Regulation of the Monomer-Dimer Equilibrium in Inducible Nitric-oxide Synthase by Nitric Oxide

Abstract

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate L-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of ϳ300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of ϳ30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys 104 and Cys 109 , located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme. Nitric-oxide synthase (NOS)4 catalyzes the formation of NO from oxygen and L-Arg via a consecutive two-step reaction using NADPH as the electron source (1-3). In the first step of the reaction, L-Arg is hydroxylated to N-hydroxyarginine; and in the second step, N-hydroxyarginine is oxidized to citrulline and NO. The three major isoforms, inducible NOS (iNOS), endothelial NOS, and neuronal NOS (found in macrophages, endothelial cells, and neuronal tissues, respectively), produce NO that functions as a cytotoxic agent, a vasodilator, and a neurotransmitter, respectively (4). The homodimeric enzyme consists of a reductase domain, which binds FMN, FAD, and NADPH, and an oxygenase domain, which binds the heme and tetrahydrobiopterin (H4B) cofactors. During catalysis, electrons flow from NADPH through FMN and FAD in the reductase domain of one subunit of the homodimer to the oxygenase domain of the other subunit (5, 6). The crystal structures of the oxygenase domain of all three isoforms have been determined (7-10). They show that the heme is coordinated by a cysteine residue on the proximal side, as in cytochrome P450-type enzymes, and that the substrate (L-Arg or N-hydroxyarginine) binds above the heme iron atom in the distal pocket, whereas the cofactor (H4B) binds along the side of the heme.It is well accepted that dimerization is essential for NOS function (1, 11). The heme group, the H4B cofactor, and the substrate have all been shown to contribute to dimer stability (12-16). In iNOS, the N-terminal region (between residues 76 and 111, comprising a ␤-hairpin hook and a CXXXC zinc-binding motif) is also believed to be important for stabilizing the dimeric structure. Crane et al. (17) reported that the N-terminal region of the iNOS oxygenase domain (iNOS oxy ) can be in either a "swapped" or an "unswapped" conformation, as illustrated in Fig. 1. In the unswapped conformation, Cys 104 and Cys 109 in the zinc-binding motif of each subunit of the dimer are tetrahedrally coordinated to a single zinc ion at the dimer interface, and the ␤-hairpin hook interac...

Interactions between substrates and the haem-bound nitric oxide of ferric and ferrous bacterial nitric oxide synthases

“…This property is reminiscent of the behaviour of many haem FeIII-NO complexes, including those of NOSs, that become reduced with time to FeII-NO complexes [34]. To obtain the resonance Raman spectra of the FeIII-NO complexes with saNOS, the acquisition time was kept as short as possible to avoid formation of the FeII-NO form.…”

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase

“…Ferric iNOS oxy -NO is unstable and spontaneously converts to a ferrous 6C NO-ligated species. This conversion may take place during loading and cooling of an FTIR sample, which typically takes ~2 h. This species may subsequently evolve further to a five-coordinate (5C) complex by dissociation of the thiolate ligand on time scales of minutes to hours, depending on the iNOS oxy oligomerization state 67, 71– 73 . Here, we can safely exclude formation of significant amounts of 5C ferrous iNOS oxy -NO because we have not observed the characteristic IR bands of this species at ~1670 cm -1 53 .…”

Liver betaine-homocysteine S-methyltransferase activity undergoes a redox switch at the active site zinc

“…A disulfide-dithiol switch also modulates the activity of inducible nitric oxide synthase (iNOS). Within the Zn binding motif of the iNOS a reversible intramolecular disulfide can be formed that inactivates the protein by avoiding its ligand-induced dimerization [24].…”