Your Inner Locomotive Revealed

Your Inner Locomotive Revealed

https://reasonandscience.catsboard.com/t2140-nadh-dehydrogenase-complex-i-in-mitochondria

NADH dehydrogenase ( Complex I ) in mitochondria 1
The molecular biological achievements of the last two decades culminated in 2010 with the deciphering of the crystal structure of another respiratory complex, the enormous (for a protein) complex I, by Walker's Cambridge colleague Leonid Sazanov (Efremov et al. 2010). Again, the structure betrays the mechanism — in this case not a rotary motor but, even more surprisingly, a lever mechanism not unlike the piston of a steam engine 6

ATP synthase operates at the end of a sequence of machines in the respiratory chain that generates chemical energy (in the form of ATP) from the food we eat (or from sunlight, in the case of plants).  The enzyme runs on proton motive force – a flow of protons that drive its carousel-like rotor.  But how does the proton gradient get established?  That’s the job of Respiratory Complex I, the first machine (enzyme) in the chain.  Complex I takes electrons from food, stored in NADH molecules, and transfers them down a chain of electron receptors to parts of the machine that pump protons across the mitochondrial membrane into the periplasm setting up a proton gradient. It now becomes evident that Complex I includes parts that move like pistons. Complex I was reported in a  Science Express paper as having a railroad-like coupling rod.

It now appears  that the we have  trillions of mechanical devices similar like those coupling rods.  They serve to transmit the energy in the food we eat into mechanical energy, driving a proton pump inside the mitochondrion.  It’s all part of an amazing series of electromechanical machines in the powerhouses of the cell.
NADH dehydrogenase, also called Mitochondrial Complex I, is an essential part of the respiration process (also called oxidative phosphorylation) that passes electrons, protons and oxygen through a sophisticated energy transport chain so that energy can be stored in ATP.Complex I, composed of four major parts and shaped somewhat like a hockey stick, produces 40% of the proton motive force used by ATP synthase to produce ATP.  Its job is to derive protons from NADH and hand them off to additional cofactors and enzymes in the transport chain that will pump the protons outside the mitochondrial membrane.  The electrical potential thus created across the membrane drives the ATP synthase rotary engine at the end of the chain.
 In discussing the paper published in Science Express 3, Science Daily contained some amazing facts about the machinery of respiration and how it delicately handles explosive ingredients:

   In a laboratory experiment, hydrogen and oxygen gas would react in an explosion and the energy contained would be released as heat.  In biological oxidation, the energy will be released by the membrane bound protein complexes of the respiratory chain in a controlled manner in small packages.  Comparable to a fuel cell, this process generates an electrical membrane potential, which is the driving force of ATP synthesis.  The total surface of all mitochondrial membranes in a human body covers about 14  square meters.  This accounts for a daily production of about 65 kg of ATP. 

That 65 kg, by the way, is near a typical human body weight.  That’s how much ATP the human body synthesizes each day – even during sleep.  At any one time, though, our body only contains the ATP equivalent of a AA battery .  The electrical potential generated across that 14 square meters of mitochondrial membrane drives the ATP synthesis that keeps us – and every living thing  alive.
What’s new and exciting  is  that a coupling rod is part of the energy transport chain.  Higher up in the enzyme, a series of iron-sulfur clusters works something like an electrical wire, transferring electrons from the first domain into the second domain.  The transport is constructed to prevent the formation of dangerous reactive oxygen species (ROS).  Between the second and third domains, it appears that the electrical energy is converted into mechanical energy via a coupling rod composed of a 6-nanometer alpha coil that is “critical for transducing conformational energy to proton-pumping elements in the distal module of the membrane arm.”  In other words, it has moving parts. 



Quips Pumping protons: a complex problem 7
Cells use a lot of ATP. An E.coli cell can use a billion ATP molecules per minute and humans use their own weight in ATP every day. Because of this, cells need an efficient mechanism to recombine ADP with phosphate to regenerate ATP, and much of the energy in our food is used in this process. ATP synthase, the enzyme that makes ATP, is located in the mitochondrial inner membrane (or the bacterial cell membrane), and is powered by a concentration difference (gradient) of protons across the membrane.

Figure 1. Schematic overview of the respiratory chain showing how complex I (blue ), complex II (green), complex III (red) and complex IV (light blue) produce the proton gradient used by ATP synthase (orange) to generate ATP. Cytochrome c is shown in magenta and shuttles electrons between complexes III and IV. Q stands for quinone.
Four different protein complexes make up the respiratory chain that pumps protons across the cell membrane: ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV). These generate the proton gradient that drives ATP synthase (Figure 1). Complex I oxidises NADH (formed in glycolysis and the Krebs cycle) to pump four protons across the membrane. Complex I also uses the reductive power of NADH to reduce quinone, which is then used as a substrate by complex III to pump two more protons across the membrane.

One ‘ell of a structure.

Figure 2. Schematic overview of T. thermophilus complex I, highlighting the 16 subunits, the iron sulfur cluster chain and the NADH and quinone (Q) binding sites.
In mammals, complex I consists of 44 different subunits and electron microscopy studies have revealed that it has an L shape. One arm of the L is inserted in the membrane (the membrane arm) and the other arm (the hydrophilic arm) projects some 100 Å into the mitochondrial matrix. Bacteria also possess a complex I, but it is a ‘cut down’ version of its eukaryotic counterpart, containing at least 14 of the subunits found in the mitochondrial enzyme. 

The crystal structure of complex I from the bacterium Thermus thermophilus (PDB entry 4hea) shows that it also has the characteristic L shape(view-1) with the hydrophilic arm projecting into the cytoplasm. 



(view-1)View 1: Overall architecture of complex I.The 16 proteins making up complex I are shown in cartoon representation. Initially, the hydrophilic (red) and membrane (cyan) arms are highlighted; subsequently, each chain is coloured separately using the same colour scheme as in Figure 2. The iron-sulfur clusters (yellow and orange) and FMN (withgreen

The complex consists of 16 protein subunits with a combined molecular weight of 536 . The complex contains seven Fe4S4 and two Fe2S2 iron-sulfur clusters and a bound flavin mononucleotide (FMN) as co-factors (Figure 2). Seven of the subunits span the membrane and contain 64 transmembrane helices in total.

Where does NADH enter complex I?

The NADH substrate-binding site is at the distal end of the hydrophilic arm in subunit Nqo1 (view-2). The site is formed from a modified Rossmann fold which also incorporates a FMN-binding site. The structure of the hydrophilic arm of T. thermophilus complex I with bound NADH (PDB entry 3iam) shed light on the interactions between FMN and NADH. The adenine ring of NADH is held in place by stacking against three phenylalanine residues. This allows the nicotinamide ring of NADH to stack against the isolloxazine ring of the bound FMN (view-2). Electron transfer then occurs between the C4N atom of the nicotinamide ring of NADH and N5 of FMN. The architecture of the binding site positions these atoms within 3.2 Å of each other for efficient electron transfer. A glutamate residue in the binding site appears to help position the two rings close together.






View 2: NADH binding and electron transfer. NADH (salmon carbons) in Nqo1 is shown in stick representation at the top of complex I, adjacent to the bound FMN molecule (green carbons). A dashed line between FMN atom N5 and C4N of NADH indicates the electron transfer path. Nqo1 residues that interact with the FMN or NADH are shown as sticks. Iron-sulfur clusters are shown as large yellow and orangespheres.

Reducing the problem.

Subunits Nqo1, Nqo3, Nqo6 and Nqo9 contain seven of the iron-sulfur clusters in the complex. These form a “wire” some 90 Å long which carries electrons from the FMN, through the hydrophilic arm to the quinone-binding site formed by Nqo4, Nqo6 and Nqo8 (view-3). The iron-sulfur clusters are at most 14 Å apart (edge to edge distance), allowing efficient electron transfer between the clusters(view-3). Nqo2 and Nqo3 contain additional iron-sulfur clusters, but the function of these clusters is unknown.

Figure 3. Menaquinone 8 used by T. thermophilus complex I.



View 3: Electrons travel down the "wire" of iron-sulfur clusters. The chain of iron-sulfur clusters down which electrons travel from NADH to the quinone-binding site is shown using spheres. Their path is highlighted in the animation by enlarging each cluster in turn.

NADH donates two electrons to the iron-sulfur cluster “wire” which are transferred one at a time along the clusters and used to reduce the bound quinone. Different organisms use different quinones, and T. thermophilus usesmenaquinone-8 (Figure 3). Menaquinone-8 binds in a 30 Å long cavity formed by subunits Nqo4, Nqo6 and Nqo8 at the interface between the two arms. The entrance to the cavity, through which the quinone has to pass, is surprisingly narrow, around 2.3 x 4.5 Å (view-4), suggesting movement in this region to allow quinone binding. The quinone-binding cavity contains hydrophilic residues along its length, which may interact with the hydrophilic head group of the quinone as it enters the cavity. Residues His38, Tyr87 and Asp139 in Nqo4, at the distal end of the cavity, probably position the quinone close to the last iron-sulfur cluster in the “wire” to allow for efficient reduction of the quinone. After being reduced, the quinone leaves complex I and is subsequently used as a substrate by complex III.





View 4: The quinone binding site. The residues that line the quinone binding site in Nqo4 (darkgreen), Nqo6 (darkred) and Nqo8 (orange) are shown in stick representation.

We all pump together.

When complex I reduces the bound quinone it also pumps four protons across the membrane. Complex I contains four potential channels within the membrane which are likely to perform this role. Subunits Nqo12, 13 and 14 are homologous to each other and to sodium/proton antiporters, indicating that they most likely form the proton-pumping channels 1, 2 and 3, respectively. Subunits Nqo8, Nqo10 and Nqo11 also form a channel across the membrane, channel 4, which links the other channels to the hydrophilic domain (view-5). 






View 5: Proton-pumping channels. The subunits containing the four proton pumping channels are highlighted, followed by the C-terminal helix in Nqo12. Subsequently, charged residues are shown as sticks, highlighting a line of charged residues in the centre of the membrane arm.

So how does reduction of the quinone result in the pumping of four protons across the membrane? The most obvious assumption is that each channel pumps a single proton. But this begs the question how the channels are coordinated, especially as the distal channel is 130 Å from the quinone binding site. Each channel contains two transmembrane helices which have a break in the middle with a charged residue at this point (view-5). It is likely that these charged residues form a path for the proton as it makes its way through the channel. Other charged residues appear to connect the proton pumping channels together, possibly providing a mechanism of coordination between the channels. The C-terminus of Nqo12 (containing channel 1) consists of a 104 Å long α-helix which runs along the surface of the membrane before terminating adjacent to Nqo14. The role of this helix is unknown, although it interacts with one of the two broken transmembrane helices in each of the channels. Additional charged residues link the quinone binding site to the adjacent channel (channel 4), suggesting that they may initiate proton pumping upon reduction of the quinone (view-5). Further work is needed to investigate the mechanism of proton pumping.

A long-distance relationship that works.

NADH is used as a reducing agent by complex I, donating two electrons that reduce a quinone molecule and drive the pumping of four protons across the membrane in four channels. Amazingly, the NADH substrate is 85 Å away from the quinone and the quinone is 140 Å removed from the most distant channel! Complex I displays a truly remarkable form of long-distance communication.

The NADH Dehydrogenase Complex Contains Separate Modules for Electron Transport and Proton Pumping
The NADH dehydrogenase complex is a massive assembly of membrane and nonmembrane proteins that receives electrons from NADH and passes them to ubiquinone. In animal mitochondria, it consists of more than 40 different protein subunits, with a molecular mass of nearly a million daltons. The x-ray structures of the NADH dehydrogenase complex from fungi and bacteria show that it is L-shaped, with both a hydrophobic membrane arm and a hydrophilic arm that projects into the mitochondrial matrix



The structure of NADH dehydrogenase. (A) The model of the mitochondrial complex shown here is based on
the x-ray structure of the smaller bacterial complex, which works in the same way. The matrix arm of NADH dehydrogenase
(also known as Complex I) contains eight iron–sulfur (FeS) clusters that appear to participate in electron transport. The
membrane contains more than 70 transmembrane helices, forming three distinct proton-pumping modules, while the matrix arm
contains the electron-transport cofactors. (B) NADH donates two electrons, via a bound flavin mononucleotide (FMN; yellow),
to a chain of seven iron–sulfur clusters (red and yellow spheres). From the terminal iron–sulfur cluster, the electrons pass to
ubiquinone (orange). Electron transfer results in conformational changes (black arrows) that are thought to be transmitted to a
long amphipathic α helix (purple) on the matrix side of the membrane arm, which pulls on discontinuous transmembrane helices
(red) in three membrane subunits, each of which resembles an antiporter (see Chapter 11). This movement is thought to change
the conformation of charged residues in the three proton channels, resulting in the translocation of three protons out of the
matrix. A fourth proton may be translocated at the interface of the two arms (dotted line). (C) This shows the symbol for NADH
dehydrogenase used throughout this chapter.

Electron transfer and proton pumping are physically separated in the NADH dehydrogenase complex, with electron transfer occurring in the matrix arm and proton pumping in the membrane arm. The NADH docks near the tip of the matrix arm, where it transfers its electrons via a bound flavin mononucleotide to a string of iron–sulfur clusters that runs down the arm, acting like a wire to carry electrons to a protein-bound molecule of ubiquinone. Electron transfer to the quinone is thought to trigger proton translocation in a set of proton pumps in the membrane arm, and for this to happen the two processes must be energetically and mechanically linked. A mechanical link is thought to be provided by a 6-nm long, amphipathic α helix that runs parallel to the membrane surface on the matrix side of the membrane arm. This helix may act like the connecting rod in a steam engine to generate a mechanical, energy-transducing power stroke that links the quinone-binding site to the proton-translocating modules in the membrane. The reduction of each quinone by the transfer of two electrons can cause four protons to be pumped out of the matrix into the crista space. In this way, NADH dehydrogenase generates roughly half of the total proton-motive force in mitochondria.

Nanomachines in the powerhouse of the cell 4
Scientists of the University of Freiburg and the University of Frankfurt have elucidated the architecture of the largest protein complex of the cellular respiratory chain.They discovered an unknown mechanism of energy conversion in this molecular complex. The mechanism is required to utilize the energy contained in food. After ten years of research work, the x-ray crystallographic analysis of the huge and most complicated protein complex of the mitochondrial respiratory chain was successful. The complex contains more than 40 different proteins, marks the entry to cellular respiration and is thus also called mitochondrial complex I. The results are published in the current online-edition of the journal “Science”.

A detailed understanding of the function of complex I is of special medical interest. Dysfunction of the complex is implicated in several neurodegenerative diseases such as Parkinson´s disease or Alzheimer´s disease, and also with the physiological processes of biological aging, in general.
The energy metabolism takes place in the so-called powerhouses of the cell, the mitochondria. They transduce the energy taken up as food into adenosine triphosphate, in short ATP, which is the universal energy currency of life. A chain of five complicated molecular machines in the mitochondrial membrane are responsible for the energy conversion. The production of ATP in mitochondria requires so many steps, as it is in principal a Knallgasreaction. In a laboratory experiment, hydrogen and oxygen gas would react in an explosion and the energy contained would be released as heat. In biological oxidation, the energy will be released by the membrane bound protein complexes of the respiratory chain in a controlled manner in small packages. Comparable to a fuel cell, this process generates an electrical membrane potential, which is the driving force of ATP synthesis. The total surface of all mitochondrial membranes in a human body covers about 14.000 square meter. This accounts for a daily production of about 65 kg of ATP.
The now presented structural model provides important and unexpected insights for the function of complex I. A special type of „transmission element“, which is not known from any other protein, appears to be responsible for the energy transduction within the complex by mechanical nanoscale coupling. Transferred to the technical world, this could be described as a power transmission by a coupling rod, which connects for instance the wheels of a steam train. This new nano-mechanical principle will now be analysed by additional functional studies and a refined structural analysis.





Key helices and residues of complex I are depicted schematically. Upon electron transfer from the Fe–S cluster N2, negatively charged quinone (or charged residues nearby) initiates a cascade of conformational changes, propagating from the E-channel (at Nqo8, Nqo10 and Nqo11) to the antiporters via the central axis (indicated by grey arrows) comprising charged and polar residues that are located around flexible breaks in key transmembrane helices (TMHs). Cluster N2-driven shifts (dashed arrows) of Nqo4 and Nqo6 helices33 (not shown) are likely to assist overall conformational changes. Helix HL and the βH element help to coordinate conformational changes by linking discontinuous TMHs between the antiporters. Key charged residues can be protonated from the cytoplasm through several possible pathways, including inter-subunit transfer (indicated by black arrows) (Fig. 3). Following the reduction of quinone and completion of conformational changes, Lys or GluTM12 in the antiporters and Glu32 from Nqo11 in the E-channel each eject a proton into the periplasm. TMHs are numbered and key charged residues (that is, GluTM5, LysTM7, Lys or HisTM8 and Lys or GluTM12 from Nqo12–Nqo14, as well as Glu67 and Glu32 from Nqo11, which interacts with Tyr59 from Nqo10, Glu213 from Nqo8 and some residues from the connection to the quinone cavity) are indicated by red circles for Glu, blue circles for Lys or His, and white circle for Tyr. FMN, flavin mononucleotide. Figure from Ref. 9, Nature Publishing Group.


The authors of the paper did not mention evolution.  The only oblique reference is that the working parts are “highly conserved” (unevolved) throughout the entire realm of life:

Fourteen central subunits are highly conserved among eukaryotes and prokaryotes.  They form the structural core of the two arms of the complex and are essential for its bioenergetic functions.  26 accessory subunits that are not found in prokaryotes  endosymbiosis hello ??!! are arranged around this core and presumably function in assembly, stabilization, regulation and additional metabolic pathways not directly linked to energy conservation.

Four other times the paper mentioned that key elements of the enzyme are conserved or highly conserved.  There was no attempt to explain how this “nano-mechanical principle” emerged or evolved.  They did mention, though, that mutations and dysfunctions in Complex I that allow the formation of ROS are implicated in debilitating conditions like Parkinson’s and Alzheimer’s disease – and perhaps in the aging process itself.

   Isn’t this wonderful information?  Now we see that the respiratory transport chain in mitochondria includes coupling rods that act like little locomotives.  Those rods must be moving incredibly fast.  They are pumping protons like gangbusters, 24x7, all the years of your life.  This mechanical wonder is only one amazing device in the first stage of a respiratory chain that includes some 40 enzymes.  The machinery dazzles and boggles the mind as it continues on its way to the climax of ATP synthase, one of the most elegant and perfect molecular machines.


Over and over again, we find researchers ignoring Darwinism as they uncover the workings of molecular machines in the cell.  Darwin himself could never have imagined that life at its foundations would be this complex, this mechanical.  It has all the appearance of Paley’s pocket watch – only more elegant, more efficient, and more beautiful at an unimaginably small scale.  And this is just one of thousands of such machines.  Remember the other locomotives, the machines that transport cargo down your molecular railroad?
Notice how scientists in a recent paper in PNAS employed “engineering models to understand the control principles” of a biological phenomenon.)  Fire the storytellers!  Train engineers! (Catch the pun?)  When science discovers powered locomotives at work in the simplest organisms, it no longer needs storytellers with loco motives.




Complex I, an enzyme found in mitochondrial and bacterial membranes, converts energy by coupling electron transfer to proton pumping. Sazanov and colleagues' crystal structures1 of bacterial complex I reveal that the transmembrane NuoL subunit of the enzyme projects a long α-helix through the adjacent NuoM and NuoN subunits. They suggest the following mechanism to explain how electron transfer drives proton pumping. a, Pairs of electrons from the metabolic intermediate NADH are transferred to a cofactor (flavin mononucleotide, FMN) and then passed along a chain of iron–sulphur clusters in the extramembrane region of complex I, eventually reaching a quinone cofactor; blue arrows indicate the electron-transfer pathway. This allows a proton (H+) to pass through complex I at the interface of the extra- and intramembrane regions. Protons can also enter channels in NuoL, NuoM and NuoN from the cytoplasm, but cannot pass through. White circles with minus signs represent negatively charged amino acids, which are key to proton transport. b, Conformational changes in the NuoA/J/K/H subunits push the long α-helix towards the other transmembrane subunits. This tilts three other helices in NuoL, NuoM and NuoN, causing the reorientation of certain residues in the subunits' channels. These local conformational changes allow protons in the channels to pass through the channels and enter the periplasm (the space between the inner and outer bacterial membranes).

Structure of the membrane domain of respiratory complex I 5
The structure of the membrane domain of respiratory complex I from E. coli is shown in colour and the aligned hydrophilic domain of complex I from Thermus thermophilus in grey. In the three antiporter-like subunits the two symmetry-related inverted domains, involved in proton translocation, are shown in shades of green. The two connecting elements are shown in yellow (helix HL) and blue (b-hairpin-helix element).  Complex I is a large molecular proton-pumping machine. Its structure is strongly suggestive of a mechanism that involves conformational coupling via connecting elements acting like a coupling rod in a steam engine, driving symmetry-related domains instead of wheels. To highlight a remarkable analogy between the independent creations of man and nature, the background shows a drawing by James Watt of his steam engine developed between 1787-1800 (The image of steam engine is from the book: "James Watt, Volume 3: Triumph through Adversity, 1785-1819" by Rev. Dr. Richard L. Hills (Landmark Publishing Ltd., 2006)). Credits: Rouslan Efremov and Leonid Sazanov, MRC.

Visualize an old locomotive train roaring down the tracks.  One of the characteristic images that surely comes to mind is the oscillating motion of the coupling rods on the wheels.  The long rods that connected the wheels provided a way to convert heat energy from the steam into mechanical energy 




1) http://creationsafaris.com/crev201009.htm
2) https://web.archive.org/web/20170708104125/http://creationsafaris.com/crev201007.htm
3) https://www.sfb746.uni-freiburg.de/Publications/Hunte/hunte-2010.pdf
4) http://www.uni-freiburg.de/news/news_050710_2_en
5) http://www.esrf.eu/news/general/respiratory-complex1/index_html
6) http://www.nature.com/scitable/topicpage/why-are-cells-powered-by-proton-gradients-14373960
7) http://www.ebi.ac.uk/pdbe/quips?story=CXI

A piston proton pump

The paper 1

R.G. Efremov et al., “The architecture of respiratory complex I,” Nature, 465:441–45, 2010. http://bit.ly/protonpump

The finding

Although the molecular machines that power ATP synthesis via trans-membrane proton gradients are well known, how the gradient is created in the first place is more mysterious. Now, a crystal structure of complex I, the first group of proteins involved in generating energy from the oxidation of glucose, shows how it uses unusual piston-shaped molecules to pump protons across the membrane.

The surprise

The mechanism proposed by Leonid Sazanov’s group at the Medical Research Council in Cambridge is “almost completely unexpected,” says Faculty Member Thomas Meier. Unlike the ATP synthase, which “drives protons across the membrane in a rotary turbine-like motion,” writes Faculty member Nathan Nelson in his review, the transfer of electrons from NADH cause a slight widening of one part of the complex, forcing the long helix to move like the a row of pistons that shove protons across the membrane.

The impact

Scientists have puzzled about the pumping mechanism of complex I for years. Faculty Member Andrea Mattevi predicts that it will become one of the most cited papers in respiratory chain research, as important to our complete understanding of energy generation as is the mechanism of ATP synthase.

The next step

Sazanov’s next task, he says, is to improve the structural resolution from the protein crystals and make site-specific mutations to verify the proposed mechanism, and to understand precisely how complex I contributes to neurodegenerative disorders such as Parkinson’s.

Pumping protons: a complex problem. 2

In mammals, complex I consists of 44 different subunits and electron microscopy studies have revealed that it has an L shape. One arm of the L is inserted in the membrane (the membrane arm) and the other arm (the hydrophilic arm) projects some 100 Å into the mitochondrial matrix. Bacteria also possess a complex I, but it is a ‘cut down’ version of its eukaryotic counterpart, containing at least 14 of the subunits found in the mitochondrial enzyme.

So 30 proteins are missing in prokaryotic cells..... thats a indication that the endosymbiotic theory is false.

The crystal structure of complex I from the bacterium Thermus thermophilus (PDB entry 4hea) shows that it also has the characteristic L shape (view-1) with the hydrophilic arm projecting into the cytoplasm.

The complex consists of 16 protein subunits with a combined molecular weight of 536 kDa. The complex contains seven Fe4S4 and two Fe2S2 iron-sulfur clusters and a bound flavin mononucleotide (FMN) as co-factors (Figure 2). Seven of the subunits span the membrane and contain 64 transmembrane helices in total.

Where does NADH enter complex I?

The NADH substrate-binding site is at the distal end of the hydrophilic arm in subunit Nqo1 (view-2). The site is formed from a modified Rossmann fold which also incorporates a FMN-binding site. The structure of the hydrophilic arm of T. thermophilus complex I with bound NADH (PDB entry 3iam) shed light on the interactions between FMN and NADH. The adenine ring of NADH is held in place by stacking against three phenylalanine residues. This allows the nicotinamide ring of NADH to stack against the isolloxazine ring of the bound FMN (view-2). Electron transfer then occurs between the C4N atom of the nicotinamide ring of NADH and N5 of FMN. The architecture of the binding site positions these atoms within 3.2 Å of each other for efficient electron transfer. A glutamate residue in the binding site appears to help position the two rings close together.

Reducing the problem.

Subunits Nqo1, Nqo3, Nqo6 and Nqo9 contain seven of the iron-sulfur clusters in the complex. These form a “wire” some 90 Å long which carries electrons from the FMN, through the hydrophilic arm to the quinone-binding site formed by Nqo4, Nqo6 and Nqo8 (view-3). The iron-sulfur clusters are at most 14 Å apart (edge to edge distance), allowing efficient electron transfer between the clusters (view-3). Nqo2 and Nqo3 contain additional iron-sulfur clusters, but the function of these clusters is unknown.

Figure 3. Menaquinone 8 used by T. thermophilus complex I. NADH donates two electrons to the iron-sulfur cluster “wire” which are transferred one at a time along the clusters and used to reduce the bound quinone. Different organisms use different quinones, and T. thermophilus uses menaquinone-8 (Figure 3). Menaquinone-8 binds in a 30 Å long cavity formed by subunits Nqo4, Nqo6 and Nqo8 at the interface between the two arms. The entrance to the cavity, through which the quinone has to pass, is surprisingly narrow, around 2.3 x 4.5 Å (view-4), suggesting movement in this region to allow quinone binding. The quinone-binding cavity contains hydrophilic residues along its length, which may interact with the hydrophilic head group of the quinone as it enters the cavity. Residues His38, Tyr87 and Asp139 in Nqo4, at the distal end of the cavity, probably position the quinone close to the last iron-sulfur cluster in the “wire” to allow for efficient reduction of the quinone. After being reduced, the quinone leaves complex I and is subsequently used as a substrate by complex III.

We all pump together.

When complex I reduces the bound quinone it also pumps four protons across the membrane. Complex I contains four potential channels within the membrane which are likely to perform this role. Subunits Nqo12, 13 and 14 are homologous to each other and to sodium/proton antiporters, indicating that they most likely form the proton-pumping channels 1, 2 and 3, respectively. Subunits Nqo8, Nqo10 and Nqo11 also form a channel across the membrane, channel 4, which links the other channels to the hydrophilic domain (view-5).

So how does reduction of the quinone result in the pumping of four protons across the membrane? The most obvious assumption is that each channel pumps a single proton. But this begs the question how the channels are coordinated, especially as the distal channel is 130 Å from the quinone binding site. Each channel contains two transmembrane helices which have a break in the middle with a charged residue at this point (view-5). It is likely that these charged residues form a path for the proton as it makes its way through the channel. Other charged residues appear to connect the proton pumping channels together, possibly providing a mechanism of coordination between the channels. The C-terminus of Nqo12 (containing channel 1) consists of a 104 Å long α-helix which runs along the surface of the membrane before terminating adjacent to Nqo14. The role of this helix is unknown, although it interacts with one of the two broken transmembrane helices in each of the channels. Additional charged residues link the quinone binding site to the adjacent channel (channel 4), suggesting that they may initiate proton pumping upon reduction of the quinone (view-5). Further work is needed to investigate the mechanism of proton pumping.

A long-distance relationship that works.

NADH is used as a reducing agent by complex I, donating two electrons that reduce a quinone molecule and drive the pumping of four protons across the membrane in four channels. Amazingly, the NADH substrate is 85 Å away from the quinone and the quinone is 140 Å removed from the most distant channel! Complex I displays a truly remarkable form of long-distance communication.





1) http://www.the-scientist.com/?articles.view/articleNo/29227/title/A-piston-proton-pump/
2) http://www.ebi.ac.uk/pdbe/quips?story=CXI

further readings:

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2011.07883.x/pdf

Piston Engine Joins Rotary Engine in Cells

a piston-driven engine has been found at work in every cell’s energy factory.

    ATP synthase operates at the end of a sequence of machines in the respiratory chain that generates chemical energy (in the form of ATP) from the food we eat (or from sunlight, in the case of plants).  The enzyme runs on proton motive force – a flow of protons that drive its carousel-like rotor.  But how does the proton gradient get established?  That’s the job of Respiratory Complex I, the first machine (enzyme) in the chain.  Complex I takes electrons from food, stored in NADH molecules, and transfers them down a chain of electron receptors to parts of the machine that pump protons across the mitochondrial membrane into the periplasm, setting up a proton gradient.  It now becomes evident that Complex I includes parts that move like pistons.

    Complex I was reported in a July Science Express paper as having a railroad-like coupling rod   This week, The Scientist described it as “A piston proton pump,” referencing a paper from Nature last May:¹  Richard P. Grant reported,

The mechanism proposed by Leonid Sazanov’s group at the Medical Research Council in Cambridge is “almost completely unexpected,” says Faculty Member Thomas Meier.  Unlike the ATP synthase, which “drives protons across the membrane in arotary turbine-like motion,” writes Faculty member Nathan Nelson in his review, the transfer of electrons from NADH cause a slight widening of one part of the complex, forcing the long helix to move like the a [sic] row of pistons that shove protons across the membrane.

Some scientists feel this important finding will rival the excitement about the discovery that a rotary engine produces ATP.  One faculty member “predicts that it will become one of the most cited papers in respiratory chain research, as important to our complete understanding of energy generation as is the mechanism of ATP synthase.”
    The original paper in Nature¹ used the same piston metaphor and contained the same enthusiasm:

The overall architecture of this large molecular machine is now clear.  F-ATPase [ATP Synthase] has been compared to a turbine.  In a similar vein, complex I seems to resemble a steam engine, where the energy of the electron transfer is used to move a piston, which then drives, instead of wheels, a set of discontinuous helices.

Tomoko Ohnishi, commenting on this paper in the same issue of Nature, continued the piston metaphor in his title, “Structural biology: Piston drives a proton pump.”²  He described how the food we eat goes through a “highly efficient process” called oxidative phosphorylation in the mitochondria, ending in the synthesis of ATP.  Complex I was known to have some distance between its electron acceptors and the transmembrane antiporters.  It was unknown how the parts were coupled.  Now, the mechanism of the first enzyme, Complex I, is becoming clear:

The membrane-spanning enzyme known as complex I couples the movement of electrons to that of protons as a way of converting energy.  Crystal structures suggest how electron transfer drives proton pumping from afar.
    Complex I is one of the energy-converting enzyme complexes found in the membranes of the cell’s fuel factories, the mitochondria, and was the last such complex without a structural portrait.  But in an epoch-making paper in this issue, Sazanov and colleagues¹ describe X-ray structures of bacterial complex I, and report that it has an unusual ‘piston’ mechanism for controlling proton movement across mitochondrial membranes (see page 441).

Both the original paper and Ohnishi’s summary contain diagrams showing how the piston mechanism works in conjunction with the connecting rod described in the 07/06/2010 entry.

    ATP Synthase was mentioned in a PNAS commentary this week.³  Stuart L. Ferguson [Oxford U] recounted the decades of effort to determine how ATP was generated.  He indicated that much remains to be learned, including why different life forms have different numbers of c-subunits in the F₀ rotor (for background, see 12/22/2003, 08/10/2004, 08/04/2010), but mentioned “the apparently universal nature of the ATP synthase” in passing, indicating that even lowly bacteria have these elegant machines.  Eukaryotes (including all plants and animals) and eubacteria, but not archaea, “are from sequence analyses very similar,” he mentioned.  Archaea also use forms of ATP synthase that differ from those of eukaryotes in some respects.

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1.  Efremov, Baradaran, and Sazamov, “The architecture of respiratory complex I,” Nature 465 (27 May 2010), pages: 441–445, doi:10.1038/nature09066.
2.  Tomoko Ohnishi, “Structural biology: Piston drives a proton pump,” Nature 465 (27 May 2010), pages 428–429, doi:10.1038/465428a.
3.  Stuart L. Ferguson, “ATP synthase: From sequence to ring size to the P/O ratio,” http://www.pnas.org/content/early/2010/09/20/1012260107.full.pdf+html>Proceedings of the National Academy of Sciences, published online before print September 21, 2010, doi: 10.1073/pnas.1012260107.

So what can proponents of naturalism do with the discovery of rotary engines and piston engines in the simplest forms of life, all the way up to humans?  They just attribute it all to the remarkable creative power of the goddess Evolution.
    A Nature Education article by Nick Lane (cf. 08/11/2010) referred to the piston paper by Efremov et al, saying “Again, the structure betrays the mechanism – in this case not a rotary motor but, even more surprisingly, a lever mechanism not unlike the piston of a steam engine ,”  But then, Lane invoked Michael Russell’s lame hydrothermal waste dump myth (02/15/2008) – you remember, the one that falsified the primordial soup myth (02/05/2010) – to draw a parallel from simple proton gradients in deep sea vents to the proton gradients that drive pistons and rotors in the cell.
    That’s like comparing rolling stones to automobiles.  Look at his convoluted reasoning to get from rolling stones to automobiles without intelligent design:


There are, of course, big open questions – not least, how the gradients might have been tapped by the earliest cells, which certainly lacked such sophisticated protein machinery as the ATP synthase,” Lane admitted.  “There are a few possible abiotic mechanisms, presently under scrutiny in Russell’s lab and elsewhere.  But thermodynamic arguments, remarkably, suggest that the only way life could have started at all is if it found a way to tap the proton gradients.


  So tell us, Nick, did Life try to tap into these gradients on purpose?  After all, if it “found a way,” it must have been looking for it.  In Lane’s vision quest, Life, in some nebulous form lacking ATP and a proton gradient, studies those deep-sea vents with furrowed brow, asking “How can we tap into that?”  But wait – without a way to tap into it already, it would have no energy to look for, discover, and harness the proton gradient.  Well, that must imply, then, that all the machinery just “arose” all together, fully formed, by chance.  Maybe it was a miracle: “the acquisition of mitochondria and the origin of complexity could be one and the same event,” he said.
    Only an naturalist gets away with this kind of nonsense in scientific lit.  But that’s not all.  Lane proceeded to extend his mythology to all complex life, with all its organs and functions, speculating how it all originated with proton gradients.  In the end, though, he had to admit the whole idea was a myth:


The question is, what kind of a cell acquired mitochondria in the first place?  Most large-scale genomic studies suggest that the answer is an archaeon – that is, a prokaryotic cell that is in most respects like a bacterium.  That begs the question, how did mitochondria get inside an archaeon?  The answer is a mystery but might go some way toward explaining why complex life derives from a single common ancestor, which arose just once in the 4 billion years of life on Earth.


Well, at least he recognized he left some “big open questions” begging.  Nothing more needs to be said.  He just shot any claim to science out from under his own feet and showed himself belonging to a “mystery” cult, along with the editors of Nature, who, by printing his speculations, became willing accomplices in promoting the mystery cult. Take Nick Lane’s freak show (08/11/2010) to Mad Magazine where it belongs.  The rest of us are enjoying this confirmation of intelligent design at the smallest scale of life.  You’re running on pistons and rotary engines.  Cool!  Lane gets a teeny bit of credit for sharing one amazing factoid in his article about the electrical potential in your body set up by these proton gradients: “A membrane potential of 150 mV across the 5-nanometer membrane gives a field strength of 30 million volts per meter – equivalent to a bolt of lightning.”  You’ve got lightning in your tank.  Hot!

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Positive Darwinian Selection in the Piston That Powers Proton Pumps in Complex I of the Mitochondria of Pacific Salmon

Discussion

This is the first study that analyzed the coding sequences in the full mitochondrial genome (except ND6), presented statistically supported evidence of positive Darwinian selection at specific amino acids sites, and related those specific amino acid residues to the 3-dimensional context of the multi-subunit protein. There have been numerous other efforts to survey mitochondrial genomes for signatures of positive selection, but most were limited in the scope of possible inferences. Limitations arose either by an analytical framework that only permitted rejection of the neutral theory of evolution jointly for a complete set of mitochondrial sequences (i.e. tests for selection at specific sites were not possible) or they sampled only a subset of mitochondrial proteins (e.g. Elson et al., 2004; Nachman et al.,1996; Wise et al., 1998; Zink 2005). Only two studies attempted what we have done here. One study compiled evidence for selection in the mitochondrial genomes of primates from numerous other studies [20]. Their focus was on selection in Complexes III and IV of the mitochondrial genome, but it did not address selection in Complex I and had minimal information for Complex V. A second study employed TreeSAAP to identify positively selected sites in 41 mammalian species. As with our work, a signal for positive selection was detected in ND5, but they were unable to place the selected sites of the ND2, ND4, and ND5 proteins within a structural-context because the structure of Complex I was not known at the time [37]. Interestingly, that study and others also detected positively selected sites in the C-terminal portion of ND5 [21], [31], [37], [69]. Finally, a study of the hydrophobicity of the mitochondrial encoded ND proteins of 11 different phyla indicated rapid evolution at the in the same location [31]. This observation, when taken together with our results, suggests the possibility that the piston arm of ND5 might have been the target of adaptive modification in a wide variety of lineages. A wider investigation of piston arm evolution appears warranted.

We observed that changes in the piston arm and, consequently, proton pumping, may have influenced fitness during the evolution of Pacific salmon species. Because these species are important for economic, conservation, and recreational reasons, a plethora of data exist that can be correlated to functional studies. Behnke provides a thorough description of the phenotypes of North American species of Pacific salmon and some life-history traits [44]. Quinn's work [70] provides a meticulous summary of life history traits in an ecological context of Pacific salmon, and Hendry and Stearns provide a detailed summary of work focused on the evolution of the species [45]. We propose that Pacific salmon species provide a unique model to study the functional consequences of these amino acid substitutions because they demonstrate diverse life history types and can be easily studied in the laboratory and in the wild.

Given that the sites of protein-protein interactions identified in this study probably coordinate the translocation of H+ ions from the matrix to the inner membrane space, it is likely that the positively selected mutations influence the electrochemical gradient, which is comprised of both a voltage potential and a difference in pH. The magnitude of the pH gradient influences respiratory control [2] and can alter the production of reactive oxygen species, which has affects on aging among other things [5], [71], [72]. When a higher resolution structure becomes available, it should be possible to determine which of the specific amino acids in the ND5 piston arm interact with which proton pumps and the nature of the changes in the interactions that result at the sites under positive selection. This might provide information to determine if the piston arm is more tightly or loosely coupled to the pumps and therefore if pumping is made more or less efficient by the amino acid substitutions.

The amuzing thing about their end remarks is that the mitochondria had to have its origin prior of cell replication, because the cell depends on ATP. So darwininan selection could not have been a possible driving force to explain the origin and emergence of this incredible, essential proton pump.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024127

A Model of the Proton Translocation Mechanism of Complex I*

Despite decades of speculation, the proton pumping mechanism of complex I (NADH-ubiquinone oxidoreductase) is unknown and continues to be controversial. Recent descriptions
of the architecture of the hydrophobic region of complex I have resolved one vital issue: this region appears to have multiple proton transporters that are mechanically interlinked. Thus, transduction of conformational changes to drive the transmembrane transporters linked by a “connecting rod” during the reduction of ubiquinone (Q) can account for two or three of the four protons pumped per NADH oxidized. The remaining proton(s) must be pumped by direct coupling at the Q-binding site. Here, we present a mixed model based on a crucial constraint: the strong dependence on the pH gradient across the membrane (
pH) of superoxide generation at the Q-binding site of complex I. This model combines direct and indirect coupling mechanisms to account for the pumping of the four protons. It explains the observed properties of the semiquinone in the Q-binding site, the rapid superoxide production from this site during reverse electron transport, its low superoxide production during forward electron transport except in the presence of inhibitory Q-analogs and high protonmotive force, and the strong dependence of both modes of superoxide production on pH.

Complex I (NADH-ubiquinone oxidoreductase) is central to energy transformation in many prokaryotes and most eukaryotes (1, 2). It establishes a protonmotive force (
p)2 by
coupling the oxidation of NADH and reduction of ubiquinone (Q) or analogous quinones to the pumping of four protons across the membrane per two electrons transferred (4H/2e)
(3–6).  Despite the importance of complex I to energy transformation, the mechanism of proton translocation remains a notable unknown in bioenergetics and is currently under intense scrutiny.

1) http://www.jbc.org/content/286/20/17579.full.pdf+html

The Evolution of Respiratory Chain Complex I from a Smaller Last Common Ancestor Consisting of 11 Protein Subunits 1

Notably, the 11-subunit version of complex I was found to be widely distributed, both in the archaeal and in the eubacterial kingdoms, whereas the 14-subunit classical complex I was found only in certain eubacterial phyla. The FpoF-containing complex I was present in Euryarchaeota but not in Crenarchaeota, which contained the 11-subunit complex I. The 11-subunit enzymes showed a primary sequence variability as great or greater than the full-size 14-subunit complex I, but differed distinctly from the membrane-bound hydrogenases. We conclude that this type of compact 11-subunit complex I is ancestral to all present-day complex I enzymes. No designated partner protein, acting as an electron delivery device, could be found for the compact version of complex I. We propose that the primordial complex I, and many of the present-day 11-subunit versions of it, operate without a designated partner protein but are capable of interaction with several different electron donor or acceptor proteins.

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144371/

The iron–sulphur protein Ind1 is required for effective complex I assembly 1

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron–sulphur (Fe–S) clusters. Little is known about the assembly of complex I and its Fe–S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe–S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe–S clusters showed that only a minor fraction (∼20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe–4S] cluster that was readily transferred to an acceptor Fe–S protein. Our data suggest that Ind1 facilitates the assembly of Fe–S cofactors and subunits of complex I.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2435133/