Dataset Information

Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase.

ABSTRACT: Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg. Chem.2011, 50, 6869-6861]. In the present work, we further investigated the role of the E546 residue in the FMN-heme interdomain electron transfer (IET), a catalytically essential step in the NOS enzymes. Laser flash photolysis was employed to directly measure the FMN-heme IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN) construct. The temperature dependence of the IET kinetics was also measured over the temperature range of 283-304 K to determine changes in the IET activation parameters. The E546N mutation was found to retard the IET by significantly raising the activation entropic barrier. Moreover, pulsed electron paramagnetic resonance data showed that the geometry of the docked FMN/heme complex in the mutant is basically the same as in the wild type construct, whereas the probability of formation of such a complex is about twice lower. These results indicate that the retarded IET in the E546N mutant is not caused by an altered conformation of the docked FMN/heme complex, but by a lower population of the IET-active conformation. In addition, the negative activation entropy of the mutant is still substantially lower than that of the holoenzyme. This supports a mechanism by which the FMN domain can modify the IET through altering probability of the docked state formation.

SUBMITTER: Li W

PROVIDER: S-EPMC3677850 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase.

Li Wenbing W Chen Li L Lu Changyuan C Elmore Bradley O BO Astashkin Andrei V AV Rousseau Denis L DL Yeh Syun-Ru SR Feng Changjian C

Inorganic chemistry 20130409 9

Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg. Chem.2011, 50, 6869-6861]. In the present work, we further investigated the role of the E546 res ...[more]

PMID: 23570607

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Project description:Nitric oxide synthase (NOS), a flavo-hemoprotein, tightly regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a key signaling molecule for vasodilatation and neurotransmission at low concentrations, and also as a defensive cytotoxin at higher concentrations. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their key biological functions by tight regulation of interdomain electron transfer (IET) process via interdomain interactions. In particular, the FMN-heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O(2) activation at the catalytic heme site. Compelling evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS through a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the domains. Another exciting recent development in NOS enzymology is the discovery of importance of the the FMN domain motions in modulating reactivity and structure of the catalytic heme active site (in addition to the primary role of controlling the IET processes). In the absence of a structure of full-length NOS, an integrated approach of spectroscopic (e.g. pulsed EPR, MCD, resonance Raman), rapid kinetics (laser flash photolysis and stopped flow) and mutagenesis methods is critical to unravel the molecular details of the interdomain FMN/heme interactions. This is to investigate the roles of dynamic conformational changes of the FMN domain and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in understanding of mechanisms of the NOS regulation that are driven by the combined approach are the focuses of this review. An improved understanding of the role of interdomain FMN/heme interaction and CaM binding may serve as the basis for the design of new selective inhibitors of NOS isoforms.

Dataset Information

Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase.

Publications

Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase.

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