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Surface charges and regulation of FMN to heme electron transfer in nitric-oxide synthase.

ABSTRACT: The nitric-oxide synthases (NOS, EC 1.14.13.39) are modular enzymes containing attached flavoprotein and heme (NOSoxy) domains. To generate nitric oxide (NO), the NOS FMN subdomain must interact with the NOSoxy domain to deliver electrons to the heme for O(2) activation during catalysis. The molecular basis and how the interaction is regulated is unclear. We explored the role of eight positively charged residues that create an electropositive patch on NOSoxy in enabling the electron transfer by incorporating mutations that neutralized or reversed their individual charges. Stopped-flow and steady-state experiments revealed that individual charges at Lys(423), Lys(620), and Lys(660) were the most important in enabling heme reduction in nNOS. Charge reversal was more disruptive than neutralization in all cases, and the effects on heme reduction were not due to a weakening in the thermodynamic driving force for heme reduction. Mutant NO synthesis activities displayed a complex pattern that could be simulated by a global model for NOS catalysis. This analysis revealed that the mutations impact the NO synthesis activity only through their effects on heme reduction rates. We conclude that heme reduction and NO synthesis in nNOS is enabled by electrostatic interactions involving Lys(423), Lys(620), and Lys(660), which form a triad of positive charges on the NOSoxy surface. A simulated docking study reveals how electrostatic interactions of this triad can enable an FMN-NOSoxy interaction that is productive for electron transfer.

SUBMITTER: Tejero J

PROVIDER: S-EPMC2930722 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Surface charges and regulation of FMN to heme electron transfer in nitric-oxide synthase.

Tejero Jesús J Hannibal Luciana L Mustovich Anthony A Stuehr Dennis J DJ

The Journal of biological chemistry 20100630 35

The nitric-oxide synthases (NOS, EC 1.14.13.39) are modular enzymes containing attached flavoprotein and heme (NOSoxy) domains. To generate nitric oxide (NO), the NOS FMN subdomain must interact with the NOSoxy domain to deliver electrons to the heme for O(2) activation during catalysis. The molecular basis and how the interaction is regulated is unclear. We explored the role of eight positively charged residues that create an electropositive patch on NOSoxy in enabling the electron transfer by ...[more]

PMID: 20592038

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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

Surface charges and regulation of FMN to heme electron transfer in nitric-oxide synthase.

Publications

Surface charges and regulation of FMN to heme electron transfer in nitric-oxide synthase.

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