Abstract
Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides 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 sulfoxide 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 molybdopterin 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 molybdopterin 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 substrate-oxidized samples. Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine. However, product-induced changes in the Mo=O 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.
Highlights
With the exception of nitrogenase, molybdenum enzymes catalyze formal oxygen atom transfer between water and substrate and contain an active site in which the molybdenum is coordinated by the dithiolene side chain of one or two molybdopterins (Fig. 1a) (1–3)
We report on resonance Raman studies of R. sphaeroides biotin sulfoxide (BSO) reductase in oxidized and reduced forms in the presence and absence of substrates and products
In the case of Me2SO reductase, resonance Raman studies indicated that the absorption features above 500 nm arise from thiolate-to-Mo(VI) charge transfer transitions, and laser excitation in this region facilitates resonant enhancement of Mo–S, MoϭO, and dithiolene ring stretching modes in the Raman spectra (16)
Summary
With the exception of nitrogenase, molybdenum enzymes catalyze formal oxygen atom transfer between water and substrate and contain an active site in which the molybdenum is coordinated by the dithiolene side chain of one or two molybdopterins (Fig. 1a) (1–3). The precise role for BSO reductase in bacterial metabolism has yet to be defined, potential physiological functions include scavenging biotin sulfoxide from the environment, reducing bound intracellular biotin that has become oxidized in an aerobic environment, and protecting the cell from oxidative damage (12) Extensive characterization of this enzyme has been limited due to the low natural abundance of the protein and its constitutive expression (13). In the other set of R. capsulatus Me2SO reductase x-ray structures, the active site is refined as being 7-coordinate in both the oxidized and dimethyl sulfide (ME2S)-reduced forms, with both molybdopterin dithiolates attached, in addition to serinate and a spectator oxo group (5, 6). In addition to the absence of any evidence for a spectator oxo group in dithioniteor Me2S-reduced samples, the vibrational frequency of the substrate- and solvent-exchangeable oxo group can only be interpreted in terms of a short MoϭO (i.e. ϳ1.7 Å)
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