Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
J Phys Chem A. 2011 Sep 22;115(37):10345-52. doi: 10.1021/jp204969d. Epub 2011 Aug 26.
Mammalian nitric oxide synthase (NOS) is a flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide. Information about the relative alignment of the heme and FMN domains of NOS is important for understanding the electron transfer between the heme and FMN centers, but no crystal structure data for NOS holoenzyme are available. In our previous work [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059-12067], the distance between the imidazole-coordinated low-spin Fe(III) heme and FMN semiquinone in a human inducible NOS (iNOS) oxygenase/FMN construct has been determined by pulsed electron paramagnetic resonance (EPR). The orientation of the Fe-FMN radius vector, R(Fe-FMN), with respect to the heme g-frame was also determined. In the present study, pulsed electron-nuclear double resonance (ENDOR) investigation of the deuterons at carbons C2 and C5 in the deuterated coordinated imidazole was used to determine the relative orientation of the heme g-frame and molecular frame, from which R(Fe-FMN) can be referenced to the heme molecular frame. Numerical simulations of the ENDOR spectra showed that the g-factor axis corresponding to the low-field EPR turning point is perpendicular to the heme plane, whereas the axis corresponding to the high-field turning point is in the heme plane and makes an angle of about 80° with the coordinated imidazole plane. The FMN-heme domain docking model obtained in the previous work was found to be in qualitative agreement with the combined experimental results of the two pulsed EPR works.
哺乳动物一氧化氮合酶（NOS）是一种黄素-血红素蛋白，可催化 L-精氨酸氧化生成一氧化氮。NOS 血红素和 FMN 结构域相对排列的信息对于理解血红素和 FMN 中心之间的电子转移非常重要，但目前还没有NOS 全酶的晶体结构数据。在我们之前的工作中 [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059-12067]，通过脉冲电子顺磁共振（EPR）测定了人诱导型 NOS（iNOS）加氧酶/FMN 结构中咪唑配位低自旋 Fe(III)血红素与 FMN 半醌之间的距离。还确定了 Fe-FMN 半径矢量 R(Fe-FMN)相对于血红素 g-框架的方向。在本研究中，通过脉冲电子-核双共振（ENDOR）研究氘代配位咪唑中的碳 C2 和 C5 上的氘核，确定了血红素 g-框架和分子框架的相对取向，由此可以将 R(Fe-FMN)参考到血红素分子框架。ENDOR 光谱的数值模拟表明，对应于低场 EPR 转折点的 g 因子轴垂直于血红素平面，而对应于高场转折点的轴在血红素平面内，与配位咪唑平面成约 80°的角度。在以前的工作中获得的 FMN-血红素结构域对接模型与两个脉冲 EPR 工作的组合实验结果定性一致。
