'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus
| Autor: | Ramona Kositzki, Holger Dau, Carola Schulzke, Stefan Reschke, Tobias Hartmann, Silke Leimkühler, Peer Schrapers, Michael Haumann |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Formates Stereochemistry Coenzymes Sulfides Ligands 010402 general chemistry Formate dehydrogenase Photochemistry 01 natural sciences Rhodobacter capsulatus Cofactor Catalysis Inorganic Chemistry 03 medical and health sciences chemistry.chemical_compound Catalytic Domain Metalloproteins Formate Cysteine Physical and Theoretical Chemistry Institut für Biochemie und Biologie 030304 developmental biology 0303 health sciences Rhodobacter biology Pteridines Active site biology.organism_classification Formate Dehydrogenases 0104 chemical sciences chemistry biology.protein Molybdenum cofactor Molybdenum Cofactors |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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