Lee H J, Basran J, Scrutton N S
Department of Biochemistry, University of Leicester, U.K.
Biochemistry. 1998 Nov 3;37(44):15513-22. doi: 10.1021/bi981853v.
Rubredoxin reductase (RR) and rubredoxin form a soluble and physiological eT complex. The complex provides reducing equivalents for a membrane-bound omega-hydroxylase, required for the hydroxylation of alkanes and related compounds. The gene (alkT) encoding RR has been overexpressed and the enzyme purified in amounts suitable for studies of eT by stopped-flow spectroscopy. The eT reactions from NADH to the flavin of RR and from reduced RR to the 1Fe and 2Fe forms of rubredoxin have been characterized by transient kinetic and thermodynamic analysis. The reductive half-reaction proceeds in a one-step reaction involving oxidized enzyme and a two-electron-reduced enzyme-NAD+ charge-transfer complex. Flavin reduction is observed at 450 nm and charge-transfer formation at 750 nm; both steps are hyperbolically dependent on NADH concentration. The limiting flavin reduction rate (180 +/- 4 s-1) is comparable to the limiting rate for charge-transfer formation (189 +/- 7 s-1) and analysis at 450 and 750 nm yielded enzyme-NADH dissociation constants of 36 +/- 2 and 43 +/- 5 microM, respectively. Thermodynamic analysis of the reductive half-reaction yielded values for changes in entropy (DeltaS = -65.8 +/- 2.2 J mol-1 K-1), enthalpy (DeltaH = 37.8 +/- 0.6 kJ mol-1) and Gibbs free energy (DeltaG = 57.5 +/- 0.7 kJ mol-1 at 298 K) during hydride ion transfer to the flavin N5 atom. Spectral analysis of mixtures of 1Fe or 2Fe rubredoxin and RR suggest that conformational changes accompany eT complex assembly. Both the 1Fe (nonphysiological) and 2Fe (physiological) forms of rubredoxin were found to oxidize two electron-reduced rubredoxin reductase with approximately equal facility. Rates for the reduction of rubredoxin are hyperbolically dependent on rubredoxin concentration and the limiting rates are 72. 7 +/- 0.6 and 55.2 +/- 0.3 s-1 for the 1Fe and 2Fe forms, respectively. Analysis of the temperature dependence of eT to rubredoxin using eT theory revealed that the reaction is not adequately described as a nonadiabatic eT reaction (HAB >> 80 cm-1). eT to both the 1Fe and 2Fe forms of rubredoxin is therefore gated by an adiabatic process that precedes the eT reaction from flavin to iron. Possible origins of this adiabatic event are discussed.
红素氧还蛋白还原酶（RR）和红素氧还蛋白形成一种可溶的生理性电子转移复合物。该复合物为膜结合的ω-羟化酶提供还原当量，这种羟化酶是烷烃及相关化合物羟基化所必需的。编码RR的基因（alkT）已被过量表达，并且已纯化出适合通过停流光谱法研究电子转移的酶量。从NADH到RR黄素以及从还原态RR到红素氧还蛋白的1Fe和2Fe形式的电子转移反应已通过瞬态动力学和热力学分析进行了表征。还原半反应以一步反应进行，涉及氧化酶和双电子还原的酶-NAD +电荷转移复合物。在450 nm处观察到黄素还原，在750 nm处观察到电荷转移形成；这两个步骤均双曲线依赖于NADH浓度。黄素还原的极限速率（180±4 s-1）与电荷转移形成的极限速率（189±7 s-1）相当，并且在450和750 nm处的分析分别得到酶-NADH解离常数为36±2和43±5 μM。还原半反应的热力学分析得出了在氢负离子转移至黄素N5原子过程中的熵变（ΔS = -65.8±2.2 J mol-1 K-1）、焓变（ΔH = 37.8±0.6 kJ mol-1）和吉布斯自由能（在298 K时ΔG = 57.5±0.7 kJ mol-1）值。对1Fe或2Fe红素氧还蛋白与RR混合物的光谱分析表明，构象变化伴随着电子转移复合物的组装。发现红素氧还蛋白的1Fe（非生理性）和2Fe（生理性）形式都以大致相同的能力氧化双电子还原的红素氧还蛋白还原酶。红素氧还蛋白还原的速率双曲线依赖于红素氧还蛋白浓度，1Fe和2Fe形式的极限速率分别为72.7±0.6和55.2±0.3 s-1。使用电子转移理论分析电子转移至红素氧还蛋白的温度依赖性表明，该反应不能充分描述为非绝热电子转移反应（HAB >> 80 cm-1）。因此，电子转移至红素氧还蛋白的1Fe和2Fe形式均由黄素到铁的电子转移反应之前的绝热过程控制。讨论了这种绝热事件的可能起源。
