10.1021/ja104461p.s001 Andrei V. Astashkin Andrei V. Astashkin Bradley O. Elmore Bradley O. Elmore Weihong Fan Weihong Fan J. Guy Guillemette J. Guy Guillemette Changjian Feng Changjian Feng Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase American Chemical Society 2010 heme iron centers IET kinetics studies crystal structure data Pulsed EPR Determination O 2 activation laser flash photolysis FMNH RIDME FMN Human Inducible Nitric Oxide SynthaseMammalian nitric oxide synthase NOS output state heme domain echo envelope modulation interdomain electron transfer 2010-09-01 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Pulsed_EPR_Determination_of_the_Distance_between_Heme_Iron_and_FMN_Centers_in_a_Human_Inducible_Nitric_Oxide_Synthase/2737873 Mammalian nitric oxide synthase (NOS) is a homodimeric flavo-hemoprotein that catalyzes the oxidation of l-arginine to nitric oxide (NO). Regulation of NO biosynthesis by NOS is primarily through control of interdomain electron transfer (IET) processes in NOS catalysis. The IET from the flavin mononucleotide (FMN) to heme domains is essential in the delivery of electrons required for O<sub>2</sub> activation in the heme domain and the subsequent NO synthesis by NOS. The NOS output state for NO production is an IET-competent complex of the FMN-binding domain and heme domain, and thereby it facilitates the IET from the FMN to the catalytic heme site. The structure of the functional output state has not yet been determined. In the absence of crystal structure data for NOS holoenzyme, it is important to experimentally determine the Fe···FMN distance to provide a key calibration for computational docking studies and for the IET kinetics studies. Here we used the relaxation-induced dipolar modulation enhancement (RIDME) technique to measure the electron spin echo envelope modulation caused by the dipole interactions between paramagnetic FMN and heme iron centers in the [Fe(III)][FMNH<sup>•</sup>] (FMNH<sup>•</sup>: FMN semiquinone) form of a human inducible NOS (iNOS) bidomain oxygenase/FMN construct. The FMNH<sup>•</sup>···Fe distance has been directly determined from the RIDME spectrum. This distance (18.8 ± 0.1 Å) is in excellent agreement with the IET rate constant measured by laser flash photolysis [Feng, C. J.; Dupont, A.; Nahm, N.; Spratt, D.; Hazzard, J. T.; Weinberg, J.; Guillemette, J.; Tollin, G.; Ghosh, D. K. <i>J. Biol. Inorg. Chem.</i> <b>2009</b>, <i>14</i>, 133−142].