Giuffrè A, Forte E, Antonini G, D'Itri E, Brunori M, Soulimane T, Buse G
Department of Biochemical Sciences, CNR Center of Molecular Biology, University of Rome "La Sapienza", Italy.
Biochemistry. 1999 Jan 19;38(3):1057-65. doi: 10.1021/bi9815389.
The kinetic properties of the ba3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70 degrees C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O2 reaction, the decay of the F intermediate is significantly slower (k' = 100 s-1 at 5 degrees C) than in the mitochondrial enzyme, with an activation energy E of 10.1 +/- 0.9 kcal mol-1. The cyanide-inhibited ba3 oxidizes cyt c522 quickly (k approximately 5 x 10(6) M-1 s-1 at 25 degrees C) and selectively, with an activation energy E of 10.9 +/- 0.9 kcal mol-1, but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c552 oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c522-ba3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba3 for CO at 20 degrees C (Keq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa3 (Keq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a32+ of ba3 (k' = 0.8 s-1 vs k' = 0.02 s-1 for beef heart aa3 at 20 degrees C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O2 tensions is discussed.
利用停流光谱法在5至70摄氏度的温度范围内研究了嗜热栖热菌ba3氧化酶的动力学特性。观察到该酶与生理底物及经典配体（CO和CN-）反应时的特殊行为。在与O2反应中，F中间体的衰减明显慢于线粒体酶（5摄氏度时k' = 100 s-1），活化能E为10.1±0.9 kcal mol-1。氰化物抑制的ba3能快速（25摄氏度时k约为5×10(6) M-1 s-1）且选择性地氧化细胞色素c522，活化能E为10.9±0.9 kcal mol-1，但氧化线粒体酶的快速电子供体六胺钌的速度较慢。细胞色素c552氧化酶活性在60摄氏度时增强，在极低离子强度下达到最大值，排除了形成高亲和力细胞色素c522 - ba3静电复合物的可能性。嗜热氧化酶对氰化物抑制的敏感性较低，尽管在周转过程中氰化物结合比完全氧化状态下快得多（数秒）（完全氧化状态下为数天）。最后，发现还原态ba3在20摄氏度时对CO的亲和力（Keq = 1×10(5) M-1）小于牛心aa3（Keq = 4×10(6) M-1），部分原因是ba3的细胞色素a32+中CO解离异常快速且强烈依赖温度（20摄氏度时k' = 0.8 s-1，而牛心aa3为k' = 0.02 s-1）。讨论了这些结果与呼吸活动适应高温和低环境O2张力的相关性。
