Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate‐liganded heme protein that hydroxylates L‐arginine to produce NO as a cellular signal

Abstract

The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4).

Recruitment of governing elements for electron transfer in the nitric oxide synthase family

“…2. Considering the possible convergent evolution of function of the NOS isoforms from simpler proteins (10,13,31), such as CYPOR, these C termini were added to the CYPOR structures, as detailed in supporting information. The purity of the preparations of these constructs and their virtually identical spectral properties are also detailed in supporting information.…”

“…The proposal that this diflavin enzyme, CYPOR, is in turn the precursor of further fusion products that gain new functions is a subject of intensive research (13)(14)(15). The family of nitric oxide synthases (NOS) combines the unique properties of heme-and flavin-containing proteins into one polypeptide chain to achieve production, by very intricately controlled processes, of the gaseous messenger, NO.…”

“…The remarkable sequence identity (58%) between the C-terminal 641 amino acids of neuronal NOS and the entire sequence of CYPOR was reported by Bredt et al (31). It is now generally accepted that the CYPOR gene was recruited into or fused with the NOS genes (13,32), which in prokaryotes produce heme domain-only proteins, resulting in efficient electron flow for NO synthesis. During catalysis by both CYPOR͞P450 and NOS isoforms, the electrons flow from NADPH to FAD, to FMN, and finally to the heme iron, where substrate monooxygenation is catalyzed (13,(32)(33)(34)(35)(36)(37)(38).…”

Inducible Nitric-oxide Synthase Generates Superoxide from the Reductase Domain

“…In cells or tissues, NO is produced by a family of NO synthases (NOSs), which utilize L-arginine, oxygen, and NADPH as substrates to synthesize NO as well as the coproduct L-citrulline (2,3). Three distinct isoforms of NOS have been cloned: neuronal NOS (nNOS, type I), inducible NOS (iNOS, type II), and endothelial NOS (eNOS, type III) (4).…”

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

“…Binding of CaM promotes electron flow from the flavins, FAD and FMN, within the reductase domain to the oxygenase haem [8]. Biochemical and functional parallels suggest that all NOS isoforms are members of the mammalian cytochrome P450 superfamily because they contain an intrinsic P450 reductase domain fused with a P450 haem domain within a single polypeptide [7,9]. However, the NOS isoforms differ primarily with respect to their expressional regulation, subcellular localization and activation mechanisms.…”