Tatsumi H, Takagi K, Fujita M, Kano K, Ikeda T
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Anal Chem. 1999 May 1;71(9):1753-9. doi: 10.1021/ac981003l.
An electrode modified with immobilized whole cells of Desulfovibrio vulgaris (Hildenborough) produces an S-shaped voltammogram with both cathodic- and anodic-catalytic-limiting currents in a methyl viologen-containing buffer saturated with H2. Methyl viologen penetrates into the bacterial cells to serve as an electron carrier in the reversible reaction of hydrogenase in the cells and functions as an electron-transfer mediator between the bacterial cells and the electrode, thus producing the catalytic currents for the evolution and consumption of H2. An equation for the catalytic current that takes into account the reversible hydrogenase reaction explains well the shape of the voltammogram. The potential at null current on the voltammogram agrees with the potential determined by potentiometry with the same electrode, which is equal to the redox potential of the H+/H2 couple in the solution--the standard potential of a hydrogen electrode at the pH of the solution. When D. vulgaris cells are suspended in an argon-saturated buffer containing methyl viologen, the suspension produces a catalytic current at a bare glassy carbon electrode for the evolution of H2. Analysis of the current by a theory for a catalytic current for a unidirectional nonlinear enzyme catalysis allows us to determine the kinetic parameters of the reaction between methyl viologen and hydrogenase in intact D. vulgaris cells. Thus we obtain the apparent Michaelis constant for methyl viologen cation radical, K'MV.+ = 0.16 mM, and the apparent catalytic constant (that is, the turnover number per D. vulgaris cell), zkcat,H+ = 1.2 x 10(7) s-1, for the H2 evolution reaction at pH 5.5 and at 25 degrees C, z being the number of hydrogenases contained in a D. vulgaris cell. The bimolecular reaction rate constant, kcat,H+/K'MV.+, of the reaction between methyl viologen cation radical and oxidized hydrogenase in intact D. vulgaris cells is estimated as 4.2 x 10(7) M-1 s-1. Similarly, the bimolecular reaction rate constant, kcat,H2/K'MV2+, of the reaction between methyl viologen and reduced hydrogenase is estimated to be 1.2 x 10(7) M-1 s-1 at pH 9.5 and 25 degrees C. Both rate constants are large enough for the reactions to be diffusion-limited processes.
用固定化的普通脱硫弧菌（希登伯勒菌株）全细胞修饰的电极，在饱和氢气的含甲基紫精缓冲液中产生S形伏安图，同时具有阴极催化极限电流和阳极催化极限电流。甲基紫精渗透到细菌细胞中，在细胞内氢化酶的可逆反应中作为电子载体，并在细菌细胞与电极之间充当电子转移介质，从而产生氢气析出和消耗的催化电流。考虑到可逆氢化酶反应的催化电流方程很好地解释了伏安图的形状。伏安图上零电流处的电位与用同一电极通过电位滴定法测定的电位一致，该电位等于溶液中H⁺/H₂电对的氧化还原电位——溶液pH值下氢电极的标准电位。当普通脱硫弧菌细胞悬浮在含甲基紫精的氩气饱和缓冲液中时，该悬浮液在裸玻碳电极上产生氢气析出的催化电流。通过单向非线性酶催化的催化电流理论对电流进行分析，使我们能够确定完整普通脱硫弧菌细胞中甲基紫精与氢化酶之间反应的动力学参数。因此，在pH 5.5和25℃下，对于氢气析出反应，我们得到甲基紫精阳离子自由基的表观米氏常数K'MV⁺ = 0.16 mM，以及表观催化常数（即每个普通脱硫弧菌细胞的周转数）zkcat,H⁺ = 1.2×10⁷ s⁻¹，z为普通脱硫弧菌细胞中所含氢化酶的数量。完整普通脱硫弧菌细胞中甲基紫精阳离子自由基与氧化态氢化酶之间反应的双分子反应速率常数kcat,H⁺/K'MV⁺估计为4.2×10⁷ M⁻¹ s⁻¹。同样，在pH 9.5和25℃下，甲基紫精与还原态氢化酶之间反应的双分子反应速率常数kcat,H₂/K'MV₂⁺估计为1.2×10⁷ M⁻¹ s⁻¹。两个速率常数都足够大，使得反应为扩散限制过程。
