Assembly of the CO Dehydrogenase Bimetallic Catalytic Site
Final Report Abstract
The enzyme CO dehydrogenase is key to the chemolithoautotrophic utilization of CO as a growth substrate by the α-proteobacterium Oligotropha carboxidovorans. The enzyme employs in its catalytic site the bimetallic [Mo+VIO2-S-Cu+1-S-Cys] cluster which is unprecedented in nature. Mo is coordinated by the ene-dithiolate of the molybdopterin cytosine dinucleotide (MCD) cofactor and Cu by the γ-S of the cysteine residue 388 which is part of the unique signature VAYRCSFR. The metal cluster matures posttranslationally while integrated into the completely folded apo-enzyme. We have studied metal cluster composition structure and functions of CO dehydogenase synthesized in mutants of O. carboxidovorans in which the genes coxD, coxE, coxF, or coxG have been disrupted by mutational insertion of a kanamycin resistance cassette. The purified apo-CO dehydrogenases were characterized by chemical analysis, electron paramagnetic resonance spectroscopy (EPR), crystallography, in vitro reconstitution experiments. and the use of sulfur compounds as probes for the accessibility of the active site and reporters of sulfur interaction with incomplete forms of the metal cluster. The recombinant proteins CoxD, CoxE, and CoxF were produced in E. coli. They appeared in inclusion bodies from where they were solubilized by urea and refolded by stepwise dilution. All three Cox-proteins formed oligomeric states. CoxD was a AAA+-ATPase able to hydrolyze MgATP and to transfer sulfur from thiosulfate to cyanide (sulfurase, rhodanese). Refolded CoxE and CoxF were unstable and rapidly precipitated. These data, along with bioinformatic evidence, led to a model on how CoxD, CoxE and CoxF accomplish the formation of the Mo-S-Cu-bridge. The posttranslational assembly of the [Mo+VIO2-S-Cu+1-S-Cys] cluster in the active site of CO dehydrogenase is a complex and highly ordered process which involves the introduction of sulfur and copper into a [MoVI(=O)2OH(2)] site. It represents the final step of cluster maturation, resulting in a catalytically active enzyme. The project revealed that cluster biosynthesis involves the functions of the genes coxD, coxE, and coxF. Cluster biosynthesis starts with the one electron reduction of [MoVI(=O)2OH(2)] to [MoV(=O)2OH(2)] by an unknown mechanism. However, since CoxD is a membrane protein, the shuttle between MoVI and MoV can be imagined to involve the bacterial electron transport system (ETS). The next step is the MgATP-dependent sulfuration of [MoV(=O)2OH(2)] yielding [MoV(=O)2OH(2)SH] which involves the AAA-ATPase chaperone CoxD. It is not known whether CoxD itself acts as a sulfurtransferase and what the actual sulfur source is. However, the reversible inactivation of of CO dehydrogenase with thiol compounds suggests that cysteine or thiosulfate would be good candidates. Other than has been concluded from x-ray structures of CO dehydrogenase, the project has established that compounds much larger than CO can freely travel through the substrate channel leading to the CO dehydrogenase active site, which would make the previously postulated chaperone function of CoxD obsolete. Chemical reconstitution and EPR established that the in vivo oxidation state of Mo in [Mo(=O)OH(2)SH] must be +VI to enable a transfer of Cu1+. The incorporation of Cu1+ involves CoxE and CoxF which is indicated by the absence of Cu in the apo-CO dehydrogenases from the mutants E::km and F::km. The exact functions of CoxE and CoxF must await studies at the protein level. However, CoxF contains signatures of a potential histidine acid phytase and a suspected Cubinding site. These could enable CoxF to release phytate-bound Cu through the hydrolysis of phosphate monoesters with the subsequent transfer to the Cu-binding site. Complex formation of soluble CoxF(Cu) and membrane bound CoxE through their RGD motif and the VWA domain establishes access to the elctron transport system for the reduction of Cu2+ to Cu1+. Finally, Cu1+ transfer from its escorting protein CoxF to [MoVI(=O)OH(2)SH] in apo-CO dehydrogenase completes the bimetallic cluster. CoxE is an integrin with a single metal ion-dependent adhesion site (MIDAS) located in its ligand-binding site. CoxE is assumed to form an intermediate ternary complex with a cation and CoxF to regulate ligand binding. The types of cations involved in this process, and particularly any role of Cu, are currently not known. Further characterization of CoxD, CoxE and CoxF at the protein level and identification of the missing factors in the maturation of the CO dehydrogenase [CuSMoO2] site are important challenges for future work.
Publications
- (2010) Interaction of CO dehydrogenase with the cytoplasmic membrane monitored by fluorescence correlation spectroscopy. ChemBioChem. 11, 2419-2423
Spreitler, F., Chr. Brock, A. Pelzmann, O. Meyer, and J. Köhler
- (2011) Biometalle für das Überleben. UBT aktuell, 2, 32
Meyer, O. und A. Pelzmann
- (2011) Complete genome sequences of the chemolithoautotrophic strains Oligotropha carboxidovorans OM4 and OM5. J. Bacteriol. 193, 5043
Volland, S., M. Rachinger, A. Strittmatter, R. Daniel, G. Gottschalk, and O. Meyer
(See online at https://doi.org/10.1128/JB.05619-11) - (2011) Trace elements in metalloenzymes determined by neutron activation analysis (NAA). Annual Report 2011 of the Scientific Cooperation at the Forschungs-Neutronenquelle Heinz Maier-Leibnitz (FRM II), 44-45
Li, X., A. Pelzmann, L. Canella, F. Mickoleit, O. Meyer, and H. Gerstenberg
- (2012) The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: Hydrolysis of nucleotidetriphosphates and oligomerization. PLoS ONE, 7, e47424, 1 - 14
Maisel, T., S. Joseph, T. Mielke, J. Bürger, S. Schwarzinger, and O. Meyer
(See online at https://doi.org/10.1371/journal.pone.0047424) - (2014) Insights into the posttranslational assembly of the Mo-, S- and Cu-containing cluster in the active site of CO dehydrogenase of Oligotropha carboxidovorans. J. Biol. Inorg. Chem., 19, 1399-1414
Pelzmann, A., F. Mickoleit, and O. Meyer
(See online at https://doi.org/10.1007/s00775-014-1201-y) - (2014) Reversible inactivation of CO dehydrogenase with thiol compounds. Biochem. Biophys. Res. Commun., 447, 413-418
Kreß, O., M. Gnida, A.M. Pelzmann, C. Marx, W. Meyer-Klaucke, and O. Meyer
(See online at https://doi.org/10.1016/j.bbrc.2014.03.147)
