Grunau Alex, Geraki Kalotina, Grossmann J Günter, Gutierrez Aldo
Department of Biochemistry, University of Leicester, UK.
Biochemistry. 2007 Jul 17;46(28):8244-55. doi: 10.1021/bi700596s. Epub 2007 Jun 20.
A combination of mutagenesis, calorimetry, kinetics, and small-angle X-ray scattering (SAXS) has been used to study the mechanism of ligand binding energy propagation through human cytochrome P450 reductase (CPR). Remarkably, the energetics of 2',5'-ADP binding to R597 at the FAD-binding domain are affected by mutations taking place at an interdomain loop located 60 A away. Either deletion of a 7 amino acid long segment (T236-G237-E238-E239-S240-S241-I242) or its replacement by poly-proline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding (DeltaHB). This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-CPR complex. Moreover, the estimated heat capacity change (DeltaCp) for this interaction changes from -220 cal mol-1 K-1 in the wild-type enzyme to -580 cal mol-1 K-1 in the deletion mutant. Pre-steady-state kinetics measurements reveal a 50-fold decrease in the microscopic rate for interdomain (FAD --> FMN) electron transfer in the deletion mutant (kobs = 0.4 s-1). Multiple turnover cytochome c reduction assays indicate that these mutations impair the ability of the FMN-binding domain to shuttle electrons from the FAD-binding domain to the cytochrome partner. Binding of 2',5'-ADP to wild-type CPR triggers a large-scale structural rearrangement resulting in the complex having a more compact domain organization, and the maximum molecular dimension (Dmax) decreases from 110 A in ligand-free enzyme to 100 A in the ligand-bound CPR. The SAXS experiments also demonstrate that what is affected by the mutations is indeed the relative diffusional motion of the domains. Furthemore, ab initio shape reconstruction and homology modeling would suggest that-in the deletion mutant-hindering of domain motion occurs concomitantly with dimerization. The results presented here show that the energetics of this highly localized interaction (2',5'-ADP binding) have a global character, and are highly sensitive to functional structural dynamics involving distal domains. These findings support early theoretical studies which postulate a single protein molecule to be a real, independent thermodynamic ensemble.
诱变、量热法、动力学和小角X射线散射（SAXS）相结合的方法已被用于研究配体结合能通过人细胞色素P450还原酶（CPR）传播的机制。值得注意的是，位于FAD结合结构域的R597与2',5'-ADP的结合能受到位于60 Å外的结构域间环上发生的突变的影响。删除一个7个氨基酸长的片段（T236-G237-E238-E239-S240-S241-I242）或将其替换为多聚脯氨酸重复序列（5个和10个残基）都会导致2',5'-ADP结合焓（ΔHB）显著增加。这伴随着配体-CPR复合物可用的热力学微状态数量的减少。此外，这种相互作用的估计热容变化（ΔCp）从野生型酶中的-220 cal mol-1 K-1变为缺失突变体中的-580 cal mol-1 K-1。稳态前动力学测量显示，缺失突变体中结构域间（FAD→FMN）电子转移的微观速率降低了50倍（kobs = 0.4 s-1）。多次周转细胞色素c还原测定表明，这些突变损害了FMN结合结构域将电子从FAD结合结构域穿梭到细胞色素伴侣的能力。2',5'-ADP与野生型CPR的结合引发了大规模的结构重排，导致复合物具有更紧凑的结构域组织，最大分子尺寸（Dmax）从无配体酶中的110 Å减小到配体结合CPR中的100 Å。SAXS实验还表明，受突变影响的确实是结构域的相对扩散运动。此外，从头形状重建和同源建模表明，在缺失突变体中，结构域运动的阻碍与二聚化同时发生。这里呈现的结果表明，这种高度局部化相互作用（2',5'-ADP结合）的能量学具有全局性，并且对涉及远端结构域的功能结构动力学高度敏感。这些发现支持了早期的理论研究，这些研究假设单个蛋白质分子是一个真实、独立的热力学整体。
