Sulfido
and Cysteine Ligation Changes at the Molybdenum
Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH)
from Rhodobacter capsulatus
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Abstract
Formate
dehydrogenase (FDH) enzymes are attractive catalysts for
potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (<i>Rc</i>FDH) binds
a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating
reversible formate (HCOO<sup>–</sup>) to CO<sub>2</sub> oxidation.
We characterized the molecular structure of the active site of wildtype <i>Rc</i>FDH 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 <i>Rc</i>FDH 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 <i>Rc</i>FDH 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 <i>Rc</i>FDH