Abstract
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.
Highlights
§ Current address: Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P
Initially there was some controversy concerning the active site structure, the crystal structure of R. sphaeroides Me2SO reductase has recently been determined at 1.3-Å resolution [17], where it is found that the oxidized enzyme is heterogeneous at the molybdenum center with two alternate conformations: a pentacoordinate dioxo monodithiolene formed when a molecule of HEPES buffer binds near the active site, as well as a hexacoordinate mono-oxo bisdithiolene ligand set
The absorption spectrum of oxidized Me2SO reductase is essentially insensitive to pH (Fig. 1A), displaying only a slight increase in absorbance near the feature observed at 350 nm in going from pH 6 to 10
Summary
Me2SO, dimethyl sulfoxide; Me2SOR, dimethyl sulfoxide reductase; Rc, Rhodobacter capsulatus; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; DMS, dimethylsulfide; PTA, phosphatriazaadamantane (1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane); MCA, multiple component analysis; MES, 4-morpholineethanesulfonic acid; TMAO, trimethylamine N-oxide. The significance of the reaction rests in the important role of DMS in modulating global solar albedo [1], DMS is one of several molecules that has been traced in recent iron-seeding experiments in the equatorial Pacific Ocean [2] In organisms such as Escherichia coli, Me2SO reductase is a membrane-bound terminal respiratory oxidase consisting of separate molybdenum- and iron-sulfur-containing subunits, as well as a membrane anchor. Significant accumulation of this intermediate is observed using Me2SO as substrate, increasing to ϳ80% late in the course of the reaction This behavior is the result of product-inhibition by DMS, which, unlike trimethylamine, is able to rebind the oxidized enzyme to yield the Ered1⁄7Me2SO complex. This behavior presumably reflects the thermodynamic stability of the Me2SObound, reduced form of the enzyme
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