ON THE IRON-SULFUR CLUSTER OF ADENOSINE PHOSPHOSULFATE REDUCTASE FROMDESULFOVIBRIO-VULGARIS (HILDENBOROUGH)

Adenosine phosphosulfate reductase from Desulfovibrio vulgaris Hildenb orough has been purified to homogeneity and was found to consist of tw o subunits. The alpha and beta subunits have molecular masses of 67.8 kDa and 25.6 kDa, respectively. The apparent molecular mass of the pro tein is dependent on the ionic strength of the buffer. At low ionic st rength, a high molecular-mass multimer is formed, which reversibly cha nges into smaller units upon addition of salt. The smallest catalytica lly active unit of the enzyme has a molecular-mass of 186 kDa, as dete rmined by gel-filtration chromatography and, therefore, an alpha(2) be ta(2) stoichiometry is proposed. The protein was found to contain 5.6/-1.1 iron and 4.4+/-0.6 acid-labile sulfur atoms/alpha beta heterodim er. The reduced protein exhibits a single, rhombic S = 1/2 signal with g values 2.070, 1.932 and 1.891. Lowering the ionic strength of the b uffer reversibly changes this spectrum into a complex EPR spectrum, in dicating intermolecular, dipolar magnetic coupling. Spin quantificatio n of the reduced protein either at low or at high ionic strength never resulted in more than 1 spin/alpha beta heterodimer. Hence, it follow s that the iron and sulfur atoms are arranged in one single cluster. T he reduction potential of the iron sulfur cluster, measured in an EPR- monitored redox titration, was found to be -19 mV versus the normal hy drogen electrode (NHE) at pH 7.5. The reduction potential of the flavi n measured in an optical titration was found to be -59 mV against NHE at pH 7.5. The flavin behaves as a two-electron-transferring group; no evidence was obtained for a stabilization of the intermediate semiqui none state in the enzyme. Determination of the kinetic parameters of a denosine 5'-phosphosulfate (Ado-PSO4) reductase for its substrates res ulted in K-m values for sulfite and AMP of 130 mu M and 50 mu M, respe ctively. It is proposed that AdoPSO(4) reductase contains a single nov el Fe/S structure, possibly with an iron-nuclearity greater than four.