Unité de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, UPR 9036, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
J Phys Chem B. 2010 Mar 11;114(9):3341-7. doi: 10.1021/jp911443y.
We examined the kinetics of nitrate reduction by periplasmic nitrate reductase (Nap) by using protein film voltammetry and solution assays. We demonstrate that, under turnover conditions, the enzyme exists as a mixture of active and inactive forms which interconvert on a time scale that is much slower than turnover. The dead-end species accumulates under mildly reducing conditions and at high nitrate concentration, resulting in substrate inhibition and in an uncommon hysteresis in the voltammetric signature. Solution assays with two electron donors having different reduction potentials fully support the electrochemical results. This illustrates the consequences of the high flexibility of the active site molybdenum coordination sphere and questions the conclusions from earlier studies in which attempts were made to trap catalytic intermediates of Nap in experiments carried out under turnover conditions at very high substrate concentration.
我们通过蛋白膜伏安法和溶液分析研究了周质硝酸盐还原酶（Nap）还原硝酸盐的动力学。我们证明，在周转条件下，该酶以活性和非活性形式的混合物形式存在，这种转化的时间尺度比周转慢得多。在温和的还原条件下和高硝酸盐浓度下，末端产物积累，导致底物抑制和伏安特征中出现不常见的滞后现象。具有不同还原电势的两种电子供体的溶液分析完全支持电化学结果。这说明了活性位点钼配位球的高灵活性的后果，并对早期研究的结论提出了质疑，这些研究试图在非常高的底物浓度下的周转条件下进行实验来捕获 Nap 的催化中间产物。
