Molybdenum Site Structure of Escherichia coli YedY, a Novel Bacterial Oxidoreductase

Abstract

We report a structural characterization using X-ray absorption spectroscopy of the molybdenum site of Escherichia coli YedY, a novel oxidoreductase related to be the sulfite oxidase family of molybdenum enzymes. We find that the enzyme can exist in Mo(V) and Mo(IV) oxidation states but cannot be readily oxidized to the Mo(VI) form. Mo(V) YedY has molybdenum coordination similar to that of sulfite oxidase, with one Mo═O at 1.71 Å, three Mo-S at 2.39 Å, and one Mo-OH at 2.09 Å, which elongates to 2.20 Å upon reduction to Mo(IV), indicating Mo-OH(2) coordination. The Mo(V) enzyme also possesses a long Mo-O coordination at 2.64 Å, which may be due to oxygen coordination by Asn-45 O(δ), with Mo-O(δ) approximately trans to the Mo═O group. A comparison with sulfite oxidase indicates that YedY possesses a much more uniform Mo-S coordination, with a maximum permitted deviation of less than 0.05 Å. Our results indicate that the YedY active site shows considerable similarity to but also important differences from that of reduced forms of sulfite oxidase.

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

“…Both dcV trumpet plot data and low-intensity FTacV harmonic currents indicate that the Mo(V/IV) redox reaction has a slow electron transfer rate, from 3 to 6 s −1 , which suggests structural reorganization. This correlates with the XAS mechanism that six-coordinate Mo(V) is reduced to a five-coordinate Mo(IV) species (9).…”

“…Varying the pH from 4 to 9 causes the electrochemically determined E m (Mo(V/IV)) value to decrease by 53 mV per pH unit, which indicates a one-electron, one-proton transition in agreement with the proposed XAS mechanism: Mo(V)−OH + 1H + + 1e − → Mo(IV)−OH 2 (9). There is a discrepancy between the midpoint potentials we measure using electrochemistry and those reported from an EPR redox titration, with respective E m, 7 values of +174 and +132 mV (6).…”

“…YedY has been structurally characterized via both X-ray crystallography and X-ray absorption spectroscopy (XAS) (3,8,9). In most mononuclear Mo enzymes, heme groups and iron sulfur clusters are found within the same protein as the Mo center, but the only metal site in YedY is Mo, making this enzyme a helpfully simple system for studying redox chemistry ( Fig.…”

“…Within the active site, the X-ray structure was interpreted to show Mo(V) in a square pyramidal environment (3), identical to other members of the "sulfite oxidase" family of mononuclear Mo enzymes. In contrast, XAS has suggested a pseudooctahedral Mo center (8,9) with an additional O (from Glu104) or N (from Asn45) axial ligand coordinating trans to the apical oxo group (Fig. 1) (8).…”

Metalloprotein active site structure determination: Synergy between X-ray absorption spectroscopy and X-ray crystallography

“…As mentioned above, EXAFS cannot generally provide information on geometry. Recently workers have attempted to supplement this by using density functional theory energy minimized structures [19], often with constraints imposed from crystal structures [9,79,80]. Density functional theory calculations of enzyme active sites are very sensitive to starting geometries, especially when one includes the local amino acid residues, where a small change in initial geometry can result in quite different answers [9].…”