALA is synthesized in the one-step condensation of succinyl-CoA and glycine catalyzed by ALA synthase .



The ALA synthase enzyme 



succinyl CoA and glycine and produces ALA by a condensation reaction ( condensation reactions are the same that bind amino acids together by amide bonds to form protein polypeptide chains ) accompanied by the liberation of Coenzyme A and CO2.

Route 2: C5 Beale pathway


Plants, algae, and most groups of bacteria, including cyanobacteria, form ALA by the C5 Beale pathway. The amino acid L-Glutamate is converted to Aminolevulinic acid (ALA) in three steps, via three different enzymes:


Glutamyl-tRNA synthetase (GluRS)
Glutamyl-tRNA reductase (GluTR)
Glutamate-1-semialdehyde aminotransferase (GSAM)


In phase one, tRNA is charged with glutamate by a Aminoacyl tRNA synthetase. Its name is Glutamyl-tRNA synthetase, to form glutamyl-tRNA.

https://www.youtube.com/watch?v=1GdTTCRw1sw&t=17s

Glutamyl-tRNA is reduced to glutamate-1-semialdehyde (GSA) by glutamyl-tRNA reductase (HemA). Then, GSA is isomerized to ALA by Glutamate-1-semialdehyde aminotransferase (GSAM)  . At physiological pH, GSA is a very unstable amino aldehyde that easily degrades generating toxic products within the cells.

Phase 1. Glutamate—tRNA ligase  catalyzes  L-Glutamate  into L-Glutamyl-tRNA(Glu)


Glutamyl-tRNA synthetase has been studied in connection with its role in protein synthesis. Like all aminoacyl-tRNA synthetases, the enzyme requires the cognate amino acid and tRNA as substrates, and the reaction requires the
energy of ATP hydrolysis. There is no evidence to suggest that the glutamyl-tRNA synthetase that charges tRNAGlu for ALA biosynthesis differs from the one involved in protein synthesis. 

Phase 2. Glutamyl-tRNA reductase (GluTR) to reduce L-Glutamyl-tRNA(Glu) to L-glutamate-semialdehyde (GSA)
Reduction of the glutamate  carboxyl group Glutamyl-tRNA reductase is the least well-understood enzyme of the five-carbon ALA biosynthetic pathway, owing to its instability in vitro, low cellular abundance, and the need to provide a relatively unstable aminoacyl-tRNA substrate. The enzyme requires NADPH as the source of the reductant for converting the tRNA-ligated  carboxyl group of glutamate to an aldehyde. An unresolved question is whether the glutamyl moiety is transferred from the tRNA to the enzyme and becomes covalently attached to the enzyme during the course of the reaction. Another question concerns the structure of the reaction product. Although most workers have assumed that the product is free L-glutamate-semialdehyde (GSA)  or its hydration product (hemiacetal), Jordan et al. (1993) have proposed that the product is the cyclic ester formed from the -carboxyl group and the hydrated aldehyde group. The cyclic structure does not contain free aldehyde or carboxylic acid functions, and is more compatible with some previously reported properties of the chemically synthesized product (stability in aqueous solution, heat stability) than the free or hydrated -aminoaldehyde. It seems probable that in solution, GSA occurs as an equilibrium mixture of the free aldehyde, hydrated form, and cyclic compound, analogously to aldose sugars.

Phase 3. Glutamate-1-semialdehyde aminotransferase (GSAM) to catalyse a transamination reaction to produce 5- aminolevulinic acid (ALA)

Activation of glutamate by ligation to form glutamyl-tRNA. Next, the activated glutamate is reduced at C-1 to form GSA. Finally, the amino group at C-2 of GSA is replaced by one at C-1, yielding ALA. The only known role of GSA is as a tetrapyrrole precursor, and therefore GSA formation can be considered to be the first committed step of the tetrapyrrole pathway in cyanobacteria. GSA supplies all of the C and N atoms of the tetrapyrrole nucleus.

This is a remarkable observation.  The only known role of GSA is as a tetrapyrrole precursor means GSA has no function by its own. How could its biosynthesis be explained by evolution, if its solely role is to be an intermediate product in the path to the final product, which requires further biosynthesis steps ? 

The pathway is a sequence of three steps for ALA formation as follows: (a) activation of the C1 of glutamate in a step requiring ATP and Mg2+; (b) reduction of the activated carboxyl group by NADPH to form glutamate 1-semialdehyde (GSA); and (c) transamination of GSA to form ALA. the activated form of glutamate is the tRNA adduct, glutamyl-tRNAglu.

Tetrapyrroles are large macrocyclic compounds. The end-product, uroporphyrinogen III

The synthesis of 5-aminoaevulinic acid in plastids, cyanobacteria, and many eubacteria proceeds by reduction of glutamate. the difference in redox potentials between a carboxylate and an aldehyde is so high that a reduction of a carboxyl group by NADPH is only possible when this carboxyl group has been previously activated (e.g., as a thioester or as a mixed phosphoric acid anhydride. In the plastid 5-aminoaevulinic acid synthesis, glutamate is activated in a very unusual way by a covalent linkage to a transfer RNA (tRNA) (Fig. 10.22). This tRNA for glutamate is encoded in the plastid genome and is involved in the plastids in the synthesis of 5-aminoaevulinic acid as well as in protein biosynthesis. As in protein biosynthesis (see Fig. 21.1), the linkage of the carboxyl group of glutamate to tRNA is accompanied by consumption of ATP. During reduction of glutamate tRNA by glutamate tRNA reductase, tRNA is liberated and in this way the reaction becomes irreversible. The glutamate 1-semi-aldehyde thus formed is converted to 5-aminoaevulinic acid by an aminotransferase with pyridoxal phosphate as a prosthetic group. This reaction proceeds according to the same mechanism as the aminotransferase reaction shown in Figure 7.4, the only difference being that the amino group (as amino donor) and the keto group (as amino acceptor) is present in the same molecule. Two molecules of 5-aminoaevulinic acid condense to form porphobilinogen. The open-chain tetrapyrrole hydroxymethylbilan is synthesized from four molecules of porphobilinogen via hydroxymethylbilan synthase. The enzyme contains a dipyrrole as cofactor. After the exchange of the two side chains on ring d the closure of the tetrapyrrole ring produces uroporphyrinogen III. Subsequently, protoporphyrin IX is formed by reaction with a decarboxylase and two oxidases (not shown in detail). Magnesium is incorporated into the tetrapyrrole ring by magnesium chelatase and the resultant Mg-protoporphyrin IX is converted by three more enzymes to protochlorophyllide. The tetrapyrrole ring of protochlorophyllide contains the same number of double bonds as protoporphyrin IX. The reduction of one double bond in ring d by NADPH yields chlorophyllide. Protochlorophyllide oxido-reductase, which catalyzes this reaction, is only active when protochlorophyllide is activated by absorption of light. The transfer of a pyrophosphate activated phytyl chain to protochlorophyllide via a prenyl transferase (chlorophyll synthetase, completes the synthesis of chlorophyll. The light dependence of the protochlorophyllide reductase allows a developing shoot to green only when it reaches the light. Also, the synthesis of the chlorophyll binding proteins of the light harvesting complexes is light-dependent. The exceptions are some gymnosperms (e.g., pine), in which protochlorophyllide reduction as well as the synthesis of chlorophyll binding proteins also progresses during darkness. Unprotected and unbound porphyrins may lead to photochemical cell damage. It is therefore important that intermediates of chlorophyll biosynthesis do not accumulate. To prevent this, the synthesis of 5-aminoaevulinic acid is light-dependent, but the mechanism of this regulation is not yet fully understood. Moreover, 5-aminoaevulinic acid synthesis is subject to feedback inhibition by chlorophyllide. The end products protochlorophyllide and chlorophyllide inhibit magnesium chelatase (Fig. 10.24). Moreover, intermediates of chlorophyll synthesis control the synthesis of light harvesting proteins (section 2.4) via the regulation of gene expression.

The porphyrin ring with its conjugated double bonds is assembled in the chloroplast from eight molecules of 5-aminolevulinic acid, a highly reactive nonprotein amino acid (5-amino, 4-keto pentanoic acid).

1. Glutamyl-tRNA synthetase
Glutamyl-tRNA (Glu-tRNA), formed by Glu-tRNA synthetase (GluRS), is a substrate for protein biosynthesis and tetrapyrrole formation by the C5 pathway. In this route Glu-tRNA is transformed to δ-aminolevulinic acid, the universal precursor of tetrapyrroles (e.g., heme and chlorophyll) by the action of Glu-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase.

In our example of solar panel production, if one of the robots of the production line ceases to work for some reason, the whole fabrication ceases, and the completion of the finished solar panels cannot be accomplished. That means, a tiny mal connection of one of the robots in the production line of the solar panel might stop the production of the solar panel, and the finished photovoltaic system cannot be produced. Nobody would project a solar panel without visualizing the higher end upfront, in the project and development stage, and based on the requirement, specify the complex functional form, shape and materials which will be useful to perform the required task. And the whole production line and each robot the right placement and sequence where each robot will be placed must be planned and implemented as well. Everything has to be projected with a higher end goal in mind.

In each moment of cell life, billions of molecules are transformed into different ones through reactions that are accelerated (catalyzed) by the so-called enzymes, most of which are represented by proteins. Even though these proteins might interact with a plethora of different molecules during their chaotic trip within the cell, they bind only to specific molecules representing their substrate, and transform it into another and different molecules called product (of the reaction). Overall, this is not true for all enzymes; each enzyme interacts with one substrate giving rise to a specific product. Hence, in each moment of cell life billions of substrates are transformed into billions of products by billions of enzyme molecules. These reactions are extremely fast, and we can imagine the cell as a viscous environment where these reactions occur in an ordered (and only apparently chaotic) fashion. The whole body of these reactions is called metabolism, a circular “entity” in the sense that molecules can be destroyed (catabolism) to obtain energy and “bricks” that are required to construct other different molecules

 Nobody would project a solar panel without visualizing the higher end upfront, in the project and development stage, and based on the requirement, specify the complex shape of the solar panel. And the whole production line and each robot the right placement and sequence where each robot will be placed must be planned and implemented as well. Everything has to be projected with a higher end goal in mind. And there is an interdependence. If one of the robots ceases to work for some reason, the whole fabrication ceases, and the completion of the finished solar panel cannot be accomplished. That means, a tiny mal connection of one of the robots in the production line of the door might stop the production of the door, and the finished solar panel cannot be produced.




The argument of irreducible complexity is obvious and clear.

The metabolic pathways to form Chlorophyll molecules is subdivided into two steps: The first consists of ten steps where the branch point is between chlorophyll and heme synthesis. And the second consists in seven steps where the end product is Chlorophyll a


 
We can further subdivide Chlorophyll biosynthesis in four distinct sections.(1) The synthesis of protoporphyrin IX from the first committed precursor, 5-aminolevulinic acid (ALA). Since protoporphyrin IX is the common precursor for Chl and protoheme, this section is called “common pathway”. In the common pathway, two molecules of ALA are condensed to form the monopyrrole, porphobilinogen, which are then sequentially polymerized linearly and subsequently to form the cyclic tetrapyrrole, called uroporphyrinogen III. The pathway is branched at this step to form siroheme which is called (“siroheme branch”). Siroheme is by the way life essential, and had to be synthesized prior life began.



The insertion of Magnesium (Mg2+) into protoporphyrin IX for Chlorophyll a biosynthesis is called “the Magnesium branch”. Later, we will give a closer look at these biosynthesis steps. Now i am giving just an overview.



The biosynthesis pathway of Chlorophyll



Cyanobacteria emerged first in the evolutionary narrative, the pathway on the left demonstrates the pathway to metabolize chlorophyll molecules of cyanobacterias, so we will give a closer look. 

Steps involving more than two enzymes are shown by two arrows. 
Two separated arrows indicate that the two enzymes are distinct, and two overlapped arrows indicate that two enzymes are isoforms.  A protein isoform, or "protein variant" is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences.
Enzymes that have an essential role under aerobic conditions are shown in red. 
Enzymes that operate mainly under microoxic conditions and the transcription of the genes encoding these upregulated enzymes are shown in blue. 
Enzymes that require oxygen for catalysis are shown by “O2” in the red background, and 
enzymes that are inactivated by oxygen are shown by red “x-O2” symbols.

There are at least four enzymatic steps that require oxygen for catalysis. On the other hand, there are also oxygen-sensitive enzymes that are readily inactivated by oxygen in this biosynthetic pathway. These enzymes may be inactivated under aerobic conditions. Thus, to cope with the circumstances of various oxygen levels, cyanobacteria have an elaborate mechanism involving two enzymes that catalyze the same reaction. One enzyme functions under aerobic conditions and the other under anaerobic/microoxic conditions. ( microxic = with very small amounts of oxygen in the atmosphere  )The expression of genes encoding these enzymes is mainly controlled at the transcriptional level in response to cellular oxygen tension.

Comment: If the atmosphere was anaerobic, without oxygen, before oxygenic photosynthesis brought the atmosphere to levels as today by the great oxygenation event, of about 20% oxygen, why would cyanobacteria evolve enzymes that only function in aerobic conditions?   Did Cyanobacteria have foresight, and know that once oxygenic photosynthesis evolved by themselves and the atmosphere would become aerobic, these enzymes that only work in aerobic conditions would be required? Evidently, that makes no sense whatsoever. And on top of that, the use of either aerobic or anaerobic enzymes is regulated by transcription factors !! How did that regulation emerge? Truth is, the existence of both enzymes, one adapted for aerobic conditions, and the other for anaerobic conditions, and both regulated to adapt the various conditions had to exist from day one. It seems that the atmosphere was always aerobic, but there are ecological niches, where oxygen is low or even non-extant, and cyanobacteria, which occupy all ecological systems on earth, were created to be able to adapt to all kind of atmospheric conditions.

There are  37 cyanobacterial representative species. The aerobic-type enzymes are ubiquitously conserved in all species. In contrast, the anaerobic/microoxic-type enzymes are missing in about half of these species, and some anaerobic enzymes, in only two species.

On the diagram of the Chlorophyll biosynthesis pathway, you see many double arrows. This has an amazing meaning.

Organisms like cyanobacteria, supposedly existed as anaerobes in a reduced atmosphere without oxygen, and according to the evolutionary narrative, bacteria, which were doing non-oxygenic photosynthesis, evolved the oxygen-evolving complex which is responsible for the production of oxygen, and oxygenated the atmosphere, giving room for the evolution of multicellular organisms which use respiration to get enough energy.

The two arrows means, in the Chlorophyll synthesis pathway, there are enzymes that function in an anoxic environment, and as well enzymes that require oxygen to function. Photosynthetic organisms populate all environments, from the oceans to the Sahara desert. In environments without oxygen supply, and with oxygen supply. So these organisms can adapt to any environment, and use either one enzyme that is oxygen sensitive or the other which requires it.  

Now imagine there was no oxygen in the archean epoch, and only oxygen sensitive enzymes in the pathway to make chlorophylls. As soon as these bacterias would have evolved oxygenic photosynthesis , the oxygen would mute these enzymes and kill their enzymatic activity.

Not only that. Oxygen is also sensitive to nitrogenase enzymes, which fix nitrogen, another essential process to fuel the supply of ammonia, which is essential for all life forms, to produce the basic building blocks of life, amino acids, dna, rna etc.

So the evolution of oxygenic photosynthesis would have brought unsurmountable problems, which - voilá - according to the evolutionary narrative, the evolution of oxygen-depending enzymes solved. Since there are oxygen dependent enzymes that do the same reaction, but in a completely different route, they use oxygen, permit the production of light capturing cholorophylls, and photosynthesis can continue. And of course, the oxygen sensitive at the same time, evolved protection mechanisms in order not to be killed.

After all, evolution is an amazing process, capable of the most demanding adaptations. Isnt it ?

So the evolution of oxygenic photosynthesis - for no reason - which rather than helping the survival, would have killed the organism, by horizontal gene transfer, for unknown reasons, produced the oxygen evolving complex, oxygenic photosynthesis, and as well enzymes that work depending on oxygen rather than an anoxic environment, and at the same time, did split cyanobacteria cells into two types, and produced heterocysts, which protect nitrogenase enzymes from oxygen.

There is more:
Researchers have long been puzzled as to how the cyanobacteria could make all that oxygen without poisoning themselves. To avoid their DNA getting wrecked by a hydroxyl radical that naturally occurs in the production of oxygen, the cyanobacteria would have had to evolve protective enzymes. But how could natural selection have led the cyanobacteria to evolve these enzymes if the need for them didn’t even exist yet? The explanations are fantasious at best.

Nick Lane describes the dilemma in the book Oxygen, the molecule that made the world:
Before cells could commit to oxygenic photosynthesis, they must have learned to deal with its toxic waste, or they would surely have been killed, as modern anaerobes are today. But how could they adapt to oxygen if they were not yet producing it? An oxygen holocaust, followed by the emergence of a new world order, is the obvious answer; but we have seen that there is no geological evidence to favor such a catastrophic history. In terms of the traditional account of life on our planet, the difficulty and investment required to split water and produce oxygen is a Darwinian paradox.

If there was a reduced atmosphere without oxygen some time back in the past ( which is btw quite controversial ) then there would be no ozone layer, and if there was no ozone layer the ultraviolet radiation would penetrate the atmosphere and would destroy the amino acids as soon as they were formed. If the Cyanobacterias however would overcome that problem ( its supposed the bacterias in the early earth lived in the water, but that would draw other unsurmountable problems ), and evolve photosynthesis, they would have to evolve at the same time protective enzymes that prevented them oxygen to damage their DNA through hydroxyl radicals. So what evolutionary advantage would there be they to do this ?  

Of course, evolution has to be significantly elastic in order to accomodante all these challenges, but hey, no matter what, its a fact after all. So who are we to question it?

But i, as a YEC, believe, God knew the essential ecological role of cyanobacterias and plants, and equipped them with the toolkit to adapt from the beginning to all environments.

Voilá. Occams Razor well applied. The simplest explanation is often the best.

Spectroscopic properties of chlorophylls



In photosynthesis of a green plant, light is collected primarily by chlorophylls, pigments that absorb light at a wavelength below 480 nm and between 550 and 700 nm. When white sunlight falls on a chlorophyll layer, the
green light with a wavelength between 480 and 550 nm is not absorbed but is reflected. This is why plant chlorophylls and whole leaves appear green.



There are several chlorophyll molecule variants. Chlorophyll-a is the central photosynthesis pigment. 



Chlorophyll b is identical to chlorophyll a except at the C-7 position, a formyl group replaces the methyl group.  In a wide range of the visible spectrum of light, chlorophyll -a does not absorb it. This non-absorbing region is named the “green window.” The absorption gap is narrowed by the light absorption of chlorophyll-b, with its first maximum at a higher wavelength than chlorophyll -a and the second maximum at a lower wavelength. As shown in the picture, the light energy absorbed by chlorophyll b (chl-b) can be transferred very efficiently to chl-a. In this way, chl-b enhances the plant’s efficiency for utilizing sunlight energy. The structure of chlorophylls has remained remarkably unchanged in the deep past. 


Bu why does chlorophyll have this dip in the area where the sun emits the most energy?  The answer is that by this, chlorophyll makes better use of the range of light that it does absorb. It does so with high efficiency. Plants and most other photosynthetic organisms achieve far higher overall photon capture rates with chlorophyll than it would be the case with any other pigments

Leaf colour is fine-tuned on the solar spectra to avoid strand direct solar radiation 
Terrestrial green plants are fine-tuned to spectral dynamics of incident solar radiation and PAR absorption ( Photosynthetically active radiation ) is increased in various structural hierarchies.



If leaves were black, plants would absorb all wavelengths of visible light. What would be the consequence? Well, the amount of thermal stress this would plants under would probably cause cells to rupture. Cell walls are already under a lot of pressure as it is; additional heat could cause rupture in the short term, or water loss in the long-term. There are some plants with dark leaves, but they occupy ecological niches where sunlight irradiation is low, and they cannot survive in the canopy of trees.



A sequence of alternating double and single bonds in rings, which form a system of conjugated bonds, is responsible for light absorption. Differences in the structure of these pigments result in certain variations in their absorption spectra.


 
Chlorophyll structure




The basic structure is a ring made of four pyrroles, a tetrapyrrole, which is also named porphyrin. Manganese Mg is present in the center of the ring as the central atom. Chlorophylls are excellent light absorbers. At ring d a  phytol. Phytol chain is attached. It consists of a long branched hydrocarbon chain. The phytol chain is not involved in light absorption, but it anchors the chlorophyll molecule in the thylakoid membrane and provides it with the right orientation. Remarkably, if just this phytol chain were not extant, chlorophyll could not be anchored in the light-harvesting complex, light could not be absorbed, photosynthesis could not occur, and no higher, more advanced and complex life forms could exist. Isn't that remarkable and amazing ?! 

The tetrapyrrole ring not only is a constituent of chlorophyll but is also employed in a variety of other biological functions.  With Copper as the center atom, it forms cobalamin (vitamin B12), which is one of the most complex Vitamins known, and life essential. With Iron instead of Manganese, as the central atom, the tetrapyrrole ring forms the basic structure of hemes, used in haemoglobin, which stores and transports oxygen in the blood of aerobic organisms.

Chlorophylls are embedded in Light-harvesting complexes


All chlorophyll-based photosynthetic organisms contain light-gathering antenna systems. These systems function to absorb light and transfer the energy in the light to a trap, which quenches or deactivates the excited state. In most cases, the trap is the reaction center itself, and the excited state is quenched by photochemistry with energy storage. In some cases, however, the quenching is by some other process, such as fluorescence or internal conversion.




The antenna pigments are arranged in well-defined, three-dimensional structures, so that only a few energy transfer steps are required to connect any two pigments in the array.


How exactly do Chlorophyll pigments “capture” the energy of light? 
The photosynthesis pathway starts with light absorption  which excites the chlorophyll molecule
When chlorophyll absorbs a photon, an electron excitation in its ring structure occurs, which moves electrons from the ground state into a higher excited state in the atoms. A sequence of alternating double and single bonds in rings, which form a system of conjugated bonds, is responsible for light absorption.






A photon can be envisioned as a very fast-moving packet of energy. When it strikes a molecule, its energy is either lost as heat or absorbed by the electrons of the molecule, boosting those electrons into higher energy levels. Whether or not the photon’s energy is absorbed depends on how much energy it carries (defined by its wavelength) and on the chemical nature of the molecule it hits. Electrons occupy discrete energy levels in their orbits around atomic nuclei. To boost an electron into a different energy level requires just the right amount of energy, just as reaching the next rung on a ladder requires you to raise your foot just the right distance. A specific atom can, therefore, absorb only certain photons of light—namely, those that correspond to the atom’s available electron energy levels.

As a result, each molecule has a characteristic absorption spectrum, the range and efficiency of photons it is capable of absorbing. Chlorophyll pigment molecules are good absorbers of light in the visible range. There are only two general types used in green plant photosynthesis: carotenoids and chlorophylls. Chlorophylls absorb photons within narrow energy ranges. Two kinds of chlorophyll in plants, chlorophylls a and b, preferentially absorb violet-blue and red light. Neither of these pigments absorbs photons with wavelengths between about 500 and 600 nanometers, and light of these wavelengths is, therefore, reflected by plants. 

When these photons are subsequently absorbed by the pigment in our eyes, we perceive them as green. Chlorophyll a is the main photosynthetic pigment and is the only pigment that can act directly to convert light energy to chemical energy.  
 
This higher energy state then can be transferred by electromagnetic interactions to its nearby adjacent pigment, moving from one molecule to another.




Remarkably, the higher energy state can result in four different reactions. It can result in a process known as fluorescence. It can from its higher energy state simply return into its ground state, and convert the excitation energy into heat, or transfer the energy to another molecule. This excited state is inherently unstable, and for that reason, any process that captures its energy must be extremely rapid. The photochemical reactions of photosynthesis are among the fastest known chemical reactions.  This extreme speed is necessary for photochemistry to compete with three other possible reactions of the excited state. 



An extremely rapid transfer of that excitation energy through the light-harvesting antenna occurs in picoseconds, to a specific chlorophyll pair in the reaction center of an enormously large super multisubunit protein-pigment complex called photosystem. Oxygenic photosynthesis uses two photosystem supercomplexes, Photosystem II and Photosytem I. (  photosystem II comes first in the pathway. )



This process is a high quantum efficiency resonance energy transfer. The distance from the donor pigment molecule to the acceptor molecule plays a crucial role in regards to the efficiency by which this energy transfer occurs. Light-harvesting complexes have their pigments specifically positioned to optimize these rates. The antenna pigments are arranged in well-defined, three-dimensional structures, so that only a few energy transfer steps are required to connect any two pigments in the array. A staggeringly high efficiency of approximately 95-99% is achieved when the energy of photons absorbed by the pigments is transferred to the reaction center and then is used in photochemistry.If chlorophylls would be arranged so that the energy had to diffuse along a linear, or one-dimensional, array of chlorophyll pigments, then the concept of energy transfer in photosynthesis would not be feasible. One-dimensional diffusion is very inefficient because many, many transfers are required to move the excitation from one point in the array to another.





A photon – a particle of light  collides with an electron in a leaf outside your window. The electron, given a serious kick by this energy boost, starts to bounce around, a little like a pinball. It makes its way through a tiny part of the leaf’s cell, and passes on its extra energy to a molecule that can act as an energy currency to fuel the plant. 

One-dimensional and three-dimensional antenna organization models.
In the one-dimensional model, excitation must be transferred by many steps before encountering a trap where photochemistry takes place. In the three-dimensional model, the trap is always no more than a few energy transfer steps from any of the pigments in the antenna complex.

Question: How could the right arrangement have emerged in a gradual, stepwise, evolutionary fashion, if only the arrangement in well-defined, three-dimensional structures, where only a few energy transfer steps are required to connect any two pigments in the array is feasible? 



Observe that electrons are not transferred from one chlorophyll pigments in the antenna, to the adjacent ones. Only the energy of the excited state of the electron is moved on until reaching the special chlorophyll pair in the reaction center. The special chlorophyll pairs are named P680 in Photosystem I, and P700 in Photosystem II.  

The absorbed energy of the photon in this special chlorophyll pair performs a process called charge separation.  In this process, an electron is removed from that special chlorophyll pair, and this highly energized electron is donated to a carrier protein called Plastoquinone, to start a journey in the electron transport chain. During these electron transport reactions, adenine triphosphate (ATP) which is the energy provider in living cells, is produced by a proton gradient and is later consumed in carbon reductions, which will produce Glucose as the end product.

That special chlorophyll pair in the reaction center of the photosystem, alone, would not be able to collect enough energy through sunlight to be able to eject that electron to start the electron transfer. For that reason, there is this antenna light-harvesting complex which hosts in average 200 to 300 chlorophyll molecules per each photosystem. These surround the special chlorophyll pair in the reaction center to focus, deliver and transmit efficiently enough energy using before mentioned resonance energy transfer, more specifically named Förster resonance energy transfer. Chlorophyll thus acts as a large light-collecting antenna and it is at the reaction centers that the photochemical event occurs.

If every chlorophyll had associated with it the entire electron transfer chain and enzymatic complement needed to finish the job of photosynthesis, then these expensive components would sit idle most of the time, only occasionally springing into action when a photon is absorbed. This would obviously be wasteful, and ultimately such an arrangement would be unworkable. It is as if a factory were to have a number of expensive manufacturing machines sitting idle most of the time while a key raw material is being brought in at a slow pace. It makes more sense to buy only a few expensive machines and somehow to improve the delivery system of raw materials. This is what antennas do for photosynthetic organisms.

My comment: Did you observe the language employed? " It makes more sense". This is teleology. Goal orientation. Why would unguided evolutionary mechanisms produce chlorophylls wherein an inadequate arrangement and chaotic position would be of no use at all? - but only, in a workable arrangement? This is once again a formidable example where only intelligent setup explains rationally the setup of the biological system in question. 

Only by the accumulating of the energy from all these chlorophylls, the charge separation occurs in this special chlorophyll pair can occur,  enough to eject an electron from one atom to the plastoquinone carrier molecule, which starts the journey of the electron in the electron transport chain.




Light-harvesting antennae do not contain only chlorophylls, but also other molecules, additional accessory pigments such as carotenoids which have a crucial,  role in photosynthesis: photoprotection through destruction of reactive oxygen species that arise as byproducts of photoexcitation, and chlorophyll biosynthesis. 

When a chlorophyll pigment absorbs a photon,  the energy of the photon excites electrons and transfers them to a higher energy level, which results in an excited state of the chromophore molecule. The energy is absorbed only in discrete quanta, resulting in discrete excitation states.


Photosynthesis is comprised of a series of redox reactions. An oxidation-reduction (redox) reaction is a type of chemical reaction that involves a transfer of electrons between two species. It is any chemical reaction in which the oxidation number of a molecule, atom, or ion changes by gaining or losing an electron.

The oxidation state of an element corresponds to the number of electrons, that an atom loses, gains, or appears to use when joining with other atoms in compounds. The chemical species from which the electron is stripped is being oxidized, while the chemical species to which the electron is added is being reduced.

Redox reactions are vital to photosynthesis in which light produces NADPH, which acts as the reducing molecule for CO2 fixation via the Calvin cycle.  

Biological energy is frequently stored and released by means of redox reactions. Photosynthesis involves the reduction of carbon dioxide which occurs in the light-independent reactions, in the second phase of photosynthesis, into glucose sugars and the oxidation of water into molecular oxygen in the light-dependent reactions, right in the beginning.  

The reverse reaction, respiration in mitochondria in eukaryotic cells, oxidizes glucose sugars ( produced through photosynthesis ) to produce carbon dioxide and water. As intermediate steps, the reduced carbon compounds are used to reduce nicotinamide adenine dinucleotide (NAD+) to NADH, which then contributes to the creation of a proton gradient, which drives the synthesis of adenosine triphosphate (ATP) and is maintained by the reduction of oxygen.

When the photosynthetic apparatus is light saturated,  the intense absorptions and the long-lived excited states render both Chls and their tetrapyrrole toxic, generating highly toxic reactive oxygen species. Biosynthesis and degradation of Chls are therefore tightly controlled and co-regulated with the other parts of the photosynthetic apparatus.


rattlesnakes have infrared detectors that give them “heat pictures” of their surroundings. To our eyes, both the male and female Indian luna moths are light green and indistinguishable from each other, but the luna moths themselves perceive the ultraviolet range of light. Therefore, to them, the female looks quite different from the male. Other creatures have difficulty seeing the moths when they rest on green leaves, but luna moths are not camouflaged to one another; rather, they see each other as brilliantly colored. Bees can also detect ultraviolet light. In fact, many flowers have beautiful patterns that bees can see to guide them to the flower. These attractive and intricate patterns are totally hidden from human perception. 


https://www.ncbi.nlm.nih.gov/pubmed/27023791/

GUANGYU E. CHEN Complete enzyme set for chlorophyll biosynthesis in Escherichia coli 26 Jan 2018 1

Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg–protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in Escherichia coli. Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning E. coli cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism.

Chlorophylls (Chls) underpin photosynthesis, which generates the oxygen that supports all complex life on Earth and the reducing potential to fix carbon dioxide as carbohydrates, the ultimate source of all the organic compounds required for life on Earth. The annual production of Chls, on land and in the oceans, is on the scale of billions of tons, yet the enzyme components of the biosynthesis pathway have neither been fully determined nor assembled to define the minimal set of genes required to make Chl. The Chl biosynthetic pathway (Fig. 1A) is a branch of tetrapyrrole biosynthesis, and it begins with protoporphyrin IX (PPIX), which is also the precursor for heme biosynthesis. Most life forms, including those able to photosynthesize, make PPIX and convert it to heme by inserting Fe2+ into the porphyrin macrocycle. Heme is a cofactor for respiratory proteins, and it is also converted into bilins, linear tetrapyrroles that are used as light-harvesting pigments in cyanobacteria.


Assembly of the Chl biosynthesis pathway in Escherichia coli.
(A) Overall reactions from PPIX to Chl a catalyzed by the enzymes introduced to E. coli. The insertion of Fe2+ into PPIX (not shown) creates a biosynthetic branchpoint (not shown) that yields heme. Colored shading denotes the chemical change(s) at each step. ATP, adenosine triphosphate; ADP, adenosine diphosphate; SAM, S-adenosine-L-methionine; SAH, S-adenosyl-L-homocysteine; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP+; Pi, inorganic phosphate; PPi, inorganic pyrophosphate. 
(B) Arrangement and relative size of each gene in the constructed plasmids. The chlI, chlD, chlH, gun4, chlM, acsF, dvr (bciB), and chlG genes were consecutively cloned into a modified pET3a vector with a single T7 promoter upstream of chlI and a ribosome-binding site upstream of each gene using the link-and-lock method (see fig. S2 for details) (40). Colors for genes correspond to those used in (A). Plasmid constructs and gene contents are IM (chlI–chlM), IA (chlI–acsF), ID (chlI–dvr), IG (chlI–chlG), BoP (BoWSCP and chlP), and DE (dxs and crtE). BoP is a pACYCDuet1-based plasmid containing a sequence encoding the BoWSCP protein with a C-terminal His10 tag (10) and the Synechocystis chlP gene. DE is a pCOLADuet1-based plasmid containing the E. coli dxs gene and the Rvi. gelatinosus crtE gene.

Chl biosynthesis is initiated when the magnesium chelatase enzyme complex (ChlIDH, Gun4) inserts Mg2+ into PPIX, so the Mg2+/Fe2+ Chl/heme branchpoint in photosynthetic bacteria, algae, and plants requires fine control to ensure that the correct amounts of hemes, bilins, and Chls are produced (1). Following the production of Mg-PPIX (MgP), six more enzymatic steps culminate in the production of Chl a, the ubiquitous pigment of oxygenic photosynthesis (see Fig. 1A). The MgP methyltransferase (ChlM) produces the substrate for the MgP monomethyl ester (MgPME) cyclase (AcsF), which produces 3,8-divinyl protochlorophyllide a (DV PChlide a); this intermediate has acquired a fifth ring that imparts the green color characteristic of Chls. Protochlorophyllide oxidoreductase (POR) produces 3,8-divinyl chlorophyllide a (DV Chlide a), which is reduced by divinyl reductase (DVR) and then esterified with geranylgeranyl pyrophosphate (GGPP) by Chl synthase (ChlG) to produce GG–Chl a. Finally, the reduction of the GG “tail,” catalyzed by GG reductase (ChlP), completes the pathway, and Chl a is produced. At this point, the pigment is hydrophobic and enters the membrane-bound assembly pathway for photosynthetic complexes, where it engages with the Sec/YidC-assembly machinery that coordinates the cotranslational insertion of nascent photosystem polypeptides with pigment production (2). Many steps of the Chl biosynthesis pathway have been studied individually in mutant strains of phototrophs, and some of the early steps have been characterized to define kinetic parameters (3), but it is not known whether these enzymes are sufficient to assemble the pathway and convert PPIX to Chl a. Apart from the global significance of Chl biosynthesis, the bottom-up construction of this pathway represents the first step in reprogramming a heterotrophic bacterium for growth under a variety of predetermined conditions of light intensity and wavelength. This metabolic engineering, which would also require genes encoding photosynthetic apoproteins, not only addresses important biological problems such as the concept of the minimal amount of genetic information required for photosynthetic life but also lays the groundwork for engineering cell factories with light-powered metabolism.





1. https://www.science.org/doi/10.1126/sciadv.aaq1407

Ryouichi Tanaka Tetrapyrrole Biosynthesis in Higher Plants 2007 1

Tetrapyrroles play vital roles in various biological processes, including photosynthesis and respiration. Higher plants contain four classes of tetrapyrroles, namely, chlorophyll, heme, siroheme, and phytochromobilin. All of the tetrapyrroles are derived from a common biosynthetic pathway. The progress consists of biochemical, structural, and genetic analyses, which contribute to our understanding of how the flow and the synthesis of tetrapyrrole molecules are regulated and how the potentially toxic intermediates of tetrapyrrole synthesis are maintained at low levels. We also describe interactions of tetrapyrrole biosynthesis and other cellular processes including the stay-green events, the cell death program, and the plastid-to-nucleus signal transduction. Finally, we present several reports on attempts for agricultural and horticultural applications in which the tetrapyrrole biosynthesis pathway was genetically modified.

Chlorophyll is a tetrapyrrole macrocycle containing Mg, a phytol chain, and a characteristic fifth ring (Figure 1).





Figure 1 The tetrapyrrole biosynthetic pathway in higher plants. Numbers correspond to the description in the text. The enzymatic steps that do not occur in higher plants are shown with blue dashed lines. The International Union of Pure and Applied Chemistry (IUPAC) numbering scheme for C atoms is shown on the structure of chlorophyll a, which is located near the bottom of the figure.

Chlorophyll serves an essential role in photosynthesis by absorbing light and transfering the light energy or electrons to other molecules. Higher plants have two chlorophyll species, chlorophyll a and b (Figure 1). The methyl group at the C7 position of chlorophyll a is replaced by a formyl group in chlorophyll b. Heme is another closed macrocycle that contains iron and it plays a vital role in various biological processes including respiration and photosynthesis. Similar to heme, siroheme also contains iron in its closed macrocycle. It is a prosthetic group of nitrite and sulfite reductase that plays central roles in nitrogen and sulfur assimilation, respectively. Phytochromobilin is a linear tetrapyrrole and a chromophore of phytochrome that perceives light and mediates its signal to the nuclei. The major site of tetrapyrrole biosynthesis in plants occurs in plastids. 

The Chlorophyll Branch 


(10) The first step of the chlorophyll branch is the ATP-dependent insertion of the Mg2+ ion into protoporphyrin IX, a reaction that is catalyzed by magnesium chelatase (MgCh). This enzyme consists of three subunits, ChlH, ChlI, and ChlD, whose average molecular weights of those subunits are 140, 40, and 70 kDa, respectively. The ChlH subunit is predicted to have the catalytic site, whereas ChlI and ChlD bind to each other to activate ChlH. 

(11) Chlorophyll synthesis subsequently proceeds to the transfer of a methyl group from S-adenosyl-L-methionine to the carboxyl group of the 13-propionate on Mgprotoporphyrin IX by Mg-protoporphyrin IX methyltransferase (MgMT), resulting in the formation of Mg-protoporphyrin IX monomethyl ester. 

(12) In the next reaction, Mg-protoporphyrin IX monomethyl ester cyclase (MgCy) incorporates an atomic oxygen (O) to Mg-protoporphyrin IX, forming 3,8-divinyl protochlorophyllide. In plants, an oxygen-dependent MgCy operates this reaction, whereas purple bacteria and cyanobacteria contain both oxygen-dependent and oxygen-independent MgCy. The oxygen-independent MgCy functions to incorporate O from H2O into Mg protoporphyrin IX monomethyl ester. It is likely that the catalytic subunit of plant MgCy is encoded by the Crd1 gene. However, the involvement of an additional membrane protein and a stromal protein in the MgCy reaction has been suggested. 

(13) The D ring of 3,8-divinyl protochlorophyllide is reduced by protochlorophyllide oxidoreductase (POR) to form 3,8-divinyl chlorophyllide. This reaction is absolutely light-dependent in angiosperms because they contain only a light-dependent POR, which belongs to the short-chain dehydrogenase family. Other plants, algae, and cyanobacteria contain both light-dependent and light-independent POR, the latter of which is related to nitrate reductase. Due to the presence of light-independent POR, those organisms can synthesize chlorophyll in darkness. 

(14) The 8-vinyl group of the B ring of this compound is reduced by divinyl chlorophyllide reductase (DVR) to form 3-vinyl chlorophyllide a (monovinyl chlorophyllide a). This enzyme can reduce 3,8- divinyl protochlorophyllide as well; however, the efficiency of this substrate is substantially lower than that of 3,8-divinyl chlorophyllide in vivo and in vitro. Hence, we revise the conventional order of reaction steps illustrated in most previously published articles, and we place the DVR reaction after the POR reaction in the scheme in Figure 1. 

(15) In the last step of the chlorophyll branch, 17-propionate on the D ring of monovinyl chlorophyllide a is esterified with phytol-pyrophosphate by chlorophyll synthase and results in the formation of chlorophyll a.

1. The biosynthesis pathway of Chlorophyll, a life-essential cofactor, consisting of 17 individual steps, has to be regulated. If the intermediate molecules of the later steps accumulate excessively, they generate toxic singlet oxygen species, (ROS) ultimately leading to cell death. On the other hand, if not enough Chlorophylls are synthesized, it will stop the entire photosynthesis pathway, and the cell dies. When chlorophyll synthesis is not active
enough, the amount of fully functional chlorophyll-binding proteins is insufficient to gain the optimal photosynthetic activities.
2. Such regulation is performed through transcriptional control of the genes encoding proteins involved in tetrapyrrole biosynthesis, and various coordination mechanisms that operate in a joint venture together.
3. That means, that the biosynthesis pathway, and its regulation, had to emerge together. This is an all-or-nothing business, and is therefore best explained by the instantiation all at once by an intelligent designer with foresight, intent, and the goal to create Chlorophyll, to permit advanced life on earth.


1. https://sci-hub.yncjkj.com/10.1146/annurev.arplant.57.032905.105448

10. Protoporphyrin IX Mg-chelatase

(1) glutamyl-tRNA synthetase; 
(2) glutamyl-tRNA reductase; 
(3) glutamate 1-semialdehyde aminotransferase;
(4) porphobilinogen synthase; 
(5) hydroxymethylbilane synthase; 
(6) uroporphyrinogen III synthase;
(7) uroporphyrinogen III decarboxylase; 
(8 ) coproporphyrinogen III oxidative decarboxylase; 
(9) protoporphyrinogen IX oxidase; 
(10) protoporphyrin IX Mg-chelatase; 
(11) S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase;
(12)–(14) Mg-protoporphyrin IX monomethyl ester oxidative cyclase; 
(15) divinyl (proto)chlorophyllide 4-vinyl reductase; 
(16) light-dependent NADPH:protochlorophyllide oxidoreductase or light-independent protochlorophyllide
reductase; 
(17) chlorophyll synthase.



Magnesium-chelatase is a three-component enzyme that catalyses the insertion of Mg2+ into protoporphyrin IX. This is the first unique step in the synthesis of chlorophyll and bacteriochlorophyll.[1][2] As a result, it is thought that Mg-chelatase has an important role in channeling intermediates into the (bacterio)chlorophyll branch 1 

The reaction: 5




Amino Acid sequence 863 aa
Nucleotide sequence:       2589 nt


https://www.genome.jp/entry/ajm:119045164

Robert D. Willows The Mg branch of chlorophyll synthesis: Biosynthesis of chlorophyll a from protoporphyrin IX 2019 1

The protein subunits are generally known as I, D and H with the prefix “Bch” and “bch” to the proteins and encoding genes, respectively, from bacteriochlorophyll-synthesizing organisms and “Chl” and “chl” to the proteins and encoding genes, respectively, from chlorophyll-synthesizing organisms

All subunits are highly conserved among bacteria and plants, with about 60 % of the amino acid positions invariant in all known sequences of BchI, 30 % in BchD and 40 % in BchH. 2

The I protein sequences belong to a unique protein family (pfam), has 41% sequence homology across the family. ( homology is similarity due to shared ancestry between a pair of structures or genes in different taxa. a )
The 2.1A˚ X-ray structure revealed that BchI belonged to the AAA+ class of proteins with a novel inverted domain structure linked by a long kinked alpha helix. 

In contrast, the D proteins are quite divergent in their sequences and they do not belong to a single pfam. The D proteins have an AAA+ (ATPases-associated with diverse cellular activities’)-N-terminal domain, similar to the I protein, which is linked to an integrin-I/von Willibrand C-terminal domain by a polyproline sequence containing 8–10 prolines followed by a low complexity highly charged domain.

The H protein sequences belong to a unique pfam, consisting of magnesium chelatase H and cobalt chelatase CobN subunits. 

The four substrates of this enzyme are ATP, protoporphyrin IX, Mg2+, and H2O; its four products are ADP, phosphate, Mg-protoporphyrin IX, and H+. This enzyme belongs to the family of ligases, specifically those forming nitrogen-D-metal bonds in coordination complexes.

The first step in the synthesis of chlorophyll from PPIX is the insertion of magnesium into the tetrapyrrole macrocycle to make magnesium proto porphyrin IX (MgPPIX). Magnesium chelatase requires a minimum of three protein subunits for activity and requires ATP hydrolysis and the substrates Mg2+ and PPIX. An accessory protein, GUN4, is also important for regulating magnesium chelatase activity in oxygenic photosynthetic organisms.



Dominique Pontier Knock-out of the Magnesium Protoporphyrin IX Methyltransferase Gene in Arabidopsis JANUARY 2007  3

Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process.

My comment: According to Wiki 4 Regulation is the management of complex systems according to a set of rules and trends. In systems theory, these types of rules exist in various fields of biology and society, but the term has slightly different meanings according to context. For example: In biology, gene regulation and metabolic regulation allow living organisms to adapt to their environment and maintain homeostasis; Was regulation of the process not something necessary right from the start ? That means, it would have had to evolve in a coordinated sense together with the enzyme activity, and afterward, somehow, brought together to work in a functional relationship?  But in order to coordinate something, foresight is necessary. Something that mindless evolutionary processes lack. 

The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes.

Filipa L. Sousa Chlorophyll Biosynthesis Gene Evolution Indicates Photosystem Gene Duplication, Not Photosystem Merger, at the Origin of Oxygenic Photosynthesis  19 December 2012 6

Mg Chelatase
The first unique intermediate of chlorophyll biosynthetic pathway, Mg-protoporphyrin IX, is generated by the insertion of Mg2+ into protoporphyrin IX. Biochemical and genetic analysis identified a Class I ATP-dependent magnesium chelatase, composed of three subunits BchH, BchI, and BchD, that catalyzes a reaction  consisting of an activation and a chelation step. In the presence of both ATP and Mg2+, an AAA+ motor complex is assembled from a hexameric or heptameric BchI ring connected to a hexameric BchD ring  (the activation step). Proto IX binds to the BchH catalytic subunit, and its transient interaction with the formed AAA+ motor complex leads to the insertion of Mg2+ in the tetrapyrrole macrocycle (the chelation step). 

The Mg-chelatase complex has both sequence and structural homology with the Class I cobalt chelatase of the O2-dependent cobalamin biosynthetic pathway. The CobN, CobS, and CobT subunits of the trimeric cobalt chelatase are homologous with the BchH/ChlH, BchI/ChlI, and BchD/ChlD subunits of magnesium chelatase, respectively. This homology has been interpreted as reflecting ancient duplication and divergence. Although there is a broad taxonomic distribution of the CobN gene among cobalamin-dependent organisms, a much narrower distribution is observed for the genes that compose the AAA+ motor complex (CobS and CobT).

The Catalytic Subunit BchH/ChlH
Chlorobaculum tepidum, similar to most members of GSB, has three paralogous genes ( Paralogous genes are genes that are related via duplication events in the last common ancestor (LCA) of the species being compared. ) for this subunit, named BchS, BchH, and BchT. Single- and double-mutant experiments showed that strains with only the BchH or the BchS gene retained sufficient Mg-chelatase activity to be viable, but its activity was maximal if the BchS gene was present together with BchH. This was also confirmed by biochemical characterization of the recombinant enzymes. However, in all mutagenesis experiments, a decrease in the production of bacteriochlorophyll c, the main photosynthetic pigment of GSB, was detected. This suggests that the different isoforms ( protein variants) function in end-product regulation and/or substrate channeling of the Bch c intermediates.

The existence of BchH/ChlH paralogs is not unique to the GSB. All chloroflexi have at least one additional gene coding this subunit, and the same is also true for some cyanobacterial species. Isoforms of the gene also occur in some eukaryotes  although, in this case, different duplication events are possibly at their origin.

The AAA+ Motor: BchI/ChlI and BchD/ChlD
The BchI/ChlI subunit is a member of the AAA+-ATPase family of proteins, which includes proteins of diverse function. With the exception of some land plants and green algae, there is a consensus in the literature that photosynthetic organisms only have one BchI/ChlI subunit. However, a study of two recombinant isoforms of BchI from Prosthecochloris vibrioformis showed that both had Mg chelatase activity in vitro, and in the recently sequenced genome of Chloroflexus aurantiacus, three genes were annotated as BchI. In our search, several isoforms belonging to both chlorobia and chloroflexi were retrieved. These enzymes are distinct from the aerobic cobalamin chelatase CobS gene and were included in the analysis.

The BchI/ChlI tree is divided into two clades, one comprising BchI/ChlI genes among all photosynthesizers and one containing the different isoforms from GSB and GNSB, which we call the GSB/GNSB clade. In the former, two major groups can be observed, one with heliobacteria, chlorobia, chloroflexi, acidobacteria, and proteobacteria (branching in that order) and a second one, where the cyanobacteria and eukaryotic sequences cluster together. The isoforms present in the GSB/GNSB clade, as in the case of G. violaceus ChlH2 sequence, have high similarity with sequences similar to hypothetical magnesium chelatases from nonphotosynthetic organisms. Although the results from P. vibrioformis indicate that both isoforms have identical activities in vitro, their sequences show higher similarity with sequences from organisms that do not synthetize bacteriochlorophyll, questioning their role in chlorophyll biosynthesis. Specifically, within delta-proteobacteria, proprionobacterales, firmicutes, and euryarchaeota, some organisms possess isoforms of the BchI/ChlI gene in addition to CobS. Other cobalamin producers possess only the BchI/ChlI gene and lack CobS. In these organisms, the BchI/ChlI genes probably substitute the missing CobNST genes. The distributions of BchI/ChlI and CobS homologs suggest a way in which pre-existing building blocks could have been recruited into the assembly of the ancestral chlorophyll and O2-dependent cobalamin pathway.

The N-terminus of BchD/ChlD gene, which is also a member of the AAA+-ATPase family of proteins, has a segment of approximately 260 amino acid residues with sequence homology to BchI/ChlI. BchD/ChlD has a proposed structural role in magnesium chelatase, functioning as a platform for the convergence of the other two subunits. The BchD/ChlD phylogenetic tree in figure 3C was rooted by two sequences of von Willebrand factor Type A, which is considered to be an ancient protein domain. The overall BchD/ChlD tree topology is very similar to the upper part of the BchH/ChlH tree with the exception of the position of C. Chloracidobacterium thermophilum. There are two distinct BchD/ChlD clades. One contains acidobacteria, the GSB/GNSB clade, and proteobacteria. In the second, heliobacteria, cyanobacteria, and eukaryotic sequences cluster. There are two eukaryotic clades, one composed of green algae isoforms that branch between heliobacteria and cyanobacteria, and a second in which at least one copy from every eukaryotic organism is represented in a sister group of the G. violaceus sequence. However, this branch is poorly supported. As in the case of BchI/ChlI, copies of this gene are also present in nonphotosynthetic organisms.

Xuemin Chen Crystal structure of the catalytic subunit of magnesium chelatase 24 August 2015 7

Tetrapyrroles, including haem and chlorophyll, play vital roles for various biological processes, such as respiration and photosynthesis, and their biosynthesis is critical for virtually all organisms. In photosynthetic organisms, magnesium chelatase (MgCh) catalyses insertion of magnesium into the centre of protoporphyrin IX, the branch-point precursor for both haem and chlorophyll, leading tetrapyrrole biosynthesis into the magnesium branch. This reaction needs a cooperated action of the three subunits of MgCh: the catalytic subunit ChlH and two AAA+ subunits, ChlI and ChlD. To date, the mechanism of MgCh awaits further elucidation due to a lack of high-resolution structures, especially for the ∼150 kDa catalytic subunit. Here we report the crystal structure of ChlH from the photosynthetic cyanobacterium Synechocystis PCC 6803, solved at 2.5 Å resolution. The active site is buried deeply inside the protein interior, and the surrounding residues are conserved throughout evolution. This structure helps to explain the loss of function reported for the cch and gun5 mutations of the ChlH subunit, and to provide the molecular basis of substrate channelling during the magnesium-chelating process. The structure advances our understanding of the holoenzyme of MgCh, a metal chelating enzyme other than ferrochelatase. 

Chlorophyll, the most abundant pigment in plants, algae and cyanobacteria, is synthesized through a multistep pathway in which protoporphyrin IX (Proto) is the branch-point precursor for both haem and chlorophyll. In contrast to the single-subunit ATP-independent ferrochelatase that catalyses insertion of a ferrous iron into the Proto ring6 , MgCh comprises three subunits, ChlH, ChlI and ChlD, and requires ATP for magnesium chelation. To complete a catalytic cycle, the three subunits cooperate in a dynamic manner with the AAA+ subunits ChlI and ChlD assembling into a two-tiered hexameric ring, and then interacting with the substrate-binding ChlH subunit to form a transient holoenzyme complex . 


Crystal structure of the ChlH subunit. 
a, Three surface models related by 90° rotation along the vertical axis. The head (I) domain is blue, the neck (II) domain in green, domain III in orange, the insertion (IV) domain in brown, domain V is magenta, and domain VI is purple. 
b, Schematic representation of Synechocystis ChlH subunit. Regions not visible in the crystal structure include residues 140–156, 221–229, 327–330, 380–391, 415–424 and 630–640. The boundary between domains I and II that cannot be defined (residues 221–229) is grey. 
c, Domains I–VI as ribbon diagrams

Joakim Lundqvist ATP-Induced Conformational Dynamics in the AAA+ Motor Unit of Magnesium Chelatase MARCH 10, 2010 8

Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg2+ into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 Å, 14 Å, and 13 Å resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.

Introduction
The enzyme magnesium chelatase (Mg-chelatase) is active in the branch point between chlorophyll and heme biosynthesis. It catalyzes the insertion of Mg2+ into protoporphyrin IX (PPIX), which is the first committed reaction of the chlorophyll biosynthesis pathway. Mg-chelatase belongs to the class of AAA+-type chelatases. This is also true for aerobic cobaltochelatase and nickel chelatase, which are active in cobalamin (vitamin B12) and coenzyme F430 biosynthesis, respectively. Magnesium chelatase is currently the most extensively studied enzyme in this class of AAA+ chelatases. Its activity requires the presence of three subunits, BchH, BchD, and BchI, which have molecular masses of approximately 140, 70, and 40 kDa, respectively. Biochemical studies have suggested that the enzymatic reaction proceeds in distinct steps.

Question: Had these steps not to be fully functional, and intermediates would be non-functional, and not selected by natural selection, since not confering any advantage of function, nor survival ?

In the first step, the AAA+ motor complex between subunits BchI and BchD is formed in the presence of ATP and Mg2+, while the largest subunit, BchH, binds PPIX by an unknown mechanism. Our recent work has suggested that subunit BchD may serve as a platform for the assembly of the complex.  Binding of PPIX to BchH induces a large conformational rearrangement in this subunit.

In the reaction step that follows, the BchH:PPIX complex interacts with the BchI:BchD complex, leading to insertion of Mg2+ into PPIX. During this part of the reaction, the BchH:PPIX complex is a substrate of the BchI:BchD complex. It has been estimated that around 15 ATP molecules may be required for each catalytic cycle. The BchI subunit, which contains the characteristic ATP binding Walker A and Walker B motifs (GX4GKSX6A and hhhhD(D/E), where h is any hydrophobic residue), is responsible for ATP hydrolysis. The X-ray crystallographic structure of Rhodobacter capsulatus BchI has been determined and it has been shown to belong to the AAA+ family of ATPases. The protein was later assigned to the pre-sensor II (PS-II) insert clade of the AAA+ family, which includes the MCM (minichromosome maintenance) family of helicases, the MoxR family of molecular chaperones, and the dynein/midacin family of ATP-dependent motors, the members of which are known to interact with microtubules and the nuclear pore complex. A characteristic feature of AAA+ proteins is the formation of oligomeric ring structures with the most common ring types consisting of 6 or 7 monomers. Electron microscopy (EM) and single-particle analysis has indeed shown that in the presence of ATP, R. capsulatus BchI and the corresponding subunit ChlI from Synechocystis sp. PCC6830 can form hexameric and heptameric ring structures, respectively.

Amino acid sequence analysis has demonstrated that subunit BchD, which is the second in size after BchH, has an AAA+ module at its N terminus with distinct homology to BchI. However, the Walker A and Walker B motifs, which are necessary for ATP hydrolyzing activity, are poorly conserved in this subunit. Despite this, BchD is still capable of forming oligomeric ring structures, even in the absence of ATP. Interestingly, the C-terminal part of BchD was found to contain a domain homologous to a class of proteins termed integrin I domains, which is a subgroup of a larger group of von Willebrand factor A domain proteins. These domains are usually found as part of larger complexes, and they are the principal receptors on the surfaces of animal cells, being involved in the binding of most extracellular matrix proteins. Integrin I domains are characterized by the MIDAS motif (metal ion-dependent adhesion site), which constitutes a unique Mg2+/Mn2+ binding site. The same type of domain is present in cobaltochelatase and in the MoxR family of AAA+ proteins. Mutation of residues in the MIDAS motif of R. capsulatus BchD (D385A and S387A) was found to abolish Mg-chelatase activity. The integrin I domain and the N-terminal AAA+ module of BchD are linked to each other by a proline and an acidic residue-rich region. These types of domains are often involved in protein-protein interactions. Based on this knowledge, the region was suggested to be involved in the stabilization of the BchI:BchD complex.



a https://en.wikipedia.org/wiki/Homology_(biology)

1. http://pfam.xfam.org/family/PF01078
2. https://www.sciencedirect.com/science/article/abs/pii/S002228360194834X
3. https://www.jbc.org/article/S0021-9258(20)72099-5/fulltext
4. https://en.wikipedia.org/wiki/Regulation
5. https://www.genome.jp/entry/R03877
6. https://academic.oup.com/gbe/article/5/1/200/729709
7. https://www.nature.com/articles/nplants2015125   https://sci-hub.yncjkj.com/10.1038/nplants.2015.125
8. https://www.cell.com/structure/fulltext/S0969-2126(10)00031-6?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0969212610000316%3Fshowall%3Dtrue

11. Mg-protoporphyrin IX methyltransferase

(1) glutamyl-tRNA synthetase;
(2) glutamyl-tRNA reductase;
(3) glutamate 1-semialdehyde aminotransferase;
(4) porphobilinogen synthase;
(5) hydroxymethylbilane synthase;
(6) uroporphyrinogen III synthase;
(7) uroporphyrinogen III decarboxylase;
(8 ) coproporphyrinogen III oxidative decarboxylase;
(9) protoporphyrinogen IX oxidase;
(10) protoporphyrin IX Mg-chelatase;
(11) S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase;
(12)–(14) Mg-protoporphyrin IX monomethyl ester oxidative cyclase;
(15) divinyl (proto)chlorophyllide 4-vinyl reductase;
(16) light-dependent NADPH:protochlorophyllide oxidoreductase or light-independent protochlorophyllide
reductase;
(17) chlorophyll synthase.

This enzyme belongs to the family of transferases, specifically those transferring one-carbon group methyltransferases. 4


In enzymology, a magnesium protoporphyrin IX methyltransferase (EC 2.1.1.11) is an enzyme that catalyzes the chemical reaction
S-adenosyl-L-methionine + magnesium protoporphyrin IX {\displaystyle \rightleftharpoons } S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester
The two substrates of this enzyme are S-adenosyl methionine and magnesium protoporphyrin IX; its two products are S-adenosylhomocysteine and magnesium protoporphyrin IX 13-methyl ester.
This enzyme belongs to the family of transferases, specifically those transferring one-carbon group methyltransferases. The systematic name of this enzyme class is S-adenosyl-L-methionine:magnesium-protoporphyrin-IX O-methyltransferase. This enzyme is part of the biosynthetic pathway to chlorophylls.^[1][2] 

Dominique Pontier Knock-out of the Magnesium Protoporphyrin IX Methyltransferase Gene in Arabidopsis JANUARY 2007 2

Protoporphyrin IX is the last common intermediate between the heme and chlorophyll biosynthesis pathways. The addition of magnesium directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the magnesium chelatase and is a highly regulated process. The corresponding product, magnesium protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. To get more information on the chlorophyll biosynthesis pathway and on magnesium protoporphyrin IX derivative functions, we have identified an magnesium protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from magnesium protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cytochrome b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that magnesium protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression. Moreover, it suggests the possible implication of magnesium protoporphyrin IX methyl ester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the magnesium chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step.

CHLM Is a Key Gene for Chlorophyll Biosynthesis and Chloroplast Development—Our results show that chlorophyll formation is totally dependent on the CHLM gene product in Arabidopsis. The inactivation of this gene prevents setting up of chlorophyll-binding proteins in the thylakoids, whereas most other proteins in the chloroplast remain relatively stable. Not only photosystem I and II with their associated light harvesting complex are affected but also the cytochrome b6f complex that contains very low amounts of chlorophyll

Xuemin Chen Structural Insights into the Catalytic Mechanism of Synechocystis Magnesium Protoporphyrin IX O-Methyltransferase (ChlM)* SEPTEMBER 2014 3

Magnesium protoporphyrin IX O-methyltransferase (ChlM) catalyzes transfer of the methyl group from S-adenosylmethionine to the carboxyl group of the C13 propionate side chain of magnesium protoporphyrin IX. This reaction is the second committed step in chlorophyll biosynthesis from protoporphyrin IX. Here we report the crystal structures of ChlM from the cyanobacterium Synechocystis sp. PCC 6803 in complex with S-adenosylmethionine and S-adenosylhomocysteine at resolutions of 1.6 and 1.7 Å, respectively. The structures illustrate the molecular basis for cofactor and substrate binding and suggest that conformational changes of the two “arm” regions may modulate binding and release of substrates/products to and from the active site. Tyr-28 and His-139 were identified to play essential roles for methyl transfer reaction but are not indispensable for cofactor/substrate binding. Based on these structural and functional findings, a catalytic model is proposed.

ChlM (E.C. 2.1.1.11) catalyzes methyl transfer from the common methyl donor SAM to the carboxyl group of the C13 propionate side chain of MgP, resulting in the formation of MgPME and SAH. Genetic studies have shown that ChlM is essential for chlorophyll biosynthesis and chloroplast development.



^{1. https://en.wikipedia.org/wiki/Magnesium_protoporphyrin_IX_methyltransferase}

2. https://www.jbc.org/article/S0021-9258(20)72099-5/fulltext
3. https://www.jbc.org/article/S0021-9258(20)31525-8/fulltext
4. https://www.creative-enzymes.com/product/magnesium-protoporphyrin-ix-methyltransferase_11878.html

12. Mg-protoporphyrin IX monomethyl ester oxidative cyclase

(1) glutamyl-tRNA synthetase;
(2) glutamyl-tRNA reductase;
(3) glutamate 1-semialdehyde aminotransferase;
(4) porphobilinogen synthase;
(5) hydroxymethylbilane synthase;
(6) uroporphyrinogen III synthase;
(7) uroporphyrinogen III decarboxylase;
(8 ) coproporphyrinogen III oxidative decarboxylase;
(9) protoporphyrinogen IX oxidase;
(10) protoporphyrin IX Mg-chelatase;
(11) S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase;
(12)–(14) Mg-protoporphyrin IX monomethyl ester oxidative cyclase;
(15) divinyl (proto)chlorophyllide 4-vinyl reductase;
(16) light-dependent NADPH:protochlorophyllide oxidoreductase or light-independent protochlorophyllide
reductase;
(17) chlorophyll synthase.

David W. Bollivar’  The Chlorophyll Biosynthetic Enzyme Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase’ 1996  3 

In aerobic chlorophyll forming organisms, the cyclizing enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase catalyzes a complex reaction that appears to consist of at least three steps (Wong et al., 1985): hydroxylation of the methylpropionate at the P-carbon atom, oxidation of the hydroxyl group to a carbonyl group, and ligation of the a-carbon of the /3-ketomethylpropionate to the 7-meso bridge carbon between the C and D pyrrole rings of the tetrapyrrole

The results indicate that Fe2+ is required for activity of the cyclase from both eukaryotic and prokaryotic sources and also suggest that the Fe2+ ion is associated with the membrane component of the enzyme. A role for Mg2+ in stabilizing the enzyme has been established, but the mechanism of this function needs further clarification.



KaoriYamanashi Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria 7 August 2015  1

The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. 

Chlorophylls (Chls) are tetrapyrrole pigments that are essential for photosynthesis. The fifth ring (E-ring or isopentanone ring) of Chl is a unique structural feature different from other tetrapyrrole pigments such as heme and vitamin B12. During the biosynthesis of Chl, the E-ring is formed by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase, which converts MPE to 3,8-divinyl protochlorophyllide (Pchlide). This reaction involves a six-electron oxidation and formation of an oxo group at C131 (Fig. 1).


Fig. 1. MPE cyclase reaction. The C13-methylpropionate in MPE and the E-ring of Pchlide are highlighted in yellow. The oxygen atom of the oxo group at C131 is derived from water and oxygen in oxygen-independent (ChlE/BchE) and -dependent (ChlA/ AcsF) MPE cyclase reactions, respectively

There are two evolutionarily unrelated MPE cyclases in photosynthetic organisms. 

My comment:  Convergence, another problem for evolution 2

Biologists are uncovering numerous examples of organisms that cluster together morphologically (structurally), and yet are genetically distinct. Frogs, lizards, or herbs that appear to be identical are actually different at the genetic level. An evolutionary interpretation of this data, then, demands that the morphologically identical organisms must have evolved independently of one another in a “repeatable” fashion.

“…No finale can be specified at the start, none would ever occur a second time in the same way, because any pathway proceeds through thousands of improbable stages. Alter any early event, ever so slightly, and without apparent importance at the time, and evolution cascades into a radically different channel.1

Stephen J. Gould, Wonderful Life: The Burgess Shale and the Nature of History (New York, NY: W.W. Norton & Company, 1989), 51.

Gould’s metaphor of “replaying life’s tape” asserts that if one were to push the rewind button, erase life’s history, and let the tape run again, the results would be completely different.2 The very essence of the evolutionary process renders evolutionary outcomes as nonreproducible (or nonrepeatable). Therefore, “repeatable” evolution is inconsistent with the mechanism available to bring about biological change.

Paleontologist J. William Schopf, one of the world’s leading authorities on early life on Earth, has made this very point in the book Life’s Origin.

Because biochemical systems comprise many intricately interlinked pieces, any particular full-blown system can only arise once…Since any complete biochemical system is far too elaborate to have evolved more than once in the history of life, it is safe to assume that microbes of the primal LCA cell line had the same traits that characterize all its present-day descendents.

http://www.upenn.edu/pennnews/news/evolution-unpredictable-and-irreversible-penn-biologists-show

Gould’s famous tape of life would be very different if replayed, even more different than Gould might have imagined.”

Evolutionary theorist Stephen Jay Gould is famous for describing the evolution of humans and other conscious beings as a chance accident of history. If we could go back millions of years and “run the tape of life again,” he mused, evolution would follow a different path.

One is an oxygen-independent MPE cyclase, BchE, named after the gene bchE of Rhodobacter capsulatus. The other is an oxygen-dependent MPE cyclase. In this report, we use the term ChlA/AcsF. BchE belongs to the radical Sadenosylmethionine (SAM) superfamily and has an ironesulfur cluster, which is extremely vulnerable to oxygen. BchE forms the C131 oxo group using oxygen from a water molecule. In contrast, ChlA/AcsF is a monooxygenase that incorporates an oxygen molecule (O2) to form the C131 oxo group. BchE and ChlA/AcsF are mainly identified in anoxygenic photosynthetic bacteria and plants, respectively. However, some photosynthetic bacteria have either both or one of the two enzymes. The MPE cyclase reactions consist of three sequential two-electron oxidation steps in both systems. The BchE MPE cyclase has been assayed in the crude extract of R. capsulatus: the results show its dependence on vitamin B12. However, no reconstituted system using purified BchE protein has been reported. The ChlA/AcsF MPE cyclase activity has been detected in lysed plastids and crude extracts of cyanobacteria. Other probable subunit proteins and a protein stimulating the activity have been identified. However, this oxygen-dependent MPE cyclase has not been reconstituted using the purified proteins either. These MPE cyclases remain the least understood of the Chl biosynthesis enzymes. Cyanobacteria are prokaryotes that perform oxygenic photosynthesis in a manner similar to the plant photosynthesis. In our previous work, we have identified two homologous genes encoding two ChlA isoforms, ChlAI (CycI) and ChlAII (CycII), in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803). The chlAI gene is constitutively expressed and essential for photoautotrophic growth under aerobic conditions, and the chlAII gene is expressed only in low-oxygen conditions to complement the ChlAI activity. There are three bchE-like genes in Synechocystis 6803.

Guangyu E. Chen How the O2-dependent Mg-protoporphyrin monomethyl ester 2 cyclase forms the fifth ring of chlorophylls 4

The decisive biosynthetic step that determines the absorption properties of chlorophyll, and more visually its green color, is the formation of the unique isocyclic fifth ring. This process involves the conversion of Mg-protoporphyrin IX monomethyl ester (MgPME) to 3,8-divinyl  protochlorophyllide a (DV PChlide a), and it requires incorporation of an oxygen atom, sourced from either water or O2, indicating the existence of two mechanistically different MgPME cyclases. Most anoxygenic phototrophic bacteria utilise an O2-sensitive radical SAM enzyme containing [4Fe-4S] and cobalamin cofactors to catalyse the reaction3 , while oxygenic phototrophs including cyanobacteria, algae and plants, as well as some purple bacteria, adopt an O2-dependent cyclase for the reaction. 

David Stuart Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin  : 8 September 2020

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR.  Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions. 

Observation: Ferredoxin and FNR are required for the catalytic activity of the enzyme that synthesizes Chlorophyll. But ferredoxin is part of the operational electron transport chain in photosynthesis. That raises a chicken and egg problem. The electron transport chain is not operational without chlorophyll funneling electrons to the PS I and II reaction center, which starts the electron transport chain. But the making of chlorophyll depends on the electron transport chain.  

1. https://www.sciencedirect.com/science/article/abs/pii/S0006291X15301789
2. https://reasonandscience.catsboard.com/t2014-convergence-another-problem-for-evolution
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC157929/pdf/1120105.pdf
4. https://eprints.whiterose.ac.uk/180884/1/Chen_et_al_2021_Nat_Plants_7__365%E2%80%93375_Author_Accepted_Manuscript..pdf