On the iron-sulfur cluster of adenosine phosphosulfate reductase from Desulfovibrio vulgaris (Hildenborough).

Adenosine phosphosulfate reductase from Desulfovibrio vulgaris Hildenborough has been purified to homogeneity and was found to consist of two subunits. The alpha and beta subunits have molecular masses of 67.8 kDa and 25.6 kDa, respectively. The apparent molecular mass of the protein is dependent on the ionic strength of the buffer. At low ionic strength, a high molecular-mass multimer is formed, which reversibly changes into smaller units upon addition of salt. The smallest catalytically active unit of the enzyme has a molecular-mass of 186 kDa, as determined by gel-filtration chromatography and, therefore, an alpha 2 beta 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 heterodimer. 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 buffer reversibly changes this spectrum into a complex EPR spectrum, indicating intermolecular, dipolar magnetic coupling. Spin quantification of the reduced protein either at low or at high ionic strength never resulted in more than 1 spin/alpha beta heterodimer. Hence, it follows that the iron and sulfur atoms are arranged in one single cluster. The reduction potential of the iron sulfur cluster, measured in an EPR-monitored redox titration, was found to be -19 mV versus the normal hydrogen electrode (NHE) at pH 7.5. The reduction potential of the flavin 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 semiquinone state in the enzyme. Determination of the kinetic parameters of adenosine 5'-phosphosulfate (Ado-PSO4) reductase for its substrates resulted in Km values for sulfite and AMP of 130 microM and 50 microM, respectively. It is proposed that AdoPSO4 reductase contains a single novel Fe/S structure, possibly with an iron-nuclearity greater than four.