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Abstract
The nickel-dependent carbon monoxide dehydrogenase (CODH) employs a unique heterometallic nickel-iron-sulfur cluster, termed the C-cluster, to catalyze the interconversion of CO and CO2 Like other complex metalloenzymes, CODH requires dedicated assembly machinery to form the fully intact and functional C-cluster. In particular, nickel incorporation into the C-cluster depends on the maturation factor CooC; however, the mechanism of nickel insertion remains poorly understood. Here, we compare X-ray structures (1.50-2.48 Å resolution) of CODH from Desulfovibrio vulgaris (DvCODH) heterologously expressed in either the absence (DvCODH-CooC) or presence (DvCODH+CooC) of co-expressed CooC. We find that the C-cluster of DvCODH-CooC is fully loaded with iron but does not contain any nickel. Interestingly, the so-called unique iron ion (Feu) occupies both its canonical site (80% occupancy) and the nickel site (20% occupancy), with addition of reductant causing further mismetallation of the nickel site (60% iron occupancy). We also demonstrate that a DvCODH variant that lacks a surface-accessible iron-sulfur cluster (the D-cluster) has a C-cluster that is also replete in iron but lacks nickel, despite co-expression with CooC. In this variant, all Feu is in its canonical location, and the nickel site is empty. This D-cluster-deficient CODH is inactive despite attempts to reconstitute it with nickel. Taken together, these results suggest that an empty nickel site is not sufficient for nickel incorporation. Based on our findings, we propose a model for C-cluster assembly that requires both CooC and a functioning D-cluster, involves precise redox-state control, and includes a two-step nickel-binding process.
Highlights
The nickel-dependent carbon monoxide dehydrogenase (CODH) employs a unique heterometallic nickel–iron–sulfur cluster, termed the C-cluster, to catalyze the interconversion of CO and CO2
We demonstrate that a Desulfovibrio vulgaris CODH (DvCODH) variant that lacks a surface-accessible iron– sulfur cluster has a C-cluster that is replete in iron but lacks nickel, despite co-expression with CooC
This split C-cluster conformation combined with the inability of the DvCODH(C301S)ϩCooC variant to incorporate nickel led us to propose that the oxidized conformation of the cluster could be an intermediate in C-cluster maturation, how this conformation may participate in the assembly process remained unclear [9]
Summary
The crystal structure of DvCODH(C301S)ϩCooC revealed a partially assembled C-cluster in which Feu adopted a split conformation: Feu was in its canonical binding site at 70% occupancy, and Feu was incorporated into the cubane portion of the cluster at 30% occupancy, taking up the canonical nickelbinding site (Fig. 1D) [9] This split C-cluster conformation combined with the inability of the DvCODH(C301S)ϩCooC variant to incorporate nickel led us to propose that the oxidized conformation of the cluster could be an intermediate in C-cluster maturation, how this conformation may participate in the assembly process remained unclear [9]. These results expand our understanding of C-cluster biogenesis, with an emphasis on the importance of accessing different cluster conformations and redox states
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