title: Sulfido and cysteine ligation changes at the molybdenum cofactor during substrate conversion by formate dehydrogenase (FDH) from Rhodobacter capsulatus.
abstract: Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO(-)) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Mo-ligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.
authors: †Institut für Experimentalphysik, Freie Universität Berlin, 14195 Berlin, Germany.
authors: Peer
authors: P
authors: Schrapers
authors: ‡Institut für Biochemie und Biologie, Molekulare Enzymologie, Universität Potsdam, 14476 Potsdam, Germany.
authors: Tobias
authors: T
authors: Hartmann
authors: †Institut für Experimentalphysik, Freie Universität Berlin, 14195 Berlin, Germany.
authors: Ramona
authors: R
authors: Kositzki
authors: †Institut für Experimentalphysik, Freie Universität Berlin, 14195 Berlin, Germany.
authors: Holger
authors: H
authors: Dau
authors: ‡Institut für Biochemie und Biologie, Molekulare Enzymologie, Universität Potsdam, 14476 Potsdam, Germany.
authors: Stefan
authors: S
authors: Reschke
authors: §Institut für Biochemie, Bioanorganische Chemie, Ernst-Moritz-Arndt-Universität Greifswald, 17487 Greifswald, Germany.
authors: Carola
authors: C
authors: Schulzke
authors: ‡Institut für Biochemie und Biologie, Molekulare Enzymologie, Universität Potsdam, 14476 Potsdam, Germany.
authors: Silke
authors: S
authors: Leimkühler
authors: †Institut für Experimentalphysik, Freie Universität Berlin, 14195 Berlin, Germany.
authors: Michael
authors: M
authors: Haumann
mesh_terms: D020134:Catalytic Domain; D003067:Coenzymes / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D005560:Formate Dehydrogenases / Q000737:chemistry*; D005561:Formates / Q000737:chemistry; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D000090006:Molybdenum Cofactors; D011621:Pteridines / Q000737:chemistry*; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013440:Sulfides / Q000737:chemistry*
chemical_list: D003067:Coenzymes; D003545:Cysteine; D005560:Formate Dehydrogenases; D005561:Formates; D008024:Ligands; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; D013440:Sulfides; C022261:molybdopterin