Verhagen M F, Kooter I M, Wolbert R B, Hagen W R
Department of Biochemistry, Wageningen Agricultural University, The Netherlands.
Eur J Biochem. 1994 Apr 15;221(2):831-7. doi: 10.1111/j.1432-1033.1994.tb18797.x.
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.
来自希登伯勒脱硫弧菌的腺苷磷酸硫酸还原酶已被纯化至同质，发现它由两个亚基组成。α亚基和β亚基的分子量分别为67.8 kDa和25.6 kDa。该蛋白质的表观分子量取决于缓冲液的离子强度。在低离子强度下，会形成高分子量的多聚体，加入盐后会可逆地转变为较小的单元。通过凝胶过滤色谱法测定，该酶最小的催化活性单元分子量为186 kDa，因此提出其化学计量比为α2β2。发现该蛋白质每个αβ异二聚体含有5.6±1.1个铁原子和4.4±0.6个酸不稳定硫原子。还原态蛋白质呈现出一个单一的菱形S = 1/2信号，g值分别为2.070、1.932和1.891。降低缓冲液的离子强度会使该光谱可逆地转变为复杂的电子顺磁共振光谱，表明存在分子间偶极磁耦合。在低离子强度或高离子强度下对还原态蛋白质进行自旋定量，结果表明每个αβ异二聚体的自旋数从不超过1个。因此，可以得出铁和硫原子排列在一个单一的簇中。在pH 7.5的条件下，通过电子顺磁共振监测的氧化还原滴定法测得铁硫簇的还原电位相对于标准氢电极（NHE）为-19 mV。在pH 7.5的条件下，通过光学滴定法测得黄素的还原电位相对于NHE为-59 mV。黄素表现为双电子转移基团；未获得该酶中稳定中间半醌态的证据。测定腺苷5'-磷酸硫酸（Ado-PSO4）还原酶对其底物的动力学参数，结果表明亚硫酸盐和AMP的Km值分别为μM和50 μM。有人提出AdoPSO4还原酶含有一种单一的新型铁硫结构，其铁核数可能大于4。
