Mechanism of cytochrome c oxidase-catalyzed reduction of dioxygen to water: evidence for peroxy and ferryl intermediates at room temperature.
Mechanism of cytochrome c oxidase-catalyzed reduction of dioxygen to water: evidence for peroxy and ferryl intermediates at room temperature.
复制标题
细胞色素c氧化酶催化双氧还原成水的机制：室温下过氧和ferryl中间体的证据。
DOI：
10.1021/bi962422k
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Einarsdottir,O
中科院分区：
文献类型：
--
作者：
Sucheta,A;Georgiadis,KE;Einarsdottir,O
在完全还原的CO结合酶光解后，在室温下研究了牛心脏细胞色素氧化酶和双氧之间的反应。光解后 50 ns 至 50 ms 的时间分辨光学吸收差异光谱由门控多通道分析仪在可见光区域 (λ = 460−720 nm) 收集。奇异值分解（SVD）分析表明存在至少七种中间体。多指数拟合给出以下表观寿命：≥1.2μs、10μs、25μs、32μs、86μs和1.3ms。在 SVD 结果和双差图的基础上，提出了一种连续动力学机制，从中确定了反应中间体的光谱和时间依赖性群体。与还原酶相比，亚铁氧络合物（化合物 A）在 595 nm 处有峰，在 612 nm 处有谷，光解后约 30 μs 达到最大浓度。它衰变成 1:6 的过氧化物混合物 (a33+-O--O-)，其中细胞色素被还原和氧化。两个物种中的细胞色素 3 与其氧化形式相比，在 606 nm 处都有一个峰值。过氧物质衰变成 Ferr1 中间体，相对于氧化酶，其峰值位于 578 nm，随后电子在 CuA 和细胞色素之间重新分配。光解后，这两种 Ferryl 物质达到最大浓度~310 μs。中间体的实验和理论光谱之间的良好一致性为室温下细胞色素氧化酶还原双氧过程中过氧和铁基物种的存在提供了明确的证据。
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 (λ = 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 μs, 10 μs, 25 μs, 32 μs, 86 μs, 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 ∼30 μs after photolysis. It decays to a 1:6 mixture of peroxy species (a33+-O--O-) in which cytochromeais reduced and oxidized. Cytochromea3in 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 CuAand cytochromea. The two ferryl species reach a maximum concentration ∼310 μs 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.