Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase

Conditions for heterologous expression of Rhodobacter sphaeroides biotin su lfoxide reductase in Escherichia coli were modified, resulting in a signifi cant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the moly bdenum as compared to that found in R. sphaeroides DMSO reductase demonstra ted the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduc ed enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-r ay absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidi zed protein possesses a Mo(VI) mono-ore site (Mo=O at 1.70 Angstrom) with a dditional coordination by approximately four thiolate ligands at 2.41 Angst rom and probably one oxygen or nitrogen at 1.95 Angstrom. The NADPH- and di thionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites wi th approximately four thiolates at 2.33 Angstrom and two different Mo-O/N l igands at 2.19 and 1.94 Angstrom.