Kirk McMillan

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.

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 EX 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 Ca2+‐calmodulin and/or tetrahydrobiopterin (BH4).—Masters, B. S. S., McMillan, K., Sheta, E. A., Nishimura, J. S., Roman, L. J., Martasek, P. Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structural studies of a cysteine thiolate‐li‐ganded heme protein that hydroxylates L‐arginine to produce NO· as a cellular signal. FASEB J. 10, 552‐558 (1996)

Substrate binding-induced changes in the EPR spectra of the ferrous nitric oxide complexes of neuronal nitric oxide synthase.

A versatile diatomic physiological messenger, nitric oxide (NO), is biosynthesized by a group of flavo-heme enzymes, the nitric oxide synthases. We have examined the active site of the neuronal isoform by EPR spectroscopy of the ferrous nitric oxide complex. The nitric oxide complex of the substrate-free enzyme exhibits a cytochrome P450-type EPR spectrum typical of a hexacoordinate NO-heme complex with a non-nitrogenous proximal axial heme ligand. The NO complex of the substrate-free enzyme is rather unstable and spontaneously converts to a cytochrome P420 type pentacoordinate denatured form. Binding of L-arginine (l-Arg) enhances the stability of the hexacoordinate NO form. The EPR spectrum of the NO adduct of the enzyme-substrate complex has an increased g-anisotropy and well-resolved hyperfine couplings due to the 14N of nitric oxide. Significant perturbations in the NO EPR spectrum were observed upon Nomega-monomethyl-L-Arg and Nomega-hydroxy-L-Arg binding. The perturbations in the EPR spectrum indicate that L-Arg and its derivatives bind on the distal site of the heme in very close proximity to the bound NO to cause alterations in the heme-NO coordination structure. Interactions between the bound NO and the substrate or its analogues appear to affect the Fe-NO geometry, resulting in the observed spectral changes. We infer that analogous interactions with oxygen might be involved in the hydroxylation events during enzyme catalysis of nitric oxide synthase.

Understanding the Structural Aspects of Neuronal Nitric Oxide Synthase (NOS) Using Microdissection by Molecular Cloning Techniques

The neuronal isoform (Type I) of nitric oxide synthase (NOS) requires Ca+2/calmodulin to catalyze the formation of NO● and citrulline from L-arginine (1) and molecular oxygen (2) with reducing equivalents from NADPH. The overall reaction is a five-electron process involving two successive monooxygenation steps with the obligatory formation of N-hydroxy-L-arginine as the oxygenated intermediate. The neuronal NOS shares with the other two known isoforms the unusual property, for a mammalian enzyme, of containing iron protoporphyrin IX (3–6), FAD and FMN (3, 7–9), and tetrahydrobiopterin (9). Bredt, et al. (10) had previously demonstrated the remarkable (58%) sequence similarity of rat brain NOS to rat liver NADPH-cytochrome P450 reductase and determined that the C-terminal 641 amino acids of NOS display consensus regions for both flavin and nucleotide binding. It was reported that carbon monoxide (CO) inhibited both macrophage and neuronal NOS activity (3–6) and these various preparations exhibited reduced-CO difference spectra with absorbance maxima in the region of 445 nm, a property shared with the members of the cytochrome P450 family. However, the low degree of sequence similarity to any of the more than 200 members of this family and other CO-binding, oxygenating heme proteins, such as chloroperoxidase or Bacillus megaterium P450 (BM-3), suggests that the homology may end with the sharing of a cysteine thiolate ligand to the heme iron at the fifth axial position. Recent studies have established that this involves cysteine415 of the neuronal NOS (11, 12), as suggested originally by McMillan, et al. (3), and cysteine184 of the endothelial NOS (13) as determined by site-directed mutagenesis of the holoenzyme (11,13) and the heme domain (12), respectively.