Giuffrè A, D'Itri E, Giannini S, Brunori M, Ubbink-Kok T, Konings W N, Antonini G
Department of Biochemical Sciences and CNR Center of Molecular Biology, University of Rome "La Sapienza," 00185 Rome, Italy.
J Biol Chem. 1996 Jun 14;271(24):13987-92. doi: 10.1074/jbc.271.24.13987.
The thermophilic bacterium Bacillus stearothermophilus possesses a caa3-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R. I. R., and Konings, W. N. (1989) Eur. J. Biochem. 178, 763-770). We have carried out extensive kinetic experiments on the purified enzyme by stopped-flow time-resolved optical spectroscopy combined with singular value decomposition analysis. The results indicate a striking similarity of behavior between this enzyme and the electrostatic complex between mammalian cytochrome c and cytochrome c oxidase. CO binding to fully reduced caa3 occurs with a second order rate constant (k = 7.8 x 10(4)M-1 s-1) and an activation energy (E* = 6.1 kcal mol-1) similar to those reported for beef heart cytochrome c oxidase. Dithionite reduces cytochrome a with bimolecular kinetics, while cytochrome a3 (and CuB) is reduced via intramolecular electron transfer. When the fully reduced enzyme is mixed with O2, cytochrome a3, and cytochrome c are rapidly oxidized, whereas cytochrome a remains largely reduced in the first few milliseconds. When cyanide-bound caa3 is mixed with ascorbate plus TMPD, cytochrome c and cytochrome a are synchronously reduced; the value of the second order rate constant (k = 3 x 10(5) M-1 s-1 at 30 degrees C) suggests that cytochrome c is the electron entry site. Steady-state experiments indicate that cytochrome a has a redox potential higher than cytochrome c. The data from the reaction with O2 reveal a remarkable similarity in the kinetic, equilibrium, and optical properties of caa3 and the electrostatic complex cytochrome c/cytochrome c oxidase.
嗜热脂肪芽孢杆菌拥有一种caa3型末端氧化酶，该酶先前已被纯化（德弗里伊，W.，海涅，R.I.R.，和科宁斯，W.N.（1989年）《欧洲生物化学杂志》178卷，763 - 770页）。我们通过停流时间分辨光谱结合奇异值分解分析对纯化后的酶进行了广泛的动力学实验。结果表明，该酶与哺乳动物细胞色素c和细胞色素c氧化酶之间的静电复合物在行为上具有显著相似性。CO与完全还原的caa3结合的二级反应速率常数（k = 7.8×10⁴M⁻¹ s⁻¹）和活化能（E* = 6.1千卡/摩尔）与报道的牛心细胞色素c氧化酶相似。连二亚硫酸盐以双分子动力学还原细胞色素a，而细胞色素a3（和CuB）则通过分子内电子转移被还原。当将完全还原的酶与O₂混合时，细胞色素a3和细胞色素c迅速被氧化，而细胞色素a在最初的几毫秒内基本保持还原状态。当与氰化物结合的caa3与抗坏血酸加TMPD混合时，细胞色素c和细胞色素a同步被还原；二级反应速率常数的值（30℃时k = 3×10⁵ M⁻¹ s⁻¹）表明细胞色素c是电子进入位点。稳态实验表明细胞色素a的氧化还原电位高于细胞色素c。与O₂反应的数据揭示了caa3与静电复合物细胞色素c/细胞色素c氧化酶在动力学、平衡和光学性质方面的显著相似性。
