Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase
Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase
复制标题
DOI：
10.1074/jbc.274.47.33403
复制
发表时间：
1999-11-19
影响因子：
4.8
通讯作者：
Yoshikawa, S
中科院分区：
文献类型：
--
作者：
Mochizuki, M;Aoyama, H;Yoshikawa, S
牛心细胞色素c氧化酶制剂中大约30%的铁通过结晶被去除，分子消光系数比迄今为止报道的高1.25-1.4倍。结晶细胞色素c氧化酶制剂的完全还原需要连二亚硫酸盐提供的六个电子当量。完全还原的酶被分子氧提供的 4 个氧化当量氧化，产生的吸收光谱与原始完全氧化形式的吸收光谱略有不同，但显着不同。 O-2-氧化酶的完全还原需要四个电子当量。当O-2-氧化形式暴露于过量的O-2时，会转化为原始氧化形式，需要6个电子才能完全还原。在不添加任何外部还原剂的情况下，O-2-氧化形式的缓慢还原表明酶中存在血红素铁的内部电子供体。这些结果表明，与 O-2 氧化形式相比，原始氧化形式中的 2 个额外氧化当量是由于 O-2 和来自内部供体的电子产生的结合过氧化物所致，与酶晶体结构中 O-2 还原位点处的过氧化物一致（Yoshikawa, S.、Shinzawa-Itoh, K.、Nakashima, R.、Yaano, R.， Yamashita, E.、Inoue, N.、Yao, M.、Fei, M. J.、Peters Libeu, C.、Mizushima, T.、Yamaguchi, H.、Tomizaki, T. 和 Tsukihara, T. (1998) Science 280, 1723-1729)。
Approximately 30% of the iron contained in a bovine heart cytochrome c oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25-1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme was oxidized with 4 oxidation equivalents provided by molecular oxygen, giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were required for complete reduction of the O-2-oxidized enzyme. The O-2-oxidized form, when exposed to excess amounts of O-2, was converted to the original oxidized form which required 6 electrons for complete reduction. A slow reduction of the O-2-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original oxidized form, compared with the O-2-oxidized form, are due to a bound peroxide produced by O-2 and electrons from the internal donors, consistently with a peroxide at the O-2 reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998) Science 280, 1723-1729).