Structure of the Molybdenum Site of Rhodobacter sphaeroides Biotin Sulfoxide Reductase

• Published in: Biochemistry (Easton) Vol. 39; no. 14; pp. 4046 - 4052
• Main Authors: Temple, Carrie A, George, Graham N, Hilton, James C, George, Martin J, Prince, Roger C, Barber, Michael J, Rajagopalan, K. V
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11-04-2000
• Subjects: Amino Acid Sequence; Biotin-S-oxide reductase; dithionate; Enzyme Stability; Escherichia coli; Molecular Sequence Data; Molybdenum; NADPH; Oxidoreductases - chemistry; Oxidoreductases - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Rhodobacter sphaeroides; Rhodobacter sphaeroides - chemistry; Rhodobacter sphaeroides - enzymology; Rhodobacter sphaeroides - genetics; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9921541

Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV−visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (MoO at 1.70 Å) with additional coordination by approximately four thiolate ligands at 2.41 Å and probably one oxygen or nitrogen at 1.95 Å. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 Å and two different Mo−O/N ligands at 2.19 and 1.94 Å.