Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO 2 , generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

A combination of EPR spectroscopy and computational approaches has provided insight in to the nature of the reaction coordinate for the one-electron reduction of nitrite by the mitochondrial amidoxime reductase component (mARC) enzyme The results show that a paramagnetic Mo (V) species is generated when reduced enzyme is exposed to nitrite, and an analysis of the resulting EPR hyperfine parameters confirms that mARC is remarkably similar to the low pH form of sulfite oxidase. Two mechanisms for nitrite reduction have been considered. The first shows a modest reaction barrier of 14 kcal/mol for the formation of •NO from unprotonated nitrite substrate. In marked contrast, protonation of the proximal substrate oxygen to Mo in the Mo(IV)-O-N-O substrate bound species results in barrierless conversion to products. A fragment orbital analysis reveals a high degree of Mo-O(H)-N-O covalency that provides a π orbital pathway for one-electron transfer to the substrate and defines orbital constraints on the Mo-substrate geometry for productive catalysis in mARC and other pyranopterin molybdenum enzymes that catalyze this one-electron transformation.

Mo K-edge X-ray absorption spectroscopy has been used to probe as-isolated structures of the MOSC family proteins pmARC-1 and HMCS-CT. The Mo K-edge near-edge spectrum of HMCS-CT is shifted ∼2.5 eV to lower energy compared to the pmARC-1 spectrum, which indicates that as-isolated HMCS-CT is in a more reduced state than pmARC-1. Extended X-ray absorption fine structure analysis indicates significant structural differences between pmARC-1 and HMCS-CT, with the former being a dioxo site and the latter possessing only a single terminal oxo ligand. The number of terminal oxo donors is consistent with pmARC-1 being in the MoVI oxidation state and HMCS-CT in the MoIV state. These structures are in accord with oxygen-atom-transfer reactivity for pmARC-1 and persulfide bond cleavage chemistry for HMCS-CT.

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