Life's Blueprint: The Essential Machinery to Start Life

Proteins / Enzymes with Metal Clusters in LUCA

[2Fe-2S] Cluster:
Biotin synthase: Contains a [2Fe-2S] cluster.
Dihydrofolate reductase (DHFR): Some forms contain a [2Fe-2S] cluster.
Dihydroorotate dehydrogenase (DHODH) (EC 1.3.3.1): Contains a [2Fe-2S] cluster.
Dihydroorotate dehydrogenase (EC 1.3.5.2): Contains a [2Fe-2S] cluster.
Ferredoxin-NADP+ reductase: Contains a [2Fe-2S] cluster.
Glutamate Dehydrogenase (GDH) (EC 1.4.1.2): Some forms contain a [2Fe-2S] cluster.
Glyceraldehyde-3-phosphate dehydrogenase: Contains a [2Fe-2S] cluster with 2 iron (Fe) atoms and 2 sulfur (S) atoms.
NADH:quinone oxidoreductase: Contains several [Fe-S] clusters.
Oxoglutarate:ferredoxin oxidoreductase: Contains [2Fe-2S] clusters.
Ribonucleotide reductase (RNR) (EC 1.17.4.1): Can contain a [2Fe-2S] cluster.
Superoxide Dismutase (SOD) (EC 1.15.1.1): Contains a [2Fe-2S] type often associated with chemolithoautotrophs.
Succinate dehydrogenase: Contains a [2Fe-2S] cluster.

[3Fe-4S] Cluster:
Superoxide Dismutase (SOD) (EC 1.15.1.1): Contains a [3Fe-4S] cluster type.
Succinate dehydrogenase: Contains a [3Fe-4S] cluster.

[4Fe-4S] Cluster:
5-aminoimidazole ribotide (AIR) carboxylase (PurK) (EC 4.1.1.21): Contains a [4Fe-4S] cluster.
Acetyl-CoA carboxylase: Contains a [4Fe-4S] cluster.
Aconitase: Contains a [4Fe-4S] cluster with 4 iron (Fe) atoms and 4 sulfur (S) atoms.
Biotin synthase: Contains a [4Fe-4S] cluster.
Coenzyme F430 biosynthetic protein FbiC: Contains a [4Fe-4S] cluster.
Formate dehydrogenase: Contains a [Mo-4Fe-4S] cluster.
Methionine adenosyltransferase (MAT): Some forms contain a [4Fe-4S] cluster.
MoaA and MoaC: Both have [4Fe-4S] clusters.
Oxoglutarate:ferredoxin oxidoreductase: Contains multiple [4Fe-4S] clusters.
Phosphomethylpyrimidine synthase (ThiC): Contains a [4Fe-4S] cluster.
Ribonucleotide reductase (RNR) (EC 1.17.4.1): Can contain a [4Fe-4S] cluster.
Succinate dehydrogenase: Contains several [Fe-S] clusters, including [3Fe-4S], [4Fe-4S], and [2Fe-2S].

[5Fe-4S] Cluster:
CO Dehydrogenase/Acetyl-CoA Synthase (CODH/ACS): Contains a [5Fe-4S] cluster.

[Ni-Fe] Cluster:
Hydrogenase: Contains a [Ni-Fe] metal center with nickel (Ni) and iron (Fe) atoms.
Carbon Monoxide Dehydrogenase: Contains a [Ni-Fe] metal center.

[NiFe-4S] Cluster:
Carbon Monoxide Dehydrogenase (CODH): Contains a [NiFe-4S] cluster.
CO Dehydrogenase/Acetyl-CoA Synthase (CODH/ACS): Contains a [NiFe-4S] cluster.
Hydrogenase: Contains a [Ni-Fe] metal center.
Carbon Monoxide Dehydrogenase: Contains a [Ni-Fe] metal center.

[Mo] Cluster:
Nitrate Reductase: Contains a [Mo] metal center.
MoaA: Contains a [Mo/W] cofactor.

[Mo-4Fe-4S] Cluster:
Formate dehydrogenase: Contains a [Mo-4Fe-4S] cluster.

[Cu] Cluster:
Laccase: Contains multiple Cu centers (often 4 copper atoms).
Superoxide dismutase [Cu-Zn]: Contains both Cu and Zn centers.
Cytochrome c oxidase (COX): Contains CuA and CuB centers.

Other Metal Centers:
Carbonic anhydrase (R10092): Typically contains a Zn²⁺ zinc ion at its active site.
Cobaltochelatase: Inserts cobalt into the corrin ring.
Ferrochelatase: Incorporates a ferrous iron (Fe²⁺) to produce heme and contains a Magnesium (Mg) metal center.
Histidine kinase (HK): Can contain iron, magnesium, or zinc.
Hydrogenase nickel incorporation protein HypB: Contains a [Ni] metal center and is associated with Ni insertion in hydrogenases.
Ketopantoate reductase: Requires metal ions like [Mn2+] or [Mg2+] for activity.
Manganese-dependent superoxide dismutase (Mn-SOD): Contains a Manganese (Mn) metal center.
Molybdenum cofactor biosynthesis protein A (MoaA): Crucial for the synthesis of the active molybdenum cofactor.
Phospholipase C (Plc): Some forms contain zinc (Zn).
UreE, UreG, UreF, UreH: Nickel metallochaperones for urease maturation and contains a [Ni] metal center.
Zinc-transporting ATPase (ZntA): Transports Zn.
ZnuA: Contains a [Zn] metal center and is a high-affinity zinc uptake protein.

Complex Metal Centers:
Carbonic anhydrase (R10092): Contains a Zn²⁺ zinc ion.
Cytochrome c oxidase: Part of the respiratory electron transport chain with multiple metal centers including CuA and CuB centers.
Ketopantoate reductase: Requires metal ions like [Mn2+] or [Mg2+].
Nitrogenase: Contains a [MoFe7S9C-homocitrate] cluster.
Nitrite Reductase [NO-forming]: Contains a [CuZ] center and [siroheme] and [Fe4S4] clusters.
Nitrous oxide reductase: Contains CuZ center.
Succinate dehydrogenase: Contains multiple clusters, including [3Fe-4S], [4Fe-4S], and [2Fe-2S].
Superoxide dismutase [Cu-Zn]: Contains both Cu and Zn centers.
Zinc-transporting ATPase (ZntA): Transports [Zn].
ZnuA: Contains a [Zn] metal center.

Total 60 enzymes

Catch-22: The Intelligent Design of CODH/ACS Metal Cluster assembly

Abstract
The CODH/ACS metal cofactor assembly presents a fascinating biochemical conundrum: a system of profound interdependence echoing the age-old "chicken and egg" dilemma. Over 32 specialized accessory proteins are involved, in the formation and maturation of metal cofactors like iron-sulfur clusters of the CODH/ACS protein complex. Remarkably, many of these essential "helper" proteins, which serve as scaffolds or transfer agents, house themselves iron-sulfur clusters — the very metal clusters they aid in maturing. This circular dependency challenges conventional narratives of linear, sequential, and gradual setups. Instead, it poses the thought-provoking question: How did such a sophisticated integrated system, where the synthesis components of enzymes depend on other enzymes, which incorporate the very own components they help to synthesize, emerge? - and they must not only be present but also synchronized in function and location !! The precision and coordination inherent to the CODH/ACS pathway raise compelling arguments against a gradualist account of its origin, pointing towards a perspective of simultaneous creation by design. 

Introduction:
Metal clusters in protein active centers are indispensable for biochemical functions. Metals, such as iron, copper, and zinc, possess unique electronic properties that allow them to accelerate biochemical reactions significantly. Their ability to transition between different oxidation states facilitates crucial electron transfer processes central to cellular activities. Metal clusters provide geometries conducive to substrate binding. Their specific coordination chemistry offers versatility in accommodating diverse substrates, ensuring effective substrate orientation and activation for chemical transformations. Additionally, these clusters are foundational in redox reactions. The electron acceptance and donation capabilities of metal clusters make them integral to biological redox processes. For instance, iron-sulfur clusters are a cornerstone of electron transfer chains in vital processes like photosynthesis. Beyond their catalytic roles, metals offer structural support to proteins. They stabilize protein configurations, ensuring optimal functionality.  Given the multifaceted roles of metal clusters, it's evident that they're foundational components of many proteins and enzymes, underscoring their centrality in life-sustaining biochemical processes. From about 1350 proteins to perform the life-essential cellular processes, at least 60 proteins are foundational and hinge on the incorporation of metal clusters within their active centers. Absent these metallic elements, the efficiency and purpose of these proteins would be compromised, underscoring the paramount role of metal clusters in life's onset. Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase (CODH/ACS) stands as a hallmark in the realm of enzymology, largely due to the intricacy of its metal cluster. This enzyme facilitates crucial reactions in both anaerobic and aerobic organisms, including the conversion of carbon monoxide into acetyl-CoA, a pivotal metabolic intermediate. At the heart of CODH/ACS lies a unique metal cluster known as the A-cluster. Comprising nickel, iron, sulfur, and other ligands, this A-cluster is a marvel of complex sophistication, exhibiting a bifunctional role. On one side, it binds, oxidizes, and channels carbon monoxide (CO) while on the other, it subsequently catalyzes the assembly of acetyl-CoA using the derived CO, a methyl group, and CoA. Its design is notably intricate, consisting of a [NiFe4S4] core where the nickel atom bridges to a unique iron site. This bridging arrangement, not commonly seen in other metalloenzymes, endows CODH/ACS with its distinct reactivity and function. The configuration of the metals and the surrounding ligands ensures that the enzyme can effectively engage with its substrates and carry out its dual function.

The assembly of Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase (CODH/ACS) metal clusters

The [NiFe-4S] and [5Fe-4S] clusters in this enzyme complex play a crucial role in the Wood-Ljungdahl pathway, which is one of the most ancient carbon fixation pathways. The pathway involves five enzymes, and the CODH/ACS complex stands at the fifth position, playing a pivotal role in the final stages of the pathway, culminating in the synthesis of acetyl-CoA. Carbon fixation is a critical biochemical process where inorganic carbon, carbon dioxide (CO₂), is transformed into organic compounds. These organic compounds serve as both an energy reservoir and a carbon source for cellular constituents. The majority of Earth's organic matter originates from this fundamental process, making it the starting point of most food chains.  The maturation and insertion of these metal clusters are complex, necessitating multiple accessory proteins. It is important for carbon fixation in early life.

Following are the CODH/ACS Metal Clusters:

1. [NiFe-4S] Cluster (C-cluster): Responsible for the reversible conversion of CO to CO2. 
2. [5Fe-4S] Cluster (A-cluster): Mediates the synthesis of acetyl-CoA from a methyl group, CO, and coenzyme A. 
3. [4Fe-4S] Clusters: Additional clusters in the enzyme for electron transfer. 
4. [Bifunctional Cluster]: Unique metal cluster connecting CODH and ACS domains, facilitating the transfer of intermediates between the two functional sites.

Total number of Clusters: 4 main types of metal clusters. 1 [NiFe-4S] cluster 1 [5Fe-4S] cluster 2-3 [4Fe-4S] clusters 1 Bifunctional cluster For a total of approximately 5-6 distinct metal clusters per CODH/ACS enzyme complex.

1. [NiFe-4S] cluster synthesis and assembly

The [NiFe-4S] cluster assembly and insertion process for the Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase (CODH/ACS) complex is intricate.  Synthesis Pathway of [NiFe] Clusters for Hydrogenases Nickel insertion and initial scaffold formation:

HypA: Initial protein involved in Ni-binding.
HypB: GTPase that provides nickel to HypA.
Iron and sulfur assembly into a cluster:
HypC: Interacts with HypD to form an Fe-S cluster scaffold.
HypD: Forms a complex with HypC and helps in Fe-S cluster assembly.
CO and CN- ligands synthesis and insertion:
HypE: In the presence of HypF, synthesizes the cyanide ligands attached to the Fe of the cluster.
HypF: Facilitates the synthesis of cyanide ligands by HypE.

2. Synthesis Pathway of [5Fe-4S] Clusters for CODH/ACS

Initial Fe-S cluster formation on a scaffold protein:
IscU: Acts as a scaffold for the initial assembly of the Fe-S cluster.
Iron delivery:
IscA: Involved in iron delivery for Fe-S cluster formation.
Sulfur delivery:
IscS: Provides sulfur for the formation of the Fe-S cluster.
Cluster transfer and insertion:
HscA: Assists in cluster transfer.
HscB: Co-chaperone in cluster transfer.

3. Synthesis Pathway of [4Fe-4S] Clusters for CODH/ACS

Initial Fe-S cluster formation on a scaffold protein:
IscU: This protein acts as a primary scaffold for the initial assembly of the iron-sulfur (Fe-S) cluster.
Iron delivery:
IscA: This is involved in iron delivery for the formation of the Fe-S cluster.
Sulfur delivery:
IscS: This cysteine desulfurase provides the necessary sulfur for the formation of the Fe-S cluster by removing sulfur from cysteine.
Cluster transfer and insertion:
HscA: This is a specialized Hsp70-type ATPase that assists in the transfer of the Fe-S cluster from the scaffold to the target proteins.
HscB: Acts as a co-chaperone in the transfer process with HscA.
Cluster stabilization:
Fdx: Ferredoxins are small iron-sulfur proteins that facilitate electron transfer in various metabolic reactions. They often play a role in maintaining the stability and integrity of [4Fe-4S] clusters.

4. Synthesis Pathway of Bifunctional Cluster for CODH/ACS

Initial Formation and Scaffold Assisted Assembly:
IscU: Similar to other Fe-S clusters, IscU may serve as an initial scaffold for the assembly of the bifunctional cluster's metal ions and sulfur atoms.
Iron and Nickel Delivery:
IscA: Assists in iron delivery for the formation of the cluster.
NikABCDE: A transport system that may facilitate nickel ion delivery specifically for this cluster, given its bifunctional nature.
Sulfur Delivery:
IscS: A cysteine desulfurase that would provide sulfur for the formation of the cluster.
Cluster Insertion into CODH/ACS:
NifU and NifS: These proteins, traditionally involved in nitrogenase maturation, might play roles in transferring the assembled cluster from scaffold proteins to CODH/ACS.
Cluster Stabilization:
Fdx: Ferredoxins, in a broader context, help maintain the stability of metal clusters. They might be involved in ensuring the stability of the bifunctional cluster.

In total, there are 17 unique proteins/enzymes involved in the maturation and formation of the four clusters. Many of the proteins involved in the maturation and assembly of metalloclusters in enzymes also contain iron-sulfur clusters themselves. These iron-sulfur clusters often play essential roles in the transfer and assembly processes.

HypD: Contains a [4Fe-4S] cluster. Essential for the synthesis of the [NiFe] center of hydrogenases. 
IscU: Binds a [2Fe-2S] cluster. Serves as a scaffold for Fe-S cluster assembly. 
IscA: Binds both [2Fe-2S] and [4Fe-4S] clusters. Acts as an intermediate carrier of Fe-S clusters. 
HscA: Contains a [4Fe-4S] cluster. Plays a role in the transfer of iron-sulfur clusters to target proteins. 
Fdx (Ferredoxins): Typically contain iron-sulfur clusters, including [2Fe-2S], [3Fe-4S], or [4Fe-4S] clusters. Facilitates electron transfer in metabolic reactions.
NifU: Contains [2Fe-2S] and [4Fe-4S] clusters. Serves as a scaffold for the formation of the metalloclusters of nitrogenase. 

Commentary: The intricacy of the systems responsible for the maturation and assembly of metal cofactors in CODH/ACS  complex exemplifies a biochemical conundrum reminiscent of the classic "chicken and egg" problem. 
Dependency on Metal Cofactors: Proteins such as HypD, IscU, IscA, HscA, Fdx (Ferredoxins), and NifU are essential for the assembly and maturation of metal cofactors in CODH/ACS. They play critical roles in scaffolding, transferring, and stabilizing the metal clusters.
Inherent Metal Clusters: Interestingly, many of these proteins themselves contain iron-sulfur clusters ([4Fe-4S], [2Fe-2S], etc.), which means their proper folding, stability, and function depend on the very metal assembly processes they facilitate. For instance: HypD requires a [4Fe-4S] cluster for its function, vital for the synthesis of the [NiFe] center of hydrogenases. IscU, which acts as a scaffold for Fe-S cluster assembly, binds a [2Fe-2S] cluster.
Ferredoxins (Fdx), which aid in electron transfer and cluster stability, contain iron-sulfur clusters, further exemplifying this recursive complexity.
Sequential Paradox: If we were to hypothesize a linear, sequential origin, a predicament arises: Without the aforementioned proteins being correctly formed and functional, the metal cofactors they help assemble can't be matured. Conversely, without these metal cofactors, these proteins themselves can't attain their functional forms. Which came first? The protein that requires the metal cofactor to function or the metal cofactor that requires the protein for its assembly? Given this interdependency, it's challenging to conceive a gradual, step-by-step development for such a system. A partial or incomplete assembly pathway wouldn't be functional, and any intermediate stage lacking critical components would result in a non-functional system, devoid of selective advantage. This intricate interplay suggests a coordinated and simultaneous emergence of both the proteins and the metal cofactors they work with. In other words, the entire system, with all its components and the sophisticated processes they facilitate, had to come into existence all at once. This perspective challenges linear developmental narratives and prompts consideration of mechanisms that can account for the coordinated emergence of such interdependent systems. Such a scenario raises questions about the origins of such intertwined biochemical systems. The CODH/ACS metal cofactor assembly and maturation pathway serve as an emblematic example of a designed setup, where understanding the full picture requires a holistic approach, recognizing the interdependence of its parts.

Insertion and maturation of metal clusters into the CODH/ACS complex

The proper insertion and maturation of metal clusters into the CODH/ACS complex is facilitated by a set of accessory and assembly proteins. 

CooC: An ATPase involved in the insertion of the nickel ion into the CODH active site. Its ATPase activity likely provides the energy for nickel insertion.
CooT: Serves as a nickel transporter to ensure the availability of nickel for CODH and other enzymes.
CoaE: Part of the CoA biosynthesis pathway, essential for the functionality of the ACS component of CODH/ACS.
Acs1: Implicated in ACS maturation in some organisms, potentially aiding in the proper insertion of metal clusters.
Acs4: Like Acs1, Acs4 is also suggested to be involved in ACS maturation.
CorA: Functions as a magnesium and cobalt efflux system, potentially playing a role in metal homeostasis critical for CODH/ACS functionality.
NikABCDE: This is a nickel transport system, which may play a role in supplying nickel ions to proteins requiring them, like CODH.
CooJ: A protein believed to be involved in the maturation of CODH, although its exact function remains to be fully elucidated.
CooF: This redox protein transfers electrons during the oxidation of carbon monoxide in the CODH reaction.

But the list does not end here. There are even more accessory proteins required:

CooH: This protein is sometimes found in carbon monoxide utilization clusters. Its function is not completely understood, but it might be associated with CODH assembly or stabilization.
CooI: This protein is involved in the oxidation of carbon monoxide. It's an accessory protein required for the synthesis of the carbon monoxide dehydrogenase complex.
CooG: It's a carbon monoxide dehydrogenase maturase. While its exact role remains to be defined, maturases often play a part in the post-translational modification or assembly of protein complexes.
CooL: While the specific function of CooL in relation to the CODH complex is not entirely clear, its presence in carbon monoxide utilization gene clusters suggests a role in the assembly or function of CODH.
CooM: Found in some carbon monoxide utilization clusters. Its precise role in relation to the CODH/ACS complex is yet to be fully elucidated.
CooN: Another protein associated with carbon monoxide utilization. Its exact function concerning CODH/ACS remains to be detailed.

Commentary: The formation and maturation of metal cofactors in the CODH/ACS complex requires at least 32 accessory and assembly proteins, underscoring a sophisticated biological process governed by intricate machinery.  The CODH/ACS metal cofactor pathway demands the presence of specialized proteins like HypD, IscU, IscA, HscA, Fdx, and NifU. The roles these proteins play in the system are crucial, and their availability is paramount. The process doesn't solely hinge on having the right components; their timely presence is equally pivotal. Components of the CODH/ACS metal cofactor assembly need to be present in a synchronized manner, allowing their collective contribution to the maturation of the metal cofactors when necessary. These components must converge at the appropriate cellular locations to facilitate efficient interactions, thereby enabling the successful synthesis and integration of the metal cofactors. Each step in the assembly and integration of metal cofactors follows a meticulous sequence. This coordination is vital to ensure the meaningful and functional assembly of all components. Beyond mere coordination, components should be compatible at their interaction points. This compatibility is evident in proteins like IscU and HypD, which not only bind to metal clusters but also engage with other proteins to transfer or stabilize them. Given these criteria, the interdependent nature of the CODH/ACS metal cofactor pathway poses significant questions about its hypothesized and presupposed unguided origins. The sheer precision and synchronization required by this system suggest that a gradual, stepwise naturalistic emergence is highly improbable. The data aligns more closely with a scenario where the system's components and processes were instantiated in a coordinated manner, indicating design and simultaneous orchestration.

Design patterns of biological cells
https://onlinelibrary.wiley.com/doi/epdf/10.1002/bies.202300188