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Pulsed EPR determination of the distance between heme iron and FMN centers in a human inducible nitric oxide synthase.

ABSTRACT: 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(2) 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(*)] (FMNH(*): FMN semiquinone) form of a human inducible NOS (iNOS) bidomain oxygenase/FMN construct. The FMNH(*)...Fe distance has been directly determined from the RIDME spectrum. This distance (18.8 +/- 0.1 A) 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. J. Biol. Inorg. Chem. 2009, 14, 133-142].

SUBMITTER: Astashkin AV

PROVIDER: S-EPMC2931817 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Pulsed EPR determination of the distance between heme iron and FMN centers in a human inducible nitric oxide synthase.

Astashkin Andrei V AV Elmore Bradley O BO Fan Weihong W Guillemette J Guy JG Feng Changjian C

Journal of the American Chemical Society 20100901 34

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(2) activation in the heme domain and the subsequent NO synthesis by NOS. The NOS output state for NO production ...[more]

PMID: 20695464

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Project description:Previous biochemical studies of nitric oxide synthase enzymes (NOSs) were conducted in diluted solutions. However, the intracellular milieu where the proteins perform their biological functions is crowded with macromolecules. The effect of crowding on the electron transfer kinetics of multidomain proteins is much less understood. Herein, we investigated the effect of macromolecular crowding on the FMN-heme intraprotein interdomain electron transfer (IET), an obligatory step in NOS catalysis. A noticeable increase in the IET rate in the bidomain oxygenase/FMN (oxyFMN) and the holoprotein of human inducible NOS (iNOS) was observed upon addition of Ficoll 70 in a nonsaturable manner. Additionally, the magnitude of IET enhancement for the holoenzyme is much higher than that that of the oxyFMN construct. The crowding effect is also evident at different ionic strengths. Importantly, the enhancing extent is similar for the iNOS oxyFMN protein with added Ficoll 70 and Dextran 70 that give the same solution viscosity, showing that specific interactions do not exist between the NOS protein and the crowder. Moreover, the population of the docked FMN-heme state is significantly increased upon addition of Ficoll 70 and the fluorescence lifetime values do not correspond to those in the absence of Ficoll 70. The steady-state cytochrome c reduction by the holoenzyme is noticeably enhanced by the crowder, while the ferricyanide reduction is unchanged. The NO production activity of the iNOS holoenzyme is stimulated by Ficoll 70. The effect of macromolecular crowding on the kinetics can be rationalized on the basis of the excluded volume effect, with an entropic origin. The intraprotein electron transfer kinetics, fluorescence lifetime, and steady-state enzymatic activity results indicate that macromolecular crowding modulates the NOS electron transfer through multiple pathways. Such a mechanism should be applicable to electron transfer in other multidomain redox proteins.

Dataset Information

Pulsed EPR determination of the distance between heme iron and FMN centers in a human inducible nitric oxide synthase.

Publications

Pulsed EPR determination of the distance between heme iron and FMN centers in a human inducible nitric oxide synthase.

Similar Datasets

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