Resonance Raman Characterization of Biotin Sulfoxide Reductase

Abstract

Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter spha-eroides biotin sulfoxide reductase expressed in Escherichia coli. On the basis of 18 O/ 16 O labeling studies involving water and the alternative substrate dimethyl sulfoxide and the close correspondence to the resonance Raman spectra previously reported for dimethyl sulfox-ide reductase (Garton, S. D., Hilton, J., Oku, H., Crouse, B. R., Rajagopalan, K. V., and Johnson, M. K. (1997) J. Am. Chem. Soc. 119, 12906-12916), vibrational modes associated with a terminal oxo ligand and the two mo-lybdopterin dithiolene ligands have been assigned. The results indicate that the enzyme cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms with both molyb-dopterin dithiolene ligands remaining coordinated in both redox states. Direct evidence for an oxygen atom transfer mechanism is provided by 18 O/ 16 O labeling studies, which show that the terminal oxo group at the molybdenum center is exchangeable with water during redox cycling and originates from the substrate in sub-strate-oxidized samples. Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine. However, product induced changes in the MoO stretching frequency provide direct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate. The results indicate that biotin sulfoxide reductase is thermodynamically tuned to catalyze the reductase reaction, and a detailed catalytic mechanism is proposed.