Sucheta A, Georgiadis K E, Einarsdóttir O
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
Biochemistry. 1997 Jan 21;36(3):554-65. doi: 10.1021/bi962422k.
The reaction between bovine heart cytochrome oxidase and dioxygen was investigated at room temperature following photolysis of the fully reduced CO-bound enzyme. Time-resolved optical absorption difference spectra were collected by a gated multichannel analyzer in the visible region (lambda = 460-720 nm) from 50 ns to 50 ms after photolysis. Singular value decomposition (SVD) analysis indicated the presence of at least seven intermediates. Multiexponential fitting gave the following apparent lifetimes: 1.2 microseconds, 10 microseconds, 25 microseconds, 32 microseconds, 86 microseconds, and 1.3 ms. On the basis of the SVD results and a double difference map, a sequential kinetic mechanism is proposed from which the spectra and time-dependent populations of the reaction intermediates were determined. The ferrous-oxy complex (compound A), with a peak at 595 nm and a trough at 612 nm versus the reduced enzyme, reaches a maximum concentration approximately 30 microseconds after photolysis. It decays to a 1:6 mixture of peroxy species (a3(3+)-O(-)-O-) in which cytochrome a is reduced and oxidized. Cytochrome a3 in both species has a peak at 606 nm versus its oxidized form. The peroxy species decay to a ferryl intermediate, with a peak at 578 nm versus the oxidized enzyme, followed by electron redistribution between CuA and cytochrome a. The two ferryl species reach a maximum concentration approximately 310 microseconds after photolysis. The excellent agreement between the experimental and theoretical spectra of the intermediates provides unequivocal evidence for the presence of peroxy and ferryl species during dioxygen reduction by cytochrome oxidase at room temperature.
在完全还原的一氧化碳结合酶光解后，于室温下研究了牛心细胞色素氧化酶与双氧的反应。在光解后50纳秒至50毫秒的可见区域（波长=460 - 720纳米），用门控多通道分析仪收集时间分辨光吸收差光谱。奇异值分解（SVD）分析表明至少存在七种中间体。多指数拟合给出了以下表观寿命：1.2微秒、10微秒、25微秒、32微秒、86微秒和1.3毫秒。基于SVD结果和双差图谱，提出了一个连续动力学机制，据此确定了反应中间体的光谱和随时间变化的丰度。亚铁 - 氧复合物（化合物A），相对于还原酶，在595纳米处有一个峰，在612纳米处有一个谷，在光解后约30微秒达到最大浓度。它衰变为过氧物种（a3(3 +)-O(-)-O-）的1:6混合物，其中细胞色素a被还原和氧化。两种物种中的细胞色素a3相对于其氧化形式在606纳米处有一个峰。过氧物种衰变为一个高铁中间体，相对于氧化酶在578纳米处有一个峰，随后在CuA和细胞色素a之间进行电子重新分布。这两种高铁物种在光解后约310微秒达到最大浓度。中间体的实验光谱和理论光谱之间的出色吻合为细胞色素氧化酶在室温下还原双氧过程中过氧和高铁物种的存在提供了明确证据。
