Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.
Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.
Bastian
N R
NR
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Kay
C J
CJ
Barber
M J
MJ
Rajagopalan
K V
KV
D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008982:Molybdenum / Q000032:analysis*; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013053:Spectrophotometry; D013816:Thermodynamics; D014969:Xanthine Oxidase / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D008982:Molybdenum; D010088:Oxidoreductases; D014969:Xanthine Oxidase; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D050862:Oxidoreductases Acting on Sulfur Group Donors; C045664:dimethyl sulfoxide reductase