An Active Site Tyrosine Influences the Ability of the Dimethyl Sulfoxide Reductase Family of Molybdopterin Enzymes to Reduce S-Oxides

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.Dimethyl sulfoxide reductase is a bacterial enzyme responsible for the reduction of dimethyl sulfoxide (Me 2 SO) 1 to dimethyl sulfide (DMS). Substrate arises via breakdown of dimethylsulfoniopropionate, the principal osmolyte of marine algae, and is relatively abundant in the environment. 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 specifically in recent iron-seeding experiments in the equatorial Pacific Ocean (2). In organisms such as Escherichia coli, Me 2 SO reductase is a membrane-bound terminal respiratory oxidase consisting of separate molybdenum-and iron-sulfur-containing subunits, as well as a membrane anchor. In organisms such as Rhodobacter sphaeroides and Rhodobacter capsulatus, on the other hand, the enzyme is a soluble periplasmic protein of 85 kDa possessing only a molybdenum center (3); it appears to be involved in dissipating excess reducing equivalents in the absence of oxygen and is maximally expressed when the organism is grown photoheterotrophically on a relatively reduced carbon source such as malate. Me 2 SO reductases from both sources are very similar, and members of the same family of mononuclear molybdenum ...

Section: Resultsmentioning

confidence: 99%

Mechanistic Studies of Rhodobacter sphaeroides Me2SO Reductase

Cobb

Conrads

Hille

2005

“…Me 2 SO is not reduced by this enzyme confirming that it belongs to a distinct group, including sulfur/polysulfide/tetrathionate/thiosulfate reductases. This result could be explained by the absence of a tyrosine residue near the active site of Sre from Aquifex, which is important for the ability to utilize sulfur oxides in Me 2 SO reductases and biotin sulfoxide reductases (63).…”

Section: Existence Of a Supercomplex Involved In Sulfur Respiration Imentioning

confidence: 95%

A Membrane-bound Multienzyme, Hydrogen-oxidizing, and Sulfur-reducing Complex from the Hyperthermophilic Bacterium Aquifex aeolicus

Guiral

Tron

Aubert

et al. 2005

104

Aquifex aeolicus is a hyperthermophilic, chemolithoautotrophic, hydrogen-oxidizing, and microaerophilic bacterium growing at 85°C. We have shown that it can grow on an H 2 /S°medium and produce H 2 S from sulfur in the later exponential phase. The complex carrying the sulfur reducing activity (electron transport from H 2 to S°) has been purified and characterized. It is a membranebound multiprotein complex containing a [NiFe] hydrogenase and a sulfur reductase connected via quinones. The sulfur reductase is encoded by an operon annotated dms (dimethyl sulfoxide reductase) that we have renamed sre and is composed of three subunits. Sequence analysis showed that it belongs to the Me 2 SO reductase molybdoenzyme family and is similar to the sulfur/polysulfide/thiosulfate/tetrathionate reductases. The study of catalytic properties clearly demonstrated that it can reduce tetrathionate, sulfur, and polysulfide, but cannot reduce Me 2 SO and thiosulfate, and that NADPH increases the sulfur reducing activity. To date, this is the first characterization of a supercomplex from a bacterium that couples hydrogen oxidation and sulfur reduction. The distinctive feature in A. aeolicus is the cytoplasmic localization of the sulfur reduction, which is in accordance with the presence of sulfur globules in the cytoplasm. Association of this sulfur-reducing complex with a hydrogen-oxygen pathway complex (hydrogenase I, bc 1 complex) in the membrane suggests that subcomplexes involved in respiratory chains in this bacterium are part of supramolecular organization.The production of biomass in extreme light-independent environments is energized by chemolithoautotrophic oxidation and reduction of inorganic compounds like elemental sulfur (S°), 2 hydrogen, and nitrate (1, 2). Sulfur and sulfur compounds are the most abundant sources of both electron acceptors and electron donors in extremophilic environments (like volcanic environments) and are used by many microorganisms to support growth (3-6). Reduction and oxidation of sulfur compounds (sulfate, sulfite, thiosulfate, organic sulfoxide, elemental sulfur, polysulfides, and organic disulfide) are vital processes for many bacteria and essential steps in the global sulfur cycle. Because of the multiple oxidation states of sulfur, the biochemistry and chemistry of this cycle are complex and still not completely understood (7,8). This problem is exacerbated by the reactivities of the sulfur species at various oxidation states toward each other (7). The ability to reduce elemental sulfur is mostly found among hyperthermophilic micro-organisms (Archaea and Bacteria) (8), the majority of which depend on S°reduc-tion for optimal growth. They use either molecular hydrogen or organic compounds as electron donors. It has been suggested that S°respiration is one of the earliest mechanisms for energy conservation, because the hyperthermophiles are the forms of life evolving the most slowly (9).The processes by which micro-organisms reduce S°to H 2 S are still unclear for most of them, and only few ...

“…Trp-116 hydrogen-bonds to the labile MoAO group of oxidized enzyme and, thus, may be directly involved in catalysis. The role of Tyr-114 is less clear, although it also is close to the molybdenum center and has been suggested to hydrogen-bond to the oxygen atom of bound Me 2 SO in the E red ⅐Me 2 SO complex (24,25). With development of recombinant expression systems (in E. coli in the case of the R. sphaeroides enzyme (21,22) or homologously in R. capsulatus (23)), both Tyr-114 (24,25) and Trp-116 (26) have been targeted for mutation.…”

Section: So] Gives a K Dmentioning

confidence: 99%

“…With development of recombinant expression systems (in E. coli in the case of the R. sphaeroides enzyme (21,22) or homologously in R. capsulatus (23)), both Tyr-114 (24,25) and Trp-116 (26) have been targeted for mutation. The Y114F mutant turns over substantially more rapidly with both Me 2 SO and trimethylamine-N-oxide (TMAO, an alternate oxidizing substrate) relative to the wildtype enzyme in steady-state assays, although this increase in rate is offset by a decrease in substrate affinity (24,25). Steadystate analysis (25) has yielded a k cat of 81.…”

Section: So] Gives a K Dmentioning

confidence: 99%

See 1 more Smart Citation

Spectroscopic and Kinetic Studies of Y114F and W116F Mutants of Me2SO Reductase from Rhodobacter capsulatus

Cobb

Hemann

Polsinelli

et al. 2007

Mutants of the active site residues Trp-116 and Tyr-114 of the molybdenum-containing Me 2 SO reductase from Rhodobacter capsulatus have been examined spectroscopically and kinetically. The Y114F mutant has an increased rate constant for oxygen atom transfer from Me 2 SO to reduced enzyme, the result of lower stability of the E red ⅐Me 2 SO complex. The absorption spectrum of this species (but not that of either oxidized or reduced enzyme) is significantly perturbed in the mutant relative to wildtype enzyme, consistent with Tyr-114 interacting with bound Me 2 SO. The as-isolated W116F mutant is only five-coordinate, with one of the two equivalents of the pyranopterin cofactor found in the enzyme dissociated from the molybdenum and replaced by a second MoAO group. Reduction of the mutant with sodium dithionite and reoxidation with Me 2 SO, however, regenerates the long-wavelength absorbance of functional enzyme, although the wavelength maximum is shifted to 670 nm from the 720 nm of wild-type enzyme. This "redoxcycled" mutant exhibits a Me 2 SO reducing activity and overall reaction mechanism similar to that of wild-type enzyme but rapidly reverts to the inactive five-coordinate form in the course of turnover.Dimethyl sulfoxide reductase catalyzes the reduction of dimethyl sulfoxide (Me 2 SO) to dimethyl sulfide (DMS) 3 and is a member of a large class of mononuclear molybdenum-containing enzymes (1-3) having an L 2 MoO(X) core (where L represents a pyranopterin cofactor coordinated to the molybdenum via an enedithiolate side chain, and X represents a ligand in most cases provided by the polypeptide, a serinate in the case of Me 2 SO reductase). Two forms of Me 2 SO reductase exist, as exemplified by the enzymes from Escherichia coli (a heterotrimer consisting of the DmsABC gene products) and Rhodobacter species (the monomeric DorA protein). Both are components of anaerobic respiratory chains and catalyze the reductive abstraction of oxygen from Me 2 SO according to the following stoichiometry using electrons derived from the respiratory pool.The Rhodobacter sphaeroides DorA enzyme is a soluble periplasmic protein of 85 kDa possessing the molybdenum cofactor as the sole redox-active center. Because of the distinctive absorption features of its molybdenum center, the DorA enzyme has become a paradigm for understanding the electronic structure of molybdenum centers of the L 2 MoO(X) variety (4 -12). In addition to steady-state kinetics under a variety of conditions, rapid reaction studies with both the Rhodobacter capsulatus (10) and R. sphaeroides enzyme (13) have also been reported. With the R. sphaeroides enzyme, the reaction of reduced enzyme with Me 2 SO at low pH is biphasic. A fast [Me 2 SO]-dependent phase (k lim ϳ 1000 s Ϫ1 ) yields a spectrally distinct E red ⅐Me 2 SO complex, with absorption maxima at 490 and 550 nm, followed by a slower oxygen abstraction step (35 s Ϫ1 ) that yields DMS and E ox (13). The hyperbolic dependence of the rapid phase on [Me 2 SO] gives a K d Me 2SO of ϳ155 M and indicates tha...