Optical and EPR characterization of Desulfovibrio vulgaris (Hildenborough) sulfite reductase and ligand adducts

Abstract

The electronic properties of the low molecular mass sulfite reductase (SiR) from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) have been examined in both oxidized and reduced forms. Two well-resolved changes in optical spectra are observed following the addition of the first and second electron equivalents. One-electron reduced SiR shows a general decrease in the optical absorbance, with retention of all features. After addition of the second electron equivalent the broad absorbance at ∼400 nm in the oxidized spectrum was observed to split and more resemble the spectral features of the larger dissimilatory sulfite reductase from the same organism. Electron paramagnetic resonance spectroscopy (EPR) has also demonstrated the stepwise reduction of siroheme and cluster in a manner consistent with the observed changes in the optical spectrum. The signal from the oxidized siroheme was found to disappear prior to the appearance of the signal arising from the reduced cluster; that is, the one-electron reduced enzyme is EPR silent. Oxidized sulfite reductase shows a rhombic low-spin hexacoordinate siroheme ( S = 1 2 , g = 2.45, 2.38, 1.78 ), and the reduced enzyme shows a characteristic ferredoxin-like Fe 4S 4 EPR spectrum ( g = 2.04, 1.93) that differs from spectra obtained with E. coli sulfite reductase (P.A. Janick and L.M. Siegel, Biochemistry 21 (1982) 3538). The AsO 2 − adduct of the fully reduced enzyme displays distinctive optical characteristics. Following oxidation, the AsO 2 − ligand remains bound, and the complex yields an optical spectrum that is again different from the native enzyme.