The Three Nitric-oxide Synthases Differ in Their Kinetics of Tetrahydrobiopterin Radical Formation, Heme-Dioxy Reduction, and Arginine Hydroxylation

Chin-Chuan Wei,Zhi-Qiang Wang,Deborah Durra,Craig Hemann,Russ Hille,Elsa D Garcin,Elizabeth D Getzoff,Dennis J Stuehr

doi:10.1074/jbc.m409737200

Abstract

The nitric-oxide synthases (NOSs) make nitric oxide and citrulline from l-arginine. How the bound cofactor (6R)-tetrahydrobiopterin (H4B) participates in Arg hydroxylation is a topic of interest. We demonstrated previously that H4B radical formation in the inducible NOS oxygenase domain (iNOSoxy) is kinetically coupled to the disappearance of a heme-dioxy intermediate and to Arg hydroxylation. Here we report single turnover studies that determine and compare the kinetics of these transitions in Arg hydroxylation reactions catalyzed by the oxygenase domains of endothelial and neuronal NOSs (eNOSoxy and nNOSoxy). There was a buildup of a heme-dioxy intermediate in eNOSoxy and nNOSoxy followed by a monophasic transition to ferric enzyme during the reaction. The rate of heme-dioxy decay matched the rates of H4B radical formation and Arg hydroxylation in both enzymes. The rates of H4B radical formation differed such that nNOSoxy (18 s(-1)) > iNOSoxy (11 s(-1)) > eNOSoxy (6 s(-1)), whereas the lifetimes of the resulting H4B radical followed an opposite rank order. 5MeH4B supported a three-fold faster radical formation and greater radical stability relative to H4B in both eNOSoxy and nNOSoxy. Our results indicate the following: (i) the three NOSs share a common mechanism, whereby H4B transfers an electron to the heme-dioxy intermediate. This step enables Arg hydroxylation and is rate-limiting for all subsequent steps in the hydroxylation reaction. (ii) A direct correlation exists between pterin radical stability and the speed of its formation in the three NOSs. (iii) Uncoupled NO synthesis often seen for eNOS at low H4B concentrations may be caused by the slow formation and poor stability of its H4B radical.

Highlights

Nitric-oxide synthases (NOSs)1 (EC 1.14.13.39) are flavoheme enzymes that catalyze the oxidation of L-arginine to nitric oxide (NO) and L-citrulline [1, 2]
Three concepts arose from our kinetic studies of the inducible NOSoxy (iNOSoxy) Arg hydroxylation reaction: (i) formation of the pterin radical is kinetically coupled to reduction of the FeIIIO2Ϫ intermediate and to the rate of Arg hydroxylation; (ii) the rate of pterin electron transfer is directly proportional to the stability of the resultant pterin radical; and (iii) a sufficiently fast rate of pterin electron transfer must be maintained for iNOSoxy to couple its FeIIIO2Ϫ formation to Arg hydroxylation [18]
We know the kinetics of H4B redox transitions and the times when transitions occur relative to other key catalytic events during Arg hydroxylation in all three NOSs

Summary

Introduction

Nitric-oxide synthases (NOSs) (EC 1.14.13.39) are flavoheme enzymes that catalyze the oxidation of L-arginine to nitric oxide (NO) and L-citrulline [1, 2]. Bound NOHA is oxidized to citrulline and NO in a second NADPH- and O2-dependent reaction Both reactions occur within a heme-containing oxygenase domain dimer of NOS (NOSoxy) that receives electrons from two attached flavoprotein domains [3, 4]. It is established that an H4B radical forms in neuronal and endothelial NOS (nNOS and eNOS) during their Arg hydroxylation reactions [19, 20], the kinetic relationships between H4B radical formation, processing of the FeIIIO2Ϫ intermediate, and product formation in these NOSs are largely unresolved Because this knowledge is needed to understand eNOS and nNOS catalysis, we measured and compared the kinetics of heme transitions, pterin radical formation, and Arg hydroxylation in single turnover reactions catalyzed by human eNOSoxy and rat nNOSoxy enzymes that contained H4B or 5MeH4B

Methods

Results

Conclusion