Wijma Hein J, Jeuken Lars J C, Verbeet Martin P, Armstrong Fraser A, Canters Gerard W
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
J Biol Chem. 2006 Jun 16;281(24):16340-6. doi: 10.1074/jbc.M601610200. Epub 2006 Apr 13.
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 [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] 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 approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.
同源三聚体含铜亚硝酸还原酶（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，并穿梭至2型位点，在该位点亚硝酸被还原为一氧化氮。为了研究NiR的催化机制，我们测定了在饱和浓度下三种不同电子供体存在时pH值和亚硝酸对周转率的影响。还通过利用直接电子转移至固定在石墨旋转圆盘电极上的酶，以电化学方式测量了NiR的活性。在所有情况下，稳态动力学都非常符合随机顺序机制，其中从1型位点到2型位点的电子转移是限速步骤。在低[NO₂⁻]时，2型位点的还原先于亚硝酸结合，在高[NO₂⁻]时则相反。在pH 6.5以下，较高亚硝酸浓度下催化活性降低，这与电子转移至亚硝酸结合的2型位点比转移至水结合的2型位点更慢一致。在pH 6.5以上，观察到底物活化，这与电子转移至亚硝酸结合的2型位点比转移至羟基结合的2型位点更快一致。为了研究1型和2型位点之间较慢电子转移的影响，使用了NiR M150T。它的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至2型位点的速度大约慢50倍。结果证实NiR采用随机顺序机制。
