Verhagen M F, Wolbert R B, Hagen W R
Department of Biochemistry, Wageningen Agricultural University, The Netherlands.
Eur J Biochem. 1994 Apr 15;221(2):821-9. doi: 10.1111/j.1432-1033.1994.tb18796.x.
An electrochemical study of the periplasmic cytochrome c553 of Desulfovibrio vulgaris (Hildenborough) is presented. The dependence of the midpoint potential on temperature and pH was studied with cyclic voltammetry. The voltammograms obtained were reversible and revealed that this cytochrome showed fast electron transfer on a bare glassy carbon electrode. The midpoint potential at pH 7.0 and 25 degrees C was found to be 62 mV versus the normal hydrogen electrode. It was observed that the temperature dependence was discontinuous with a transition temperature at 32 degrees C. The standard reaction entropy at the growth temperature of the organism (37 degrees C) was calculated to be delta S degree ' = -234 J mol-1 K-1. The pH dependence of the midpoint potential could be described with one pK of the oxidized form with a value of 10.6. The second-order rate constant for electron transfer between cytochrome c553 and the Fe-hydrogenase from D. vulgaris (H) was also determined with cyclic voltammetry. The equivalent rate constant for cytochrome c3 and hydrogenase was measured for comparison. The second-order rate constants are 2 x 10(7) M-1 s-1 for cytochrome c553 and 2 x 10(8) M-1 s-1 for cytochrome c3. The kinetic parameters of the hydrogenase for both cytochromes were determined using the spectrophotometric hydrogen consumption assay. With cytochrome c553 this resulted in a Km of 46 microM and a maximum turnover number of 7.1 x 10(2) s-1 in the H2 consumption assay. The values with cytochrome c3 were 17 microM and 6.4 x 10(2) s-1, respectively. The importance of the different kinetic parameters for contrasting models proposed to describe the function of the Fe-hydrogenase are discussed.
本文介绍了对普通脱硫弧菌（希登伯勒菌株）周质细胞色素c553的电化学研究。采用循环伏安法研究了中点电位对温度和pH的依赖性。所获得的伏安图是可逆的，表明该细胞色素在裸玻碳电极上显示出快速电子转移。在pH 7.0和25℃时，相对于标准氢电极，中点电位为62 mV。观察到温度依赖性是不连续的，转变温度为32℃。计算出该生物体生长温度（37℃）下的标准反应熵为ΔS°' = -234 J mol-1 K-1。中点电位的pH依赖性可用氧化形式的一个pK值（10.6）来描述。还采用循环伏安法测定了细胞色素c553与普通脱硫弧菌（H）的铁氢化酶之间电子转移的二级速率常数。测量了细胞色素c3和氢化酶的等效速率常数以作比较。细胞色素c553的二级速率常数为2×10(7) M-1 s-1，细胞色素c3的二级速率常数为2×10(8) M-1 s-1。使用分光光度法氢消耗测定法测定了两种细胞色素的氢化酶动力学参数。对于细胞色素c553，在氢消耗测定中，这导致Km为46 μM，最大周转数为7.1×10(2) s-1。细胞色素c3的值分别为17 μM和6.4×10(2) s-1。讨论了不同动力学参数对于描述铁氢化酶功能的对比模型的重要性。
