Mechanism of nitric oxide synthase regulation: Electron transfer and interdomain interactions

Abstract

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 O2 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.

Neuronal Nitric Oxide Synthase

“…The nitrogen atom of NO is derived from the guanidino group of the l-Arg substrate, and the oxygen atom is derived from dioxygen [10,14]. NO can activate some transduction pathways, such as, activation of guanylyl cyclase and conversion of Guanosine-5'-triphosphate (GTP) into Cyclic guanosine monophosphate (cGMP) and Figure 2.…”

Raman mode‐selective spectroscopic imaging of coenzyme and enzyme redox states

“…Activated by calmodulin (CaM), NO is synthesized by NOS in a reaction of L-arginine, NADPH, and oxygen to citrulline and NADP [37] , where the conformation of the FMN domain changes from its shielded electron-accepting state to a new electron-donating state [38][39][40][41] . In the two states, the FMN domain is engaged in distinctly different inter-domain interactions and the module swings back and forth to contact the NADP + -FAD reductase module and the NOS heme domain [35,36,40,42,43] . Flavins are biologically important redox co-enzyme molecules, such as, in NOS.…”

“…Nevertheless, the mechanisms by which the FMN domain functions in the NOS catalysis and the electron transfer coupling to substrate oxidation are unclear and remain a major challenge in current NOS studying [43] . The challenge mainly rests on (1) the complex multiple electron transfer redox centers in the enzyme matrix Raman frequencies shown in parentheses are calculated using density functional theory (DFT) by Gaussian 09 with B3LYP/6-31G (d) basis functions and scaled by a factor of 0.9614.…”

Retrograde response in axotomized motoneurons: Nitric oxide as a key player in triggering reversion toward a dedifferentiated phenotype

“…In this process, NOS uses the redox cofactor tetrahydrobiopterin (H 4 B), utilizes NADPH as electron donor, and requires O 2 to carry out the reaction. Electron transfer reactions performed by NOS are regulated by the Ca 2+ -binding protein calmodulin (reviewed in Stuehr et al, 2004;Feng, 2012). Three major isoforms of NOS have been identified so far, and their nomenclature attends to three factors: the cloning order, the cell type that mainly expresses them, and their constitutive or inducible expression.…”