Hirota S, Svensson-Ek M, Adelroth P, Sone N, Nilsson T, Malmström B G, Brzezinski P
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
J Bioenerg Biomembr. 1996 Dec;28(6):495-501. doi: 10.1007/BF02110439.
The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase from Bacillus PS3 in the presence of O2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochrome c subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 microseconds, 30 microseconds, 100 microseconds, and I ms at 445 nm. The initial, 10-microsecond absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving heme a and CuA are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochrome c, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O2 were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from heme a3 to a in PS3 oxidase compared to the bovine enzyme.
在有氧气存在的情况下，对来自嗜热栖热菌（Bacillus PS3）的完全还原态羧基细胞色素氧化酶进行闪光光解后，在445、550、605和830纳米处跟踪吸光度变化的时间进程，并将结果与牛细胞色素氧化酶的相应变化进行比较。PS3酶有一个共价结合的细胞色素c亚基，因此完全还原态的物种容纳五个电子，而牛酶中容纳四个电子。在牛酶中，CO解离后，在445纳米处观察到四个阶段，时间常数分别约为10微秒、30微秒、100微秒和1毫秒。两种酶在445纳米处最初10微秒的吸光度变化相似。在PS3酶中，在445纳米处未观察到随后涉及血红素a和CuA的阶段，因为这些氧化还原中心被共价结合的细胞色素c重新还原，550纳米处的吸光度变化表明了这一点。提出了一个与实验观察结果一致的反应方案。此外，在闪光诱导混合价酶中的CO解离后，研究了在没有氧气的情况下的内部电子转移反应。混合价氧化酶和完全还原态氧化酶中CO重组速率的比较表明，与牛酶相比，PS3氧化酶中从血红素a3向a转移了更多电子。
