A database overview of metal-coordination distances in metalloproteins.

Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.

Upgrading of the general AMBER force field 2 for fluorinated alcohol biosolvents: A validation for water solutions and melittin solvation.

The standard GAFF2 force field parameterization has been refined for the fluorinated alcohols 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and 1,1,1,3,3,3-hexafluoropropan-2-one (HFA), which are commonly used to study proteins and peptides in biomimetic media. The structural and dynamic properties of both proteins and peptides are significantly influenced by the biomimetic environment created by the presence of these cosolvents in aqueous solutions. Quantum mechanical calculations on stable conformers were used to parameterize the atomic charges. Different systems, such as pure liquids, aqueous solutions, and systems formed by melittin protein and cosolvent/water solutions, have been used to validate the new models. The calculated macroscopic and structural properties are in agreement with experimental findings, supporting the validity of the newly proposed models.

Hunting down zinc(II)-binding sites in proteins with distance matrices.

MOTIVATION: In recent years, high-throughput sequencing technologies have made available the genome sequences of a huge variety of organisms. However, the functional annotation of the encoded proteins often still relies on low-throughput and costly experimental studies. Bioinformatics approaches offer a promising alternative to accelerate this process. In this work, we focus on the binding of zinc(II) ions, which is needed for 5%-10% of any organism's proteins to achieve their physiologically relevant form. RESULTS: To implement a predictor of zinc(II)-binding sites in the 3D structures of proteins, we used a neural network, followed by a filter of the network output against the local structure of all known sites. The latter was implemented as a function comparing the distance matrices of the Cα and Cß atoms of the sites. We called the resulting tool Master of Metals (MOM). The structural models for the entire proteome of an organism generated by AlphaFold can be used as input to our tool in order to achieve annotation at the whole organism level within a few hours. To demonstrate this, we applied MOM to the yeast proteome, obtaining a precision of about 76%, based on data for homologous proteins. AVAILABILITY AND IMPLEMENTATION: Master of Metals has been implemented in Python and is available at https://github.com/cerm-cirmmp/Master-of-metals.

Ruthenium(II) Polypyridyl Complexes and Metronidazole Derivatives: A Powerful Combination in the Design of Photoresponsive Antibacterial Agents Effective under Hypoxic Conditions.

Ruthenium(II) polypyridyl complexes (RPCs) are gaining momentum in photoactivated chemotherapy (PACT), thanks to the possibility of overcoming the classical reliance on molecular oxygen of photodynamic therapy while preserving the selective drug activation by using light. However, notwithstanding the intriguing perspectives, the translation of such an approach in the development of new antimicrobials has been only barely considered. Herein, MTZH-1 and MTZH-2, two novel analogues of metronidazole (MTZ), a mainstay drug in the treatment of anaerobic bacterial infections, were designed and inserted in the strained ruthenium complexes [Ru(tpy)(dmp)(MTZ-1)]PF6 (Ru2) and [Ru(tpy)(dmp)(MTZ-2)]PF6 (Ru3) (tpy = terpyridine, dmp = 2,9-dimethyl-1,10-phenanthroline) (Chart 1). Analogously to the parental compound [Ru(tpy)(dmp)(5NIM)]PF6 (Ru1) (5-nitroimidazolate), the Ru(II)-imidazolate coordination of MTZ derivatives resulted in promising Ru(II) photocages, capable to easily unleash the bioactive ligands upon light irradiation and increase the antibacterial activity against Bacillus subtilis, which was chosen as a model of Gram-positive bacteria. The photoreleased 5-nitroimidazole-based ligands led to remarkable phototoxicities under hypoxic conditions (<1% O2), with the lead compound Ru3 that exhibited the highest potency across the series, being comparable to the one of the clinical drug MTZ. Besides, the chemical architectures of MTZ derivatives made their interaction with NimAunfavorable, being NimA a model of reductases responsible for bacterial resistance against 5-nitroimidazole-based antibiotics, thus hinting at their possible use to combat antimicrobial resistance. This work may therefore provide fundamental knowledge in the design of novel photoresponsive tools to be used in the fight against infectious diseases. For the first time, the effectiveness of the "photorelease antimicrobial therapy" under therapeutically relevant hypoxic conditions was demonstrated.

Metal-induced structural variability of mononuclear metal-binding sites from a database perspective.

Metalloproteins are ubiquitous in all kingdoms of life. Their role and function are tightly related to the local structure of the metal-binding site. In this regard, the MetalPDB database is an invaluable tool since it stores the 3D structure of metal-binding sites and of their corresponding apo forms. In this work, we exploited MetalPDB to compute extensive statistics over >3000 clusters of mononuclear sites about the rearrangements occurring upon change in metalation state. For each cluster, we matched the holo and apo sites so that it was possible to average the distances between all possible pairs of Cα and donor atoms and thus quantitatively assess structural variations by computing the Δ values (mean apo distance - mean holo distance). For most of the structures the backbone is rigid with little to no rearrangement, while donor atoms experience significant changes of their relative position when the metal is removed. Sodium and potassium sites are an exception to this general observation. This is most likely caused by their preference for coordination by the main-chain oxygen atoms, making the rearrangement of donor atoms superimposable to that of the backbone. Magnesium and calcium show a different behavior, despite their chemical similarity: calcium sites undergo a larger reorganization upon metalation although both metals have similar percentage of backbone oxygen as donor atoms. We ascribe this observation to the structural and energetic factors regulating the selectivity for calcium over magnesium.

Structural Bioinformatics and Deep Learning of Metalloproteins: Recent Advances and Applications.

All living organisms require metal ions for their energy production and metabolic and biosynthetic processes. Within cells, the metal ions involved in the formation of adducts interact with metabolites and macromolecules (proteins and nucleic acids). The proteins that require binding to one or more metal ions in order to be able to carry out their physiological function are called metalloproteins. About one third of all protein structures in the Protein Data Bank involve metalloproteins. Over the past few years there has been tremendous progress in the number of computational tools and techniques making use of 3D structural information to support the investigation of metalloproteins. This trend has been boosted by the successful applications of neural networks and machine/deep learning approaches in molecular and structural biology at large. In this review, we discuss recent advances in the development and availability of resources dealing with metalloproteins from a structure-based perspective. We start by addressing tools for the prediction of metal-binding sites (MBSs) using structural information on apo-proteins. Then, we provide an overview of the methods for and lessons learned from the structural comparison of MBSs in a fold-independent manner. We then move to describing databases of metalloprotein/MBS structures. Finally, we summarizing recent ML/DL applications enhancing the functional interpretation of metalloprotein structures.

Learning to Identify Physiological and Adventitious Metal-Binding Sites in the Three-Dimensional Structures of Proteins by Following the Hints of a Deep Neural Network.

Thirty-eight percent of protein structures in the Protein Data Bank contain at least one metal ion. However, not all these metal sites are biologically relevant. Cations present as impurities during sample preparation or in the crystallization buffer can cause the formation of protein-metal complexes that do not exist in vivo. We implemented a deep learning approach to build a classifier able to distinguish between physiological and adventitious zinc-binding sites in the 3D structures of metalloproteins. We trained the classifier using manually annotated sites extracted from the MetalPDB database. Using a 10-fold cross validation procedure, the classifier achieved an accuracy of about 90%. The same neural classifier could predict the physiological relevance of non-heme mononuclear iron sites with an accuracy of nearly 80%, suggesting that the rules learned on zinc sites have general relevance. By quantifying the relative importance of the features describing the input zinc sites from the network perspective and by analyzing the characteristics of the MetalPDB datasets, we inferred some common principles. Physiological sites present a low solvent accessibility of the aminoacids forming coordination bonds with the metal ion (the metal ligands), a relatively large number of residues in the metal environment (≥20), and a distinct pattern of conservation of Cys and His residues in the site. Adventitious sites, on the other hand, tend to have a low number of donor atoms from the polypeptide chain (often one or two). These observations support the evaluation of the physiological relevance of novel metal-binding sites in protein structures.

A New Paradigm of Multiheme Cytochrome Evolution by Grafting and Pruning Protein Modules.

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochrome c involved in metabolic pathways that contribute to the nitrogen, sulfur, and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant octaheme nitrite reductase and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analyzed here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

The zinc proteome of SARS-CoV-2.

Zinc is an essential element for human health. Among its many functions, zinc(II) modulates the immune response to infections and, at high concentrations or in the presence of ionophores, inhibits the replication of various RNA viruses. Structural biology studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed that zinc(II) is the most common metal ion that binds to viral proteins. However, the number of zinc(II)-binding sites identified by experimental methods is far from exhaustive, as metal ions may be lost during protein purification protocols. To better define the zinc(II)-binding proteome of coronavirus, we leveraged the wealth of deposited structural data and state-of-the-art bioinformatics methods. Through this in silico approach, 15 experimental zinc(II) sites were identified and a further 22 were predicted in Spike, open reading frame (ORF)3a/d, ORF8, and several nonstructural proteins, highlighting an essential role of zinc(II) in viral replication. Furthermore, the structural relationships between viral and eukaryotic sites (typically zinc fingers) indicate that SARS-CoV-2 can compete with human proteins for zinc(II) binding. Given the double-edged effect of zinc(II) ions, both essential and toxic to coronavirus, only the complete elucidation of the structural and regulatory zinc(II)-binding sites can guide selective antiviral strategies based on zinc supplementation.

Basic Iron-Sulfur Centers.

Iron-sulfur clusters are ubiquitous protein cofactors composed of iron and inorganic sulfur. These cofactors are among the most ancient ones and may have contributed to the birth of life on Earth. Therefore, they are found even today in many enzymes central to metabolic processes like nitrogen fixation, respiration, and DNA processing and repair. Due to the toxicity associated with iron and sulfur ions, living organisms evolved dedicated machineries to synthetize and then transfer iron-sulfur clusters into client proteins. The iron-sulfur cluster (ISC) machinery is responsible for iron-sulfur cluster biogenesis in prokaryotes and in the mitochondrion of eukaryotes; the sulfur mobilization (SUF) machinery is present in prokaryotes and in the chloroplasts of plants; finally, the cytosolic iron-sulfur assembly (CIA) machinery is only present in the cytoplasm of eukaryotes. Genome analysis allowed the prediction of the proteins containing iron-sulfur clusters across a broad variety of living organisms, establishing links between the size and composition of iron-sulfur proteomes and the types of organisms that encode them. For example, the iron-sulfur proteomes of aerobes are generally smaller than those of anaerobes with similar genome size; furthermore, aerobes are enriched in [2Fe-2S] proteins compared to anaerobes, which predominantly use [4Fe-4S] proteins. This relates to the lower bioavailability of iron and the higher lability of [4Fe-4S] clusters within aerobic environments. Analogous considerations apply to humans, where the occurrence and functions of iron-sulfur proteins depend on the cellular compartment where they are localized. For example, an emerging primary role for nuclear iron-sulfur proteins is in DNA maintenance. Given their key functions in metabolism, dysfunctions of mutations in iron-sulfur proteins, or in proteins participating in iron-sulfur cluster biogenesis, are associated with serious human diseases.

Upgraded AMBER Force Field for Zinc-Binding Residues and Ligands for Predicting Structural Properties and Binding Affinities in Zinc-Proteins.

We developed a novel force field in the context of AMBER parameterization for glutamate and aspartate zinc(II)-binding residues. The interaction between the zinc ion and the coordinating atoms is represented by a spherical nonbonded parameterization. The polarization effect due to the zinc ion has been taken into account by redefining the atomic charges on the residues through accurate quantum mechanical calculations. The new zinc-binding ASP and GLU residues, along with the CYS and HIS zinc-binding residues, parameterized in a recent work [Macchiagodena M.;J. Chem. Inf. Model.2019, 59, 3803-3816], allow users to reliably simulate 96% of the Zn-proteins available in the Protein Data Bank. The upgraded force field for zinc(II)-bound residues has been tested performing molecular dynamics simulations with an explicit solvent and comparing the structural information with experimental data for five different proteins binding zinc(II) with GLU, ASP, HIS, and CYS. We further validated our approach by evaluating the binding free energy of (R)-2-benzyl-3-nitropropanoic acid to carboxypeptidase A using a recently developed nonequilibrium alchemical method. We demonstrated that in this setting it is crucial to take into account polarization effects also on the metal-bound inhibitor.

HIV-1 Tat Protein Enters Dysfunctional Endothelial Cells via Integrins and Renders Them Permissive to Virus Replication.

Previous work has shown that the Tat protein of Human Immunodeficiency Virus (HIV)-1 is released by acutely infected cells in a biologically active form and enters dendritic cells upon the binding of its arginine-glycine-aspartic acid (RGD) domain to the α5ß1, αvß3, and αvß5 integrins. The up-regulation/activation of these integrins occurs in endothelial cells exposed to inflammatory cytokines that are increased in HIV-infected individuals, leading to endothelial cell dysfunction. Here, we show that inflammatory cytokine-activated endothelial cells selectively bind and rapidly take up nano-micromolar concentrations of Tat, as determined by flow cytometry. Protein oxidation and low temperatures reduce Tat entry, suggesting a conformation- and energy-dependent process. Consistently, Tat entry is competed out by RGD-Tat peptides or integrin natural ligands, and it is blocked by anti-α5ß1, -αvß3, and -αvß5 antibodies. Moreover, modelling-docking calculations identify a low-energy Tat-αvß3 integrin complex in which Tat makes contacts with both the αv and ß3 chains. It is noteworthy that internalized Tat induces HIV replication in inflammatory cytokine-treated, but not untreated, endothelial cells. Thus, endothelial cell dysfunction driven by inflammatory cytokines renders the vascular system a target of Tat, which makes endothelial cells permissive to HIV replication, adding a further layer of complexity to functionally cure and/or eradicate HIV infection.

Upgrading and Validation of the AMBER Force Field for Histidine and Cysteine Zinc(II)-Binding Residues in Sites with Four Protein Ligands.

We developed and validated a novel force field in the context of the AMBER parameterization for the simulation of zinc(II)-binding proteins. The proposed force field assumes nonbonded spherical interactions between the central zinc(II) and the coordinating residues. A crucial innovative aspect of our approach is to account for the polarization effects of the cation by redefining the atomic charges of the coordinating residues and an adjustment of Lennard-Jones parameters of Zn-interacting atoms to reproduce mean distance distributions. The optimal transferable parametrization was obtained by performing accurate quantum mechanical calculations on a training set of high-quality protein structures, encompassing the most common folds of zinc(II) sites. The addressed sites contain a zinc(II) ion tetra-coordinated by histidine and cysteine residues and represent about 70% of all physiologically relevant zinc(II) sites in the Protein Data Bank. Molecular dynamics simulations with explicit solvent, carried out on several zinc(II)-binding proteins not included in the training set, show that our model for zinc(II) sites preserves the tetra-coordination of the metal site with remarkable stability, yielding zinc(II)-X mean distances similar to experimental data. Finally, the model was tested by evaluating the zinc(II)-binding affinities, using the alchemical free energy perturbation approach. The calculated dissociation constants correlate satisfactorily with the experimental counterpart demonstrating the validity and transferability of the proposed parameterization for zinc(II)-binding proteins.

Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii.

Calprotectin (CP) inhibits bacterial viability through extracellular chelation of transition metals. However, how CP influences general metabolism remains largely unexplored. We show here that CP restricts bioavailable Zn and Fe to the pathogen Acinetobacter baumannii, inducing an extensive multi-metal perturbation of cellular physiology. Proteomics reveals severe metal starvation, and a strain lacking the candidate ZnII metallochaperone ZigA possesses altered cellular abundance of multiple essential Zn-dependent enzymes and enzymes in de novo flavin biosynthesis. The ΔzigA strain exhibits decreased cellular flavin levels during metal starvation. Flavin mononucleotide provides regulation of this biosynthesis pathway, via a 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) fusion protein, RibBX, and authentic RibB. We propose that RibBX ensures flavin sufficiency under CP-induced Fe limitation, allowing flavodoxins to substitute for Fe-ferredoxins as cell reductants. These studies elucidate adaptation to nutritional immunity and define an intersection between metallostasis and cellular metabolism in A. baumannii.

The cellular economy of the Saccharomyces cerevisiae zinc proteome.

Zinc is an essential cofactor for many proteins. A key mechanism of zinc homeostasis during deficiency is "zinc sparing" in which specific zinc-binding proteins are repressed to reduce the cellular requirement. In this report, we evaluated zinc sparing across the zinc proteome of Saccharomyces cerevisiae. The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. Protein abundance was determined by mass spectrometry. In zinc-replete cells, we detected over 2500 proteins among which 229 were zinc proteins. Based on copy number estimates and binding stoichiometries, a replete cell contains ∼9 million zinc-binding sites on proteins. During zinc deficiency, many zinc proteins decreased in abundance and the zinc-binding requirement decreased to ∼5 million zinc atoms per cell. Many of these effects were due at least in part to changes in mRNA levels rather than simply protein degradation. Measurements of cellular zinc content showed that the level of zinc atoms per cell dropped from over 20 million in replete cells to only 1.7 million in deficient cells. These results confirmed the ability of replete cells to store excess zinc and suggested that the majority of zinc-binding sites on proteins in deficient cells are either unmetalated or mismetalated. Our analysis of two abundant zinc proteins, Fba1 aldolase and Met6 methionine synthetase, supported that hypothesis. Thus, we have discovered widespread zinc sparing mechanisms and obtained evidence of a high accumulation of zinc proteins that lack their cofactor during deficiency.

The human iron-proteome.

Organisms from all kingdoms of life use iron-proteins in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of iron-proteins. We separated iron-proteins based on the chemical nature of their metal-containing cofactors: individual iron ions, heme cofactors and iron-sulfur clusters. We found that about 2% of human genes encode an iron-protein. Of these, 35% are proteins binding individual iron ions, 48% are heme-binding proteins and 17% are iron-sulfur proteins. More than half of the human iron-proteins have a catalytic function. Indeed, we predict that 6.5% of all human enzymes are iron-dependent. This percentage is quite different for the various enzyme classes. Human oxidoreductases feature the largest fraction of iron-dependent family members (about 37%). The distribution of iron proteins in the various cellular compartments is uneven. In particular, the mitochondrion and the endoplasmic reticulum are enriched in iron-proteins with respect to the average content of the cell. Finally, we observed that genes encoding iron-proteins are more frequently associated to pathologies than the all other human genes on average. The present research provides an extensive overview of iron usage by the human proteome, and highlights several specific features of the physiological role of iron ions in human cells.

MetalPDB in 2018: a database of metal sites in biological macromolecular structures.

MetalPDB (http://metalweb.cerm.unifi.it/) is a database providing information on metal-binding sites detected in the three-dimensional (3D) structures of biological macromolecules. MetalPDB represents such sites as 3D templates, called Minimal Functional Sites (MFSs), which describe the local environment around the metal(s) independently of the larger context of the macromolecular structure. The 2018 update of MetalPDB includes new contents and tools. A major extension is the inclusion of proteins whose structures do not contain metal ions although their sequences potentially contain a known MFS. In addition, MetalPDB now provides extensive statistical analyses addressing several aspects of general metal usage within the PDB, across protein families and in catalysis. Users can also query MetalPDB to extract statistical information on structural aspects associated with individual metals, such as preferred coordination geometries or aminoacidic environment. A further major improvement is the functional annotation of MFSs; the annotation is manually performed via a password-protected annotator interface. At present, ∼50% of all MFSs have such a functional annotation. Other noteworthy improvements are bulk query functionality, through the upload of a list of PDB identifiers, and ftp access to MetalPDB contents, allowing users to carry out in-depth analyses on their own computational infrastructure.

To what extent do structural changes in catalytic metal sites affect enzyme function?

About half of known enzymatic reactions involve metals. Enzymes belonging to the same superfamily often evolve to catalyze different reactions on the same structural scaffold. The work presented here investigates how functional differentiation, within superfamilies that contain metalloenzymes, relates to structural changes at the catalytic metal site. In general, when the catalytic metal site is unchanged across the enzymes of a superfamily, the functional differentiation within the superfamily tends to be low and the mechanism conserved. Conversely, all types of structural changes in the metal binding site are observed for superfamilies with high functional differentiation. Overall, the catalytic role of the metal ions appears to be one of the most conserved features of the enzyme mechanism within metalloenzyme superfamilies. In particular, when the catalytic role of the metal ion does not involve a redox reaction (i.e. there is no exchange of electrons with the substrate), this role is almost always maintained even when the site undergoes significant structural changes. In these enzymes, functional diversification is most often associated with modifications in the surrounding protein matrix, which has changed so much that the enzyme chemistry is significantly altered. On the other hand, in more than 50% of the examples where the metal has a redox role in catalysis, changes at the metal site modify its catalytic role. Further, we find that there are no examples in our dataset where metal sites with a redox role are lost during evolution. SYNOPSIS: In this paper we investigate how functional diversity within superfamilies of metalloenzymes relates to structural changes at the catalytic metal site. Evolution tends to strictly conserve the metal site. When changes occur, they do not modify the catalytic role of non-redox metals whereas they affect the role of redox-active metals.

Identification of the zinc, copper and cadmium metalloproteome of the protozoon Tetrahymena thermophila by systematic bioinformatics.

Tetrahymena thermophila (T. thermophila) is a ciliated protozoon that can detect freshwater pollution by heavy metals ("whole-cell biosensor"). This work employed a systematic bioinformatics approach to predict and analyze the metalloproteome of T. thermophila for the essential Zn, Cu and the non-essential Cd. 3784 metal-binding proteins were identified compared to the 456 annotated so far in UniProt. The localization, functional classification, and the functionally enriched network of the newly identified metalloproteome are presented. Cd toxicity could be explained in terms of the metal replacing Cu and especially Zn in MAPKs, transporters and antioxidant enzymes. The predicted results for Cd toxicity and responses reflect those observed experimentally in different organisms after their exposure to Cd.

The Relationship between Environmental Dioxygen and Iron-Sulfur Proteins Explored at the Genome Level.

About 2 billion years ago, the atmosphere of the Earth experienced a great change due to the buildup of dioxygen produced by photosynthetic organisms. This transition caused a reduction of iron bioavailability and at the same time exposed living organisms to the threat of oxidative stress. Iron-sulfur (Fe-S) clusters require iron ions for their biosynthesis and are labile if exposed to reactive oxygen species. To assess how the above transition influenced the usage of Fe-S clusters by organisms, we compared the distribution of the Fe-S proteins encoded by the genomes of more than 400 prokaryotic organisms as a function of their dioxygen requirements. Aerobic organisms use less Fe-S proteins than the majority of anaerobic organisms with a similar genome size. Furthermore, aerobes have evolved specific Fe-S proteins that bind the less iron-demanding and more chemically stable Fe2S2 clusters while reducing the number of Fe4S4-binding proteins in their genomes. However, there is a shared core of Fe-S protein families composed mainly by Fe4S4-binding proteins. Members of these families are present also in humans. The distribution of human Fe-S proteins within cell compartments shows that mitochondrial proteins are inherited from prokaryotic proteins of aerobes, whereas nuclear and cytoplasmic Fe-S proteins are inherited from anaerobic organisms.