Biological carbon dioxide (CO2) reduction is an important step by which organisms obtain energy and carry out metabolic processes. The photosynthetic bacterium Rhodobacter capsulatus encodes a Mo-dependent and NAD+-consuming formate dehydrogenase (FDH) that is proposed to catalyze formate oxidation and CO2 reduction in the cytoplasm at a bound bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. The paramagnetic Mo(V) oxidation state is generally accepted to represent a coordination geometry similar to the structurally characterized Mo(VI) oxidation state, with the active site Cys386 ligand and a sulfido ligand directly coordinated to the Mo. However, the nature of Cys386 and substrate/product coordination in the two-electron reduced Mo(IV) state is less clear, which has hindered assignment of proton and hydride transfer events to active site components. To provide mechanistic insight regarding the coordination geometry of the Mo(IV) oxidation state, the paramagnetic Mo(V) state has been reinvestigated to identify parameters having potential origin to the diamagnetic Mo(IV) oxidation state. For example, the inhibitors azide and nitrate similarly couple a solvent-exchangeable proton, but distinctly alter the Mo(V) g-tensor, while binding of the former results in coupling of an additional solvent-nonexchangeble proton. Further, we have shown that the latter serves additionally as a substrate to produce nitrite. These observations, while indirect, implicate an altered coordination of the Cys386 ligand that may originate from the reduced Mo(IV) state. Collectively, these results support our proposed mechanistic model of FDH, by which a C1-intermediate binds directly to the Mo(IV) oxidation state.

Refining catalytic insights toward the chemical mechanism of R. capsulatus formate dehydrogenase via EPR spectroscopy

2019 Spring National Meeting

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