Catalytic protein film voltammetry from a respiratory nitrate reductase provides evidence for complex electrochemical modulation of enzyme activity
Catalytic protein film voltammetry from a respiratory nitrate reductase provides evidence for complex electrochemical modulation of enzyme activity
复制标题
DOI：
10.1021/bi002706b
复制
发表时间：
2001-09-25
期刊：
影响因子：
2.9
通讯作者：
Butt, JN
中科院分区：
文献类型：
--
作者：
Anderson, LJ;Richardson, DJ;Butt, JN
泛养副扇贝中硝酸盐呼吸还原为氮素的第一步是由喹酚-硝酸氧化还原酶NarGHI催化的。这个膜锚定的蛋白质将电子从膜锚定NARL处的苯二酚氧化引导到膜外源[Fe-S]簇和含二聚体的Mo-bi-MGd中的硝酸盐还原位置。从膜上解放出来，NAGH保持其硝酸还原酶的活性，并在石墨和金电极上形成膜，在膜内电极和酶之间发生直接和容易的电子交换。蛋白质膜伏安法被用来确定NaGH在电位域的催化行为，并且解决了可逆的、硝酸盐浓度依赖的活性调节的复杂模式。在低硝酸盐浓度下，在催化电流-电势分布中观察到的局部最大值揭示了NaT-GH如何通过两条具有不同特异性常数的途径催化硝酸盐还原，即k(CAT)(OBS)/K-M(OBS)。在NAGH内发生的电化学事件引导催化通过其中一条路径发生。随着硝酸盐浓度的增加，催化电流中的局部极大值变得不明显，催化波形呈逐渐增大的S形。在立体化学不同的底物氯酸盐的还原过程中，观察到了类似于硝酸盐还原时观察到的伏安模式。通过EPR监测的电位滴定，确定了氧化态变化可能定义NaGH新催化伏安法的中心，并讨论了Mo-双-MGd或[Fe-S]团簇的电化学解释所观察到的行为的机理。
The first step in the respiratory reduction of nitrate to dinitrogen in Paracoccus pantotrophus is catalyzed by the quinol-nitrate oxidoreductase NarGHI. This membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, Narl, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH. Liberated from the membrane, NarGH retains its nitrate reductase activity and forms films on graphite and gold electrodes within which direct and facile exchange of electrons between the electrode and the enzyme occurs. Protein film voltammetry has been used to define the catalytic behavior of NarGH in the potential domain and a complex pattern of reversible, nitrate concentration dependent modulation of activity has been resolved. At low nitrate concentrations the local maximum observed in the catalytic current-potential profile reveals how Nat-GH can catalyze nitrate reduction via two pathways having distinct specificity constants, k(cat)(obs)/K-M(obs). Catalysis is directed to occur via one of the pathways by an electrochemical event within NarGH. On increasing the nitrate concentration, the local maximum in the catalytic current becomes less distinct, and the catalytic waveform adopts an increasingly sigmoidal form. A pattern of voltammetry similar to that observed during nitrate reduction is observed during reduction of the stereochemically distinct substrate chlorate. Centers whose change of oxidation state may define the novel catalytic voltammetry of NarGH have been identified by EPR-monitored potentiometric titrations and mechanisms by which the electrochemistry of Mo-bis-MGD or [Fe-S] clusters can account for the observed behavior are discussed.