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J. Biol. Chem., Vol. 280, Issue 12, 11007-11017, March 25, 2005

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Mechanistic Studies of Rhodobacter sphaeroides Me₂SO Reductase^*

Nathan Cobb, Thomas Conrads, and Russ Hille¶

From the Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210-1218

Studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides have yielded new insight into its catalytic mechanism. A series of reductive titrations, performed over the pH range 6–10, reveal that the absorption spectrum of reduced enzyme is highly sensitive to pH. The reaction of reduced enzyme with dimethyl sulfoxide is found to be clearly biphasic throughout the pH range 6–8 with a fast, initial substrate-binding phase and substrate-concentration independent catalytic phase. The intermediate formed at the completion of the fast phase has the characteristic absorption spectrum of the established dimethyl sulfoxide-bound species. Quantitative reductive and oxidative titrations of the enzyme demonstrate that the molybdenum center takes up only two reducing equivalents, implying that the two pyranopterin equivalents of the molybdenum center are not formally redox active. Finally, the visible spectrum associated with the catalytically relevant "high-g split" Mo(V) species has been determined. Spectral deconvolution and EPR quantitation of enzyme-monitored turnover experiments with trimethylamine N-oxide as substrate reveal that no substrate-bound intermediate accumulates and that Mo(V) content remains near unity for the duration of the reaction. Similar experiments with dimethyl sulfoxide show that significant quantities of both the Mo(V) species and the dimethyl sulfoxide-bound complex accumulate during the course of reaction. Accumulation of the substrate-bound complex in the steady-state with dimethyl sulfoxide arises from partial reversal of the physiological reaction in which the accumulating product, dimethyl sulfide, reacts with oxidized enzyme to yield the substrate-bound intermediate, a process that significantly slows turnover.

Received for publication, October 25, 2004 , and in revised form, December 29, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S4.

Current address: Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P. O. Box B, Frederick, MD 21702-1201.

^¶ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, The Ohio State University, 333B Hamilton, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-292-3545; Fax: 614-292-4118; E-mail: hille.1{at}osu.edu.

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				N. Cobb, C. Hemann, G. A. Polsinelli, J. P. Ridge, A. G. McEwan, and R. Hille Spectroscopic and Kinetic Studies of Y114F and W116F Mutants of Me2SO Reductase from Rhodobacter capsulatus J. Biol. Chem., December 7, 2007; 282(49): 35519 - 35529. [Abstract] [Full Text] [PDF]