NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa -: Characterization of reaction intermediates that appear in the single turnover cycle
NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa -: Characterization of reaction intermediates that appear in the single turnover cycle
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DOI：
10.1074/jbc.m409996200
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发表时间：
2004-12-31
影响因子：
4.8
通讯作者：
Shiro, Y
中科院分区：
文献类型：
--
作者：
Kumita, H;Matsuura, K;Shiro, Y
硝化细菌的一氧化氮还原酶（NOR）在由高自旋血红素B（3）和非血红素Fe-B组成的双核催化中心催化NO还原为N2 O。利用光学吸收光谱和电子顺磁共振波谱结合自行设计的冷冻淬灭装置，研究了铜绿假单胞菌NOR还原NO单次翻转过程中反应中间体的结构.在样品的EPR谱中，其中将完全还原的NOR与NO溶液混合并在混合后在0.5ms淬灭，观察到亚铁Fe-B- NO和五配位亚铁血红素B（3）- NO物质的两个特征信号。CO对其形成的抑制表明，在这种状态下，两个NO分子同时分布到酶同一双核中心的两个离子中。时间和温度依赖性的EPR光谱变化表明，出现在0.5 ms的物种是一个短暂的反应中间体之前，N2 O的形成，在良好的协议与所谓的“反式”机制。研究还发现，酶在单一周转周期中的最终状态是完全氧化状态，其中在其双核中心的两个铁之间不存在μ-氧代桥接配体，这与分离的NOR的静息形式不同。在此基础上，提出了一种新的NO还原反应机理。
Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N2O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe-B. The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe-B - NO and the penta-coordinated ferrous heme b(3) - NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N2O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.