Abstract
Arsenite oxidase is thought to be an ancient enzyme, originating before the divergence of the Archaea and the Bacteria. We have investigated the nature of the molybdenum active site of the arsenite oxidase from the Alphaproteobacterium Rhizobium sp. str. NT-26 using a combination of X-ray absorption spectroscopy and computational chemistry. Our analysis indicates an oxidized Mo(VI) active site with a structure that is far from equilibrium. We propose that this is an entatic state imposed by the protein on the active site through relative orientation of the two molybdopterin cofactors, in a variant of the Rây-Dutt twist of classical coordination chemistry, which we call the pterin twist hypothesis. We discuss the implications of this hypothesis for other putatively ancient molybdopterin-based enzymes.
Highlights
The group 6 transition metals molybdenum and tungsten are the only second and third row transition elements with known functions in biology[1]
During the catalytic cycle the oxygen that is transferred to arsenite is thought to arise from an Mo = O group bound to molybdenum, and the molybdenum is reduced from the Mo(VI) to the Mo(IV) oxidation state
We report a combined Mo K-edge X-ray absorption spectroscopy (XAS) and density functional theory (DFT) study of the molybdenum active site of the NT-26 Arsenite oxidase (Aio) and show that the enzyme possesses a novel cis-dioxo structure in the oxidized Mo(VI) form
Summary
Our analysis indicates an oxidized Mo(VI) active site with a structure that is far from equilibrium. NT-26 (alphaproteobacterium)[9] show a number of common structural features, both being heterodimers with the molybdenum site and an [3Fe-4S] cluster in the larger A subunit, and a Rieske [2Fe-2S] cluster in the smaller B subunit (Fig. 2)[7, 9] Both enzymes lack significant detectible Mo(V) EPR signals, and in agreement with this observation cyclic voltammetry of the A. faecalis enzyme shows an unusual highly cooperative two-electron Mo(IV)/Mo(VI) redox couple centred at 292 mV vs SHE at pH 5.910. We report a combined Mo K-edge XAS and density functional theory (DFT) study of the molybdenum active site of the NT-26 Aio and show that the enzyme possesses a novel cis-dioxo structure in the oxidized Mo(VI) form. We show that this form of the oxidized enzyme must arise from a geometry that is far from the minimum energy for the Mo site and discuss the implications for the catalytic mechanism
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