Ratnam K, Shiraishi N, Campbell W H, Hille R
Department of Medical Biochemistry, Ohio State University, Columbus, Ohio 43210, USA.
J Biol Chem. 1997 Jan 24;272(4):2122-8. doi: 10.1074/jbc.272.4.2122.
The recombinant NADH-cytochrome c reductase fragment of spinach NADH-nitrate reductase (EC 1.6.6.1), consisting of the contiguous heme-containing cytochrome b domain and flavin-containing NADH-cytochrome b reductase fragment, has been characterized spectroscopically and kinetically. Reductive titration with sodium dithionite indicates heme reduction takes place prior to flavin reduction, which correlates well with the reduction potentials for enzyme-bound heme (15 mV) and FAD (-280 mV). Reductive titration with NADH also indicates that the reduced enzyme forms a charge-transfer complex with NAD+. The circular dichroism spectrum of the oxidized fragment is primarily due to the flavin, whereas the ferrous heme dominates the circular dichroism spectrum of reduced enzyme. Three kinetic phases are observed in the course of the reaction of the enzyme with NADH, each with a distinct spectral signature. The fast phase represents flavin reduction, concomitant with the formation of a charge-transfer complex between reduced flavin and NAD+, and exhibits hyperbolic dependence on NADH concentration with a Kd of 3 microM and a limiting rate constant of 560 s-1. Electron transfer from reduced flavin to heme with a rate constant of 12 s-1 is the intermediate phase, which is rate-limited by breakdown of the charge-transfer complex between NAD+ and reduced flavin. The slow phase is dismutation of a pair of molecules of two-electron reduced enzyme (generated at the end of the second phase of the reaction) to give one molecule each of one- and three- electron reduced enzyme, with a second order rate constant of 2 x 10(6) M-1 s-1. In the presence of excess NADH, this dismutation reaction is followed by the rapid reaction of the one-electron reduced enzyme with a second equivalent of NADH to generate fully reduced enzyme. On the basis of this work, it appears that dissociation of NAD+ from the reduced flavin site rate limits electron transfer to the cytochrome and likely represents the overall rate-limiting step of catalysis.
菠菜硝酸还原酶（EC 1.6.6.1）的重组NADH-细胞色素c还原酶片段，由相邻的含血红素的细胞色素b结构域和含黄素的NADH-细胞色素b还原酶片段组成，已通过光谱学和动力学进行了表征。连二亚硫酸钠的还原滴定表明血红素还原发生在黄素还原之前，这与酶结合血红素（15 mV）和FAD（-280 mV）的还原电位很好地相关。用NADH进行的还原滴定还表明，还原酶与NAD+形成电荷转移复合物。氧化片段的圆二色光谱主要归因于黄素，而亚铁血红素主导还原酶的圆二色光谱。在酶与NADH反应过程中观察到三个动力学阶段，每个阶段都有独特的光谱特征。快速阶段代表黄素还原，同时在还原黄素和NAD+之间形成电荷转移复合物，对NADH浓度呈双曲线依赖性，Kd为3 microM，极限速率常数为560 s-1。从还原黄素到血红素的电子转移速率常数为12 s-1是中间阶段，该阶段受NAD+与还原黄素之间电荷转移复合物分解的速率限制。缓慢阶段是两电子还原酶的一对分子（在反应的第二阶段结束时产生）歧化为一电子和三电子还原酶各一分子一次，二级速率常数为2×10(6) M-¹ s-¹。在过量NADH存在下，这种歧化反应之后是一电子还原酶与第二个当量的NADH快速反应生成完全还原的酶。基于这项工作，似乎NAD+从还原黄素位点的解离速率限制了向细胞色素的电子转移，并且可能代表催化的总体速率限制步骤。
