Structure of the molybdenum site of dimethyl sulfoxide reductase

Molybdenum K-edge X-ray absorption and Mo(V) electron paramagentic resonanc e (EPR) spectroscopies have been used to probe the metal coordination in ox idized and reduced forms of both wildtype and a site-directed mutant of Rho dobacter sphaeroides dimethyl sulfoxide (DMSO) reductase, We confirm our ea rlier findings (George, G. N.; Hilton, J.; Rajagopalan, K. V. J. Am. Chem. Sec. 1996, 118, 1113-1117) that the molybdenum site of the oxidized Mo(VI) enzyme possesses one terminal oxygen ligand (Mo=O) at 1.68 Angstrom, four t hiolate ligands at: 2.44 Angstrom, and one oxygen at 1.92 Angstrom and that the dithionite-reduced Mo(IV) enzyme possesses a desoxo species with three or:four Mo-S at 2.33 Angstrom and two different Mo-O ligands at 2.16 and 1 .92 Angstrom. Mo(V) EPR indicates the presence of one exchangeable oxygen l igand, most likely an Mo-OH, in the signal-giving species; probably origina ting from the Mo=O of the oxidized enzyme (E-m8.5(IV/V) + 37 mV, E-m8.5(V/V I) = +83 mV). The addition of dimethyl sulfide,in the reverse of the physio logical reaction, reduces the enzyme. In this form, the enzyme contains a d esoxo active site with four Mo-S at 2.36 If and two different Mo-O ligands at 1.94 and 2.14 Angstrom. Recombinant wild-type R. sphaeroides DMSO reduct ase expressed in Escherichia coli. initially has a dioxo structure (two Mo= O at 1.72 Angstrom and four Mo-S at 2.48 Angstrom) but assumes the wild-typ e Mo(VI) structure after a cycle of reduction and reoxidation. The site-dir ected Ser147-->Cys mutant possesses a monooxo active site in the oxidized s tate (Mo=O at 1.70 Angstrom) with five sulfur ligands (at 2.40 Angstrom), c onsistent with cysteine 147 coordination to Mo. The dithionite reduced form of the mutant possesses a desoxo site also with five. Mo-S ligands (at 2.3 7 Angstrom) and one Mo-O at 2.12 Angstrom. The variant has substantially di fferent Mo(V) EPR and electrochemistry (E-m8.5(IV/V) -43 mV, E-m8.5(V/VI) = +106 mV). The active-site structure and catalytic mechanism of DMSO reduct ase are discussed in the light of the-se results.