The FMN−heme interdomain electron transfer (IET) in nitric oxide synthase (NOS) is a key stage of the electron transport chain, which supplies the catalytic heme site(s) with the NADPH-derived electrons. While there is a recognition that this IET depends on both the electron tunneling and the conformational dynamics, the detailed mechanism remains unclear. In this work, the IET kinetics were measured by laser flash photolysis for a bidomain oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS) over the ionic strength range from 0.1 to 0.5 M. The forward (heme → FMN, k_ETf) and backward (FMN → heme, k_ETb) intrinsic IET rate constants were determined from the analysis of the observed IET rates using the additional information regarding the conformational dynamics obtained from the FMN fluorescence lifetime measurements and theoretical estimates. Both k_ETf and k_ETb exhibit a bell-shaped dependence on the ionic strength, I, with the maximum rates corresponding to I ~ 0.2 M. This dependence was explained using a new model, which considers the effect of formation of pairs between the protein surface charged residues and solution ions on the docked state dynamics. The trial simulations of the intrinsic IET rate dependences using this model show that the data can be reproduced using reasonable energetic, structural, and chemical parameters. The suggested model can explain both the monophasic and biphasic ionic strength dependences and can be used to rationalize the interprotein/interdomain electron transfer rates for other types of protein systems where the docked state is sufficiently long-lived.

一氧化氮合酶（NOS）中的FMN-血红素域间电子转移（IET）是电子传输链的关键阶段，它为催化血红素位点提供NADPH衍生的电子。尽管人们认识到该IET既依赖于电子隧穿又依赖于构象动力学，但其详细机理仍不清楚。在这项工作中，通过激光闪光光解法在离子强度范围为0.1到0.5 M的人类诱导型NOS（iNOS）的双域加氧酶/ FMN（oxyFMN）构建体中测量了IET动力学。正向（血红素→FMN，k _ETf）和向后（FMN→血红素，k _ETb）固有的IET速率常数是通过使用FMN荧光寿命测量和理论估计获得的有关构象动力学的附加信息，通过对观察到的IET速率的分析来确定的。既ķ _ETF和ķ _ETB表现出对离子强度，钟形依赖我，与对应于最大速率我 〜0.2M。使用新模型解释了这种依赖性，该模型考虑了蛋白质表面带电残基和溶液离子之间成对的形成对接态动力学的影响。使用该模型对内在IET速率依赖性的试验模拟表明，可以使用合理的能量，结构和化学参数来再现数据。建议的模型可以解释单相和双相离子强度的依赖性，并且可以用于合理化对接状态足够长寿的其他类型蛋白质系统的蛋白质间/域间电子转移速率。