A new approach for studying fast biological reactions involving dioxygen: the reaction of fully reduced cytochrome c oxidase with O2.
A new approach for studying fast biological reactions involving dioxygen: the reaction of fully reduced cytochrome c oxidase with O2.
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研究涉及双氧的快速生物反应的新方法：完全还原的细胞色素c氧化酶与O2的反应。
DOI：
10.1021/bi000955u
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Einarsdóttir,O
中科院分区：
文献类型：
--
作者：
VanEps,N;Szundi,I;Einarsdóttir,O
我们描述了一种研究涉及双氧的快速生物反应的新方法。这种方法基于合成笼状分子氧载体的光解，可快速产生分子氧。该方法用于研究 (μ-过氧)(μ-羟基)双[双(联吡啶基)钴(III)]络合物光解后细胞色素氧化酶在室温下将分子氧还原为水的情况。事实上，分子氧在纳秒或更快的时间尺度上原位产生，避免了与传统 CO 流动闪蒸实验中光解 CO 的命运相关的潜在并发症。该钴配合物在室温、厌氧条件下稳定，在355 nm照射下释放分子氧，量子产率为0.04。该复合物在实验的混合时间内不与还原型细胞色素氧化酶或其还原剂发生反应，并且其光产物不干扰双氧还原的动力学。在钴络合物光解离后，使用门控光谱多通道分析仪在 500 至 750 nm 之间监测还原型细胞色素氧化酶的氧化。使用奇异值分解和全局指数拟合对数据进行分析，解析两个表观寿命（380 ± 50 μs 和 1.7 ± 0.2 ms），并与传统 CO 流动闪蒸实验的结果进行比较。结果表明，单次激光脉冲可以产生~90 μM 分子氧，这种方法可用于研究涉及 O2 的其他快速生物反应。
We describe a new method for studying rapid biological reactions involving dioxygen. This approach is based on the photolysis of a synthetic caged dioxygen carrier, which produces dioxygen on a fast time scale. The method was used to investigate the reduction of dioxygen to water by cytochromecoxidase at room temperature following photolysis of a (μ-peroxo)(μ-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex. The fact that dioxygen is generated in situ on a nanosecond or faster time scale avoids potential complications related to the fate of photodissociated CO in a conventional CO flow-flash experiment. The cobalt complex is stable at room temperature under anaerobic conditions and releases dioxygen upon irradiation at 355 nm with a quantum yield of 0.04. The complex does not react with reduced cytochrome oxidase or its reducing agents within the mixing time of the experiment, and its photoproducts do not interfere with the kinetics of the dioxygen reduction. The oxidation of the reduced cytochrome oxidase was monitored between 500 and 750 nm using a gated optical spectrometric multichannel analyzer following photodissociation of the cobalt complex. The data were analyzed using singular value decomposition and global exponential fitting, and two apparent lifetimes (380 ± 50 μs and 1.7 ± 0.2 ms) were resolved and compared to results from a conventional CO flow-flash experiment. The results show that ∼90 μM dioxygen can be generated upon a single laser pulse and that this approach can be used to study other fast biological reactions involving O2.