Using direct electrochemistry to probe rate limiting events during nitrate reductase turnover.
Using direct electrochemistry to probe rate limiting events during nitrate reductase turnover.
复制标题
使用直接电化学来探测硝酸还原酶周转期间的限速事件。
DOI：
10.1039/b000946f
复制
发表时间：
2000
影响因子：
3.4
通讯作者：
J. Butt
中科院分区：
文献类型：
--
作者：
L. J. Anderson;D. Richardson;J. Butt
NAGH在稳态条件下催化硝酸盐还原的蛋白质膜伏安法提供了催化循环期间酶内发生的事件的信息。在这次讨论中，我们重点探索了两种简单的催化方案重现NAGH伏安响应的能力；通过界面电子交换(方案1)或通过电子中继中心操作的分子内电子传递(方案2)描述了电子到酶活性位置的转移。当酶的两个电子被还原，在催化循环的非限速步骤中从被氧化的形式产生时，无法重现NAGH的伏安行为。相反，在所研究的所有条件下，发现活性中心从半还原状态到完全还原状态的一个电子还原对于酶的催化循环是至关重要的。活性中心的催化相关的半还原和全还原氧化态很可能分别对应于Mo(MGd)2中心的Mo(V)和Mo(IV)态，但不能排除它们与其他酶中观察到的基于钼杂蝶呤的氧化态相对应的可能性。我们认为，半还原活性中心内的构象重排或分子内电子传递到活性中心的速率构成了NAGH催化循环的一个决定性特征，并导致了NAPP大约1种催化波形的出现。
Protein film voltammetry of NarGH catalysing nitrate reduction under steady state conditions provides information on events occurring within the enzyme during the catalytic cycle. In this discussion we have focused on exploring the ability of two simple catalytic schemes to reproduce the voltammetric response of NarGH; electron transfer to the enzyme's active site being described either by interfacial electron exchange (Scheme 1) or intramolecular electron delivery via the operation of an electron relay centre (Scheme 2). When the two electron reduced, catalytically competent active site of the enzyme is generated from the oxidised form in 'rapid', non-rate limiting steps of the catalytic cycle, the voltammetric behaviour of NarGH cannot be reproduced. Rather under all the conditions investigated, one electron reduction of the active site from a semi-reduced to a fully-reduced state is found to be crucial to progression through the enzyme's catalytic cycle. The catalytically relevant semi- and fully-reduced oxidation states of the NarGH active site are most likely to correspond to the Mo(V) and Mo(IV) states of the Mo(MGD)2 centre, respectively, although it is not possible to rule out the possibility that they correspond to molybdopterin based oxidation states as observed in other enzymes. We suggest that the rate of either conformational rearrangement within the semi-reduced active site or intramolecular electron delivery to the active site constitutes a defining feature in the catalytic cycle of NarGH and results in the napp approximately 1 appearance of the catalytic waveform.