Proton and electron pathways in the bacterial nitric oxide reductase
Proton and electron pathways in the bacterial nitric oxide reductase
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DOI：
10.1021/bi0121050
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发表时间：
2002-02-19
期刊：
影响因子：
2.9
通讯作者：
Verkhovsky, MI
中科院分区：
文献类型：
--
作者：
Hendriks, JHM;Jasaitis, A;Verkhovsky, MI
细菌]一氧化氮还原酶（NOR）中的电子和质子转移反应已通过光谱学和静电测量法进行了研究。在脂质体中，NOR 在稳态转换过程中不会产生任何电势。这种电中性意味着在催化过程中质子与电子从膜的同一侧被吸收。部分还原的CO结合酶光解后的分子内电子重新分布表明，NOR中的电子转移与血红素铜氧化酶具有相同的途径。电子从受体位点血红素 c 通过低自旋血红素 b 转移到双核活性位点（血红素 b(3)/Fe-B）。血红素 c 和 b 之间的电子转移率为 (3 +/- 2) x 10(4) s(-1)。血红素 b 和 b(3) 之间的电子转移速率太快，无法解析 (> 10(6) s(-1))。只有血红素 c 和血红素 b 之间的电子转移与电势的产生相关。这意味着 NOR 中氧化还原中心的拓扑结构与血红素铜细胞色素氧化酶中的相似。光学和电测量可以识别完全还原的酶转换过程中形成的中间状态，以及与 NO 还原相关的质子和电子运动。第一相 (k = 5 x 10(5) s(-1)) 是电静默的，其特征是 433 nm 处吸光度消失并在 410 nm 处出现宽峰。我们将此阶段指定为血红素 b(3) 的亚铁 NO 加合物的形成。 NO 结合之后是电荷分离阶段 (k = 2.2 x 10(5) s(-1))。我们认为，这种与显着光学变化无关的中间体的形成涉及活性位点附近带电侧链的移动。下一步创建一个负电势，其速率常数类似于 3 x 10(4) s(-1) 和弱光学特征。接下来是一个速率常数为 5 x 10(3) s(-1) 的电静默阶段，导致第一次周转的最后一个中间阶段（速率常数类似于 10(3) s(-1)）。完全还原的酶有四个电子，足以进行两个完整的催化循环。然而，第二次周转的质子必须从本体中取出，从而导致分两步产生正电势。光学测量还验证了低自旋血红素氧化的两个阶段。基于这些结果，我们提出了 NOR 还原 NO 的机制模型。结果可以用反式机制而不是涉及 Fe-B 的顺式模型来解释。此外，数据揭示了 NOR 采用 P450 型机制的可能性，其中只有血红素 b(3) 在周转期间充当 NO 结合位点。
Electron- and proton-transfer reactions in bacteria] nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b(3)/Fe-B). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b(3) is too fast to be resolved (> 10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b(3). NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of similar to3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of similar to10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving Fe-B. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b(3) functions as the NO binding site during turnover.