A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase
A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase
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DOI：
10.1074/jbc.m601610200
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发表时间：
2006-06-16
影响因子：
4.8
通讯作者：
Canters, Gerard W.
中科院分区：
文献类型：
--
作者：
Wijma, Hein J.;Jeuken, Lars J. C.;Canters, Gerard W.
同源三聚体含铜亚硝酸盐还原酶（NiR）每个单体含有一个1型和一个2型铜中心。电子通过1型位点进入，并穿梭到2型位点，在那里亚硝酸盐被还原为一氧化氮。为了研究NiR的催化机理，在饱和浓度下，在三种不同的电子供体存在下，测量了pH和亚硝酸盐对周转率的影响。NiR的活性也通过利用直接电子转移到固定在石墨旋转盘电极上的酶的电化学测量。在所有情况下，稳态动力学拟合良好的随机顺序机制，其中电子转移从1型到2型网站是限速的。在低[ NO2-]还原的类型-2网站之前亚硝酸盐结合，在高[ NO2-]发生相反。低于pH 6.5，在较高的亚硝酸盐浓度的催化活性降低，与电子转移慢的亚硝酸盐结合的2型网站比水结合的2型网站。在pH 6.5以上，观察到底物活化，与电子转移到亚硝酸盐结合的2型网站比电子转移到羟基结合的2型网站更快。为了研究1型和2型位点之间较慢的电子转移的影响，使用NiR M150 T。它有一个1型位点，中点电位高125 mV，重组能高0.3 eV，导致分子内电子转移到2型位点的速度慢50倍。结果证实，NiR采用随机顺序机制。
The homotrimeric copper-containing nitrite reductase ( NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [ NO2-] reduction of the type-2 site precedes nitrite binding, at high [ NO2-] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an similar to 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.