Life relies on countless chemical reactions, almost all of which need to be sped up by enzymes. About half of all enzymes carry metal ions that expand the range of the reactions that they can catalyze. In some enzymes these metal ions assemble with sulfur ions to form so-called metalloclusters. These structures can carry out many different types of reactions, including converting simple forms of elements like nitrogen and carbon into other forms that can be used to make more complicated biological molecules.

One enzyme that contains metalloclusters is carbon monoxide dehydrogenase. Known as CODH for short, this enzyme uses a metallocluster called the “C-cluster” to interconvert two gases: the pollutant carbon monoxide and the greenhouse gas carbon dioxide. CODH enzymes are found inside certain bacteria, but they are also of interest for humans, who wish to use them to remove the harmful gases from the environment. But this is not as simple as it may at first seem: CODH enzymes usually become inactive when exposed to air because the metalloclusters fall apart in the presence of oxygen. One CODH enzyme from a widespread bacterium called Desulfovibrio vulgaris, however, is an attractive target for industrial use because it can tolerate oxygen better. Yet, it is still unclear why this enzyme does not get inactivated the way other CODHs do.

Wittenborn et al. have now characterized the CODH enzyme from D. vulgaris in more depth via a technique called X-ray crystallography, which can reveal the location of individual atoms within a molecule. By a happy accident, the structures revealed that the C-cluster can adopt a dramatically different arrangement of metal and sulfur ions after being exposed to oxygen. This rearrangement is fully reversible; when oxygen is removed, the metal and sulfur ions move back to their normal positions. This ability to flip between different arrangements appears to protect the metallocluster from losing its metal ions when exposed to oxygen.

By providing structural snapshots of how CODH responds to oxygen these results provide a more complete understanding of an enzyme that plays a key role in the global carbon cycle. This understanding could help scientists to develop bioremediation tools to remove carbon monoxide and carbon dioxide from the atmosphere and to engineer bacteria to capture carbon to make biofuels.

Roughly half of all enzymes make use of metal centers to expand their chemical repertoire (Waldron et al., 2009). Among the most fascinating of the metallocofactors used for such purposes are Fe-S clusters, which are thought to be the most ancient biological cofactors and which enable chemical transformations ranging from simple electron transfer events to the formation and cleavage of carbon-carbon bonds (Rees and Howard, 2003; Beinert et al., 1997). Complex Fe-S clusters, containing alternative metal ions and/or expanded metal frameworks, such as the FeMo-cofactor of nitrogenase, the H-cluster of Fe-Fe hydrogenase, and the C-cluster of Ni-dependent carbon monoxide dehydrogenase (CODH), catalyze fundamental redox conversions that are thought to have enabled early life on Earth (Rees and Howard, 2003; Rees, 2002). Given their structural complexity and the essential nature of the reactions they catalyze, these clusters have collectively been termed the ‘great clusters’ of biology (Rees, 2002).

The great clusters, and the proteins that house them, have become the focus of extensive mechanistic and structural investigation in the hopes of yielding new applications in clean energy production and bioremediation. In particular, CODH catalyzes the interconversion of the gaseous pollutant CO and the greenhouse gas CO₂, leading to the removal of an estimated 10⁸ tons of CO from the lower atmosphere each year and making it an attractive remediation tool (Bartholomew and Alexander, 1979). The anaerobic, Ni-dependent CODH has a homodimeric structure containing a total of five metalloclusters, called the B-, C-, and D-clusters. The C-cluster is the site of CO/CO₂ interconversion and is composed of a [Ni-3Fe-4S] cubane connected through a linking sulfide (S_L) to a unique iron site (Fe_u) (Figure 1) (Drennan et al., 2001; Dobbek et al., 2001). Comprehensive spectroscopic analyses have revealed the basic redox states and kinetic properties of this complex metallocluster, and crystal structures with substrates and inhibitors bound have provided snapshots along the reaction pathway (Figure 2) (Jeoung and Dobbek, 2007; Gong et al., 2008; Kung et al., 2009; Jeoung and Dobbek, 2009; Fesseler et al., 2015; Lindahl et al., 1990; Kumar et al., 1993; Anderson and Lindahl, 1994; Anderson and Lindahl, 1996; Seravalli et al., 1997; Fraser and Lindahl, 1999; Chen et al., 2003; Seravalli and Ragsdale, 2008; Drennan and Peters, 2003). These studies have revealed the C-cluster to take on four discrete redox states termed C_ox, C_red1, C_int, and C_red2 (Lindahl et al., 1990; Kumar et al., 1993; Anderson and Lindahl, 1994; Anderson and Lindahl, 1996; Seravalli et al., 1997; Fraser and Lindahl, 1999). The most widely accepted mechanism of CO oxidation involves a one-electron reductive activation of the inactive C_ox state to C_red1 followed by a catalytic cycle involving conversion between C_red1 and C_red2 (Figure 2) (Lindahl et al., 1990; Kim et al., 2004; Lindahl, 2008). Despite this relatively unified understanding of CO oxidation activity, there are still many gaps in our understanding of this complicated enzyme that are limiting with regard to both our understanding of CODH biochemistry and potential applications of CODH in industrial settings. In particular, there has been a push to characterize enigmatic redox states and also to probe the effects of molecular oxygen on enzyme activity (Merrouch et al., 2015; Wang et al., 2015; Domnik et al., 2017). Here, we report the crystal structure of the CODH from Desulfovibrio vulgaris (DvCODH) (Hadj-Saïd et al., 2015), which reveals a surprising and unprecedented conformational rearrangement of metal ions in the C-cluster and provides the first visualization of the cluster in an oxidized state. Through combined structural and spectroscopic data, we show that conversion between the oxidized and reduced states of the cluster is reversible, consistent with previous electrochemical investigations (Merrouch et al., 2015). We further consider the implications of these findings in terms of oxygen sensitivity and cluster assembly and with respect to the other great clusters in biology.

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The overall fold and cluster placement of DvCODH is highly similar to other structurally characterized CODHs (Figure 1—figure supplement 1) (Drennan et al., 2001; Dobbek et al., 2001; Domnik et al., 2017; Doukov et al., 2002; Darnault et al., 2003). One noteworthy difference with respect to other CODHs is the identity of the D-cluster, a solvent-exposed Fe-S cluster at the dimer interface that serves as an electron conduit to the surface of the protein. Instead of the expected [4Fe-4S] cluster, (Drennan et al., 2001; Dobbek et al., 2001; Domnik et al., 2017; Doukov et al., 2002; Darnault et al., 2003) the electron density is consistent with a [2Fe-2S] cluster (Figure 1—figure supplement 2) as is the placement of cysteine residues in the primary structure. CODH sequence alignments reveal that instead of a C-X₇-C D-cluster binding motif, DvCODH, as well as several uncharacterized CODHs, have a C-X₂-C motif (Figure 1—figure supplement 2). This shortened C-X₂-C motif appears to constrain the geometry of the ligating cysteine residues such that coordination to a [4Fe-4S] cluster is not possible. Instead, the cysteine positions are ideally suited for coordination of a [2Fe-2S] cluster.

In the present work, we determined two structures of as-isolated DvCODH using two independent protein batches and much to our surprise, we observe different conformations of the C-cluster in each structure. A 2.50 Å resolution structure of as-isolated DvCODH (determined using protein batch 1) displays the canonical [Ni-3Fe-4S]-Fe_u C-cluster (Figure 3a; Figure 3—figure supplement 1a; Supplementary file 1) (Drennan et al., 2001; Gong et al., 2008; Doukov et al., 2002; Darnault et al., 2003); the [Ni-3Fe-5S]-Fe_u state that was observed in structures of Carboxydothermus hydrogenoformans CODH-II (Dobbek et al., 2001; Dobbek et al., 2004) is no longer thought to be catalytically relevant (Jeoung and Dobbek, 2007; Kung et al., 2009; Drennan and Peters, 2003; Feng and Lindahl, 2004). The ligation of the C-cluster is conserved in DvCODH (Drennan et al., 2001; Dobbek et al., 2001; Gong et al., 2008; Domnik et al., 2017; Doukov et al., 2002; Darnault et al., 2003) with four cysteines ligating the cubane portion of the cluster (Cys519 is the Ni ligand); one histidine (His266) and one cysteine (Cys302) ligate Fe_u (Figure 3a; Figure 3—figure supplement 1a).

The second, higher-resolution (1.72 Å) structure of as-isolated DvCODH (determined using protein batch 2) reveals a novel arrangement of ions within the C-cluster that was confirmed using anomalous diffraction data (Figure 3b; Supplementary file 1). In this structure, Ni, Fe_u, and S_L are shifted, accompanied by conformational changes of several amino acid side chains, while the positions of the remaining three Fe and three S ions are unchanged. The Ni ion is bound in the site formerly occupied by Fe_u, coordinated by His266 and Cys302 (Figure 3b; Figure 3—figure supplement 1b). The Ni is additionally ligated by Cys519 and Lys556 (Figure 3b; Figure 3—figure supplement 1b). As mentioned above, Cys519 serves as a ligand to Ni in the canonical C-cluster and here adopts an alternative rotamer conformation such that coordination to Ni is maintained (Figure 3b; Figure 3—figure supplement 1b). The occupancy of the alternative Cys519 conformation correlates with the occupancy of Ni, and both have been refined at an atomic occupancy of 70%, in general agreement with the metal analysis result of 0.5 Ni per monomer for the sample that was crystallized (Supplementary file 2). Lys556 is highly conserved and does not normally coordinate to the C-cluster, but is instead the proposed general base catalyst for deprotonation of water during CO oxidation (Figure 2) (Drennan et al., 2001; Dobbek et al., 2001; Kim et al., 2004). Here, the lysine amine group comes within 2.5 Å of Ni. Together, His266, Cys302, Cys519, and Lys556 ligate the Ni in this altered cluster in a highly distorted tetrahedral coordination geometry that is reminiscent of the geometry of the Ni site in Ni-Fe hydrogenases (Volbeda et al., 1995).

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Concomitant with the shift of the Ni ion, Fe_u and S_L also undergo changes in their coordination environments while remaining associated with the [3Fe-3S] partial cubane (Figure 3b; Figure 3—figure supplement 1b). In addition to interaction with S_L, Fe_u is coordinated by Cys302, which forms a bridging interaction with Ni, and by a conserved cysteine residue (Cys301) that does not normally serve as a ligand to the C-cluster, resulting in an apparent three-coordinate geometry around Fe_u (Figure 3b; Figure 3—figure supplement 1b). The shift in the positions of Fe_u and S_L results in the loss of an interaction between S_L and a second Fe ion of the cubane (Fe₁), leaving Fe₁ with three coordinating ligands, Cys340 and two cubane sulfides. A small peak of residual electron density at a site bridging Fe_u and Fe₁ is present in one of the protein chains in the asymmetric unit (Figure 3—figure supplement 2). The occupancy of this site is low (<30%) precluding identification of the atom/ion. However, if fully occupied, the atom/ion would complete the tetrahedral geometry around these Fe atoms. In addition to changes in Fe coordination, the altered conformation of the C-cluster also involves changes in sulfide coordination state. In particular, the two cubane S ions that coordinate Ni in the canonical cluster are left in a possibly unstable state in which they could be susceptible to protonation or loss as free sulfide ions (Crack et al., 2006). In our structure, however, we see no evidence of degradation at these sites, likely due to inaccessibility to solvent or other protective features of the protein environment, in analogy to the case of stable [3Fe-4S] clusters.

We next investigated whether this altered C-cluster state is redox dependent. Pre-formed crystals of as-isolated DvCODH (batch 2, containing the altered cluster) were incubated with the reductant sodium dithionite. Strikingly, the resulting crystal structure displays the canonical C-cluster with Ni, Fe_u, and S_L rearranged into their catalytically-relevant positions (Figure 4a,b; Supplementary file 1). To examine whether this metal rearrangement is reversible upon oxidation, crystals of reduced DvCODH were taken from the anaerobic chamber and incubated under ambient atmospheric conditions. Remarkably, oxidation of the reduced C-cluster by exposure to O₂ results in reformation of the unusual cluster architecture (Figure 4c; Supplementary file 1), whereas both the D- and B-clusters remain intact (Figure 4—figure supplement 1). Together, these results suggest that this altered cluster is an oxidized form of the C-cluster and that this multi-metal ion rearrangement is reversible (Figure 4d, Figure 4—video 1). Most likely, a fortuitous oxidation event, affecting DvCODH batch 2, initially allowed us to obtain the first visualization of an oxidized state of the C-cluster.

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Given the apparent ability of DvCODH to undergo fully reversible oxidation/reduction events in crystallo, we used electron paramagnetic resonance (EPR) spectroscopy to determine the effect of oxidation on the enzyme in solution. First, an EPR spectrum was recorded on a sample of dithionite-reduced DvCODH. This spectrum exhibits resonances characteristic of the one-electron reduced B- and D-clusters (centered around g ~ 2) and of the C_red1 and C_red2 forms of the C-cluster (g_av ~ 1.83 and 1.87, respectively; see Figure 2), as has been previously observed for DvCODH (Hadj-Saïd et al., 2015) (Figure 4e, black trace). A parallel dithionite-reduced sample was incubated under ambient atmospheric conditions to mimic treatment of the DvCODH crystals (EPR-silent, data not shown). The oxygen-exposed sample was then re-reduced with dithionite and the EPR spectrum was recorded, revealing full recovery of the previously observed signals (Figure 4e, red trace). Combined, our crystal structures and EPR data reveal that the metalloclusters of DvCODH are not degraded upon oxidation and that the C-cluster avoids degradation by adopting an alternative, stable, oxidized conformation. Consistent with these results, DvCODH was recently shown to regain activity upon chemical or electrochemical reduction following exposure to molecular oxygen (Merrouch et al., 2015). In this respect, the rearranged C-cluster scaffold can be thought of as a ‘safety net’ for retaining cluster ions upon oxygen exposure, and may explain, at least in part, the ability of certain CODHs to recover activity following oxidation in air (Merrouch et al., 2015; Wang et al., 2015; Domnik et al., 2017). This kind of safety net could improve the ability of an organism to rapidly recover CODH activity after transient exposure to oxic conditions.

One of the more noteworthy aspects of the oxidized C-cluster is that cluster ligation involves one residue (Cys301) that is strictly conserved in CODHs but is not a ligand to the canonical C-cluster. Interestingly, previous work on the heterotetrameric CODH/acetyl-CoA synthase from Moorella thermoacetica (MtCODH/ACS) showed that mutation of the equivalent cysteine residue (Cys316) to serine resulted in an inactive CODH that appeared to lack an intact C-cluster (Kim et al., 2004).

To probe the effect of Cys301 in DvCODH, a DvCODH(C301S) variant was produced and characterized. Similar to what was observed with MtCODH/ACS (Kim et al., 2004), DvCODH(C301S) is inactive and does not contain Ni, as assessed by CO oxidation activity assays and inductively coupled plasma optical emission spectroscopy (ICP-OES), respectively (Supplementary file 2). Unlike wild-type DvCODH, DvCODH(C301S) cannot be activated by Ni under reducing conditions, analogous to what has been observed previously for DvCODH samples grown in the absence of the C-cluster maturation factor CooC (Hadj-Saïd et al., 2015; Merrouch et al., 2018).

To investigate the architecture of a non-activatable C-cluster, we determined the crystal structure of DvCODH(C301S) to 2.0 Å resolution (Supplementary file 1) and discovered an intact [3Fe-4S] C-cluster core with an Fe_u ion that adopts alternative conformations (Figure 5). Approximately 70% of Fe_u is in its canonical position coordinated by His266 and Cys302, whereas approximately 30% is in the Ni-cubane site (see Methods; Figure 5—figure supplement 1). Thus, in the absence of the non-canonical Fe_u-ligand Cys301, the cluster cannot be activated by Ni and Fe_u appears to be free to occupy multiple sites. Taken together, these data suggest that the novel structure of the C-cluster that we observe here, with Fe_u coordinated by Cys301, is relevant to processes beyond oxidation, specifically Ni incorporation.

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Regardless of its role(s), the dramatic rearrangement observed for the C-cluster adds to a growing appreciation that the ions of great clusters are in fact mobile. The oxidized P-cluster of nitrogenase adopts an open conformation relative to its reduced form through the outward movement of two Fe ions by 1.4 and 0.9 Å with accompanying loss of S coordination in a rearrangement that is proposed to couple proton transfer to electron transfer (Figure 6a) (Peters et al., 1997). More recently, Rees and coworkers demonstrated that both the inhibitor CO and an artificially-incorporated Se atom are able to displace a S atom of the cluster and furthermore that, under turnover conditions, the Se atom migrates around the cluster, sampling S positions (Figure 6b) (Spatzal et al., 2014; Spatzal et al., 2015). Very recently, it was also revealed that the same S atom of the VFe-cofactor (containing V in place of Mo) is displaced by a NH ligand, suggesting that cluster dynamics are likely key in catalysis (Sippel et al., 2018). Additionally, an O₂-tolerant membrane-bound hydrogenase was shown to have a novel [4Fe-3S] cluster that undergoes redox-dependent structural changes as part of an O₂-tolerance mechanism (Fritsch et al., 2011; Shomura et al., 2011); one Fe ion moves ~1.6 Å upon cluster oxidation and becomes coordinated by a protein backbone amide group (Figure 6c) (Shomura et al., 2011). In the present work, the metal migration of C-cluster atoms is more dramatic than in these other examples, with Ni moving ~3 Å and adopting an entirely new coordination environment, and Fe_u and S_L moving ~1.9 and 2.6 Å, respectively, with Fe_u also taking on a new coordination environment. This C-cluster rearrangement from oxidized to reduced appears to involve what amounts to a ‘molecular cartwheel’ with Ni, Fe_u, and S_L following the same trajectory to end up in their canonical positions (Figure 4d, Figure 4—video 1).

Figure 6

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In summary, X-ray crystallography has provided views of the ‘great clusters’ of biology, allowing us to marvel at these incredible metallic frameworks that capture and make use of CO, H₂, and N₂ gases. We are increasingly finding that these frameworks should not be thought of as rigid scaffolds, but rather as labile assemblies of metal with sulfide. The full nature and significance of this metallocluster lability is just now beginning to emerge and the roles appear to be diverse, including catalysis, electron transfer, protection from oxygen damage, and possibly cluster assembly. The one consistency is that these great clusters continue to surprise us.

Crystallographic model coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under the following accession codes: 6B6V (as-isolated DvCODH, batch 1), 6B6W (as-isolated DvCODH, batch 2), 6B6X (reduced DvCODH, batch 2), 6B6Y (reduced/O2-exposed DvCODH, batch 2), and 6DC2 (DvCODH(C301S)).

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Author details

1. Elizabeth C Wittenborn

   Department of Chemistry, Massachusetts Institute of Technology, Cambridge, United States

   Present address

   California Institute for Quantitative Biosciences, University of California, Berkeley, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing, Performed the crystallographic experiments, Analyzed the crystallographic data, Wrote the manuscript

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8473-0814

2. Mériem Merrouch

   Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille, France

   Contribution

   Formal analysis, Validation, Investigation, Purified protein and performed activity assays

   Competing interests

   No competing interests declared

3. Chie Ueda

   Department of Biochemistry, Brandeis University, Waltham, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Prepared EPR samples, Analyzed the EPR data

   Competing interests

   No competing interests declared

4. Laura Fradale

   Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille, France

   Contribution

   Formal analysis, Validation, Investigation, Purified protein and performed activity assays

   Competing interests

   No competing interests declared

5. Christophe Léger

   Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille, France

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing—review and editing, Assisted with editing the manuscript

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8871-6059

6. Vincent Fourmond

   Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Project administration, Writing—review and editing, Assisted with editing the manuscript

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9837-6214

7. Maria-Eirini Pandelia

   Department of Biochemistry, Brandeis University, Waltham, United States

   Contribution

   Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Writing—review and editing, Collected and analyzed the EPR data, Assisted in preparing the manuscript

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6750-1948

8. Sébastien Dementin

   Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines, Marseille, France

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Writing—review and editing, Purified protein, Performed activity assays, Assisted in editing the manuscript

   For correspondence

   dementin@imm.cnrs.fr

   Competing interests

   No competing interests declared

9. Catherine L Drennan

   1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, United States
   2. Department of Biology, Massachusetts Institute of Technology, Cambridge, United States
   3. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, United States
   4. Bio-inspired Solar Energy Program, Canadian Institute for Advanced Research, Toronto, Canada

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing, Helped analyze the crystallographic data, Wrote the manuscript

   For correspondence

   cdrennan@mit.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5486-2755

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