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

David Li,Eric Y Hayden,Koustubh Panda,Dennis J Stuehr,Haiteng Deng,Denis L Rousseau,Syun-Ru Yeh

doi:10.1074/jbc.m507328200

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 approximately 300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of approximately 30 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 Cys104 and Cys109, 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.

Highlights

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 [1,2,3]
To evaluate the effect of NO on the dimeric interactions in iNOS oxygenase domain (iNOSoxy), the substrate- and cofactor-free ferric enzyme was subjected to NO, and the reactions were monitored by optical absorption spectroscopy as a function of time
The data presented here clearly demonstrate that, in the absence of H4B, the ferric derivative of iNOSoxy is in equilibrium between a monomeric state and a loose dimeric state

Summary

Introduction

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 [1,2,3]. These data indicate that the NO-induced monomerization of the loose dimer is coupled to an intramolecular disulfide bond formation between cysteine residues at positions 104 and 109 and that the monomerization process exposes peptide fragments 82–97 and 393– 404 to solvent, 8200 JOURNAL OF BIOLOGICAL CHEMISTRY

Results

Conclusion