Kobayashi K, Tagawa S, Suzuki S
The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan.
J Biochem. 1999 Aug;126(2):408-12. doi: 10.1093/oxfordjournals.jbchem.a022465.
Electron transfer over 12.6 A from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu. In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A. xylosoxidans decreased 1/2 fold to 9 x 10(2) s-1 (20 degrees C, pH 7.0) as compared to that for the same process in the absence of nitrite. However, the rate constant increased with decreasing pH to become the same (2 x 10(3) s-1) as that in the absence of nitrite at pH 6.0. A similar result was obtained for the enzyme from A. cycloclastes. The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction. This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite. The difference in redox potential (DeltaE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer. The pH-dependence of DeltaE was in accord with the equation: DeltaE = DeltaE(0)+0.058 log (Kr[H+]+[H+]2)/(K(0)+[H+]), where K(r) and K(0) are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively. These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu.
在对两种反硝化细菌（木糖氧化产碱菌GIFU 1051和环裂无色杆菌IAN 1013）含铜亚硝酸还原酶中从1型铜（T1Cu）到2型铜（T2Cu）的12.6埃电子转移进行研究时，先对T1Cu进行了脉冲辐射还原。在有亚硝酸盐存在的情况下，与无亚硝酸盐时相比，木糖氧化产碱菌的该酶分子内电子转移速率常数降低了1/2，降至9×10² s⁻¹（20℃，pH 7.0）。然而，随着pH降低，速率常数增大，在pH 6.0时与无亚硝酸盐时相同（2×10³ s⁻¹）。环裂无色杆菌的酶也得到了类似结果。两种酶在有亚硝酸盐存在时的pH曲线与亚硝酸还原酶活性的pH曲线几乎相同。这表明分子内电子转移过程与随后的亚硝酸盐催化还原过程紧密相连。根据电子转移的平衡数据计算了T2Cu减去T1Cu的氧化还原电位差（ΔE）。ΔE对pH的依赖性符合以下方程：ΔE = ΔE(0)+0.058 log (Kr[H⁺]+[H⁺]²)/(K(0)+[H⁺])，其中Kr和K(0)分别是T2Cu氧化态和还原态的质子解离常数。这些结果增加了一种可能性，即通过T2Cu的氧化还原连接的氨基酸残基在酶反应中起重要作用，它们位于T2Cu附近。
