Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome c nitrite reductase
Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome c nitrite reductase
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DOI：
10.1074/jbc.m200495200
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发表时间：
2002-06-28
影响因子：
4.8
通讯作者：
Butt, JN
中科院分区：
文献类型：
--
作者：
Angove, HC;Cole, JA;Butt, JN
细胞色素c亚硝酸盐还原酶通过催化亚硝酸盐六电子还原成铵，在生物氮循环中起着关键作用。涂有大肠杆菌细胞色素c亚硝酸盐还原酶的石墨电极，置于含有亚硝酸盐（pH 7）的溶液中，在低于0 V的电位下，循环伏安法显示出大的催化还原电流。在没有细胞色素c亚硝酸盐还原酶的情况下，没有观察到这些催化电流，并且表明这些催化电流起源于与电极直接进行电子交换的酶膜。从底物限制到酶限制的亚硝酸盐还原过程中观察到的催化电流-电位谱显示了与羟胺还原过程中观察到的催化行为不同的指纹，羟胺是酶在双电子过程中还原为铵的替代底物。细胞色素c亚硝酸盐还原酶与这两种底物的相互作用明显不同。然而，随着亚硝酸盐或羟胺浓度的增加，伏安反应的发展也具有类似的特征。这些特征与活性位点的协调双电子还原一致，表明这两种底物的还原机制是由共同的速率定义过程支撑的。
The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium. Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V. These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode. The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process. Cytochrome c nitrite reductase clearly interacts differently with these two substrates. However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration. These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.