Arsenite oxidase from Alcaligenes faecalis is used to reduce the toxicity of arsenite by converting it to arsenate. The active site contains a molybdenum center coordinated to two molybdopterin ligands, making this enzyme structurally similar to DMSO reductase. However,previous characterization by EPR has not revealed a Mo(V) oxidation state for this enzyme [1], as has been observed for DMSO reductase [2]. This suggests that the oxidation of Mo(IV) to Mo(VI) in arsenite oxidase proceeds in a cooperative, two-electron manner.

Protein film voltammetry (PFV) has been used to verify that the enzyme does indeed possess a two-electron signal corresponding to a reversible Mo(IV/VI) couple. However, voltammetry in the presence of substrate indicates that substrate binding can stabilize a Mo(V) intermediate in the oxidation of the enzyme to the catalytically active Mo(VI) state. This result is obtained by careful consideration of the shape of the catalytic wave, using a model that incorporates the possibility of multiple adsorption conformations for the enzyme on the electrode surface[3]. These results further demonstrate the efficacy of PFV in studying redox centers that may be unclear or even silent when probed by other methods.

This work is in collaboration with Russ Hille at The Ohio State University.

References:

[1] Anderson, G. L., Williams, J., Hille, R., J. Biol. Chem., 267 (1992) 23674

[2] Heffron, K.,Léger, C., Rothery, R. A., Weiner, J. H., Armstrong, F. A., Biochemistry, 40 (2001) 3117.

[3] Léger,., Jones, A. K., Albracht, S. P. J., Armstrong, F. A., J. Phys. Chem.B, in press.