Interaction between Neuronal Nitric-Oxide Synthase and Tetrahydrobiopterin Revisited: Studies on the Nature and Mechanism of Tight Pterin Binding

Christian L Heine,Antonius C F Gorren,Bernd Mayer,Ernst R Werner,Bernd Kolesnik,Renate Schmidt

doi:10.1021/bi401307r

Christian L Heine, Antonius C F Gorren + Show 4 more

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https://doi.org/10.1021/bi401307r

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Journal: Biochemistry	Publication Date: Feb 17, 2014
Citations: 10	License type: cc-by

Affiliation: University of Graz, Innsbruck Medical University

Abstract

Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10–7–10–6 M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10–7 M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-dihydrobiopterin or 4-amino-dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 μM BH4 (2 min with 1 μM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.

Highlights

We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry
We found that both enzyme species exhibited a similar EC50 for added BH4 with no indication of biphasic behavior for EConNOS, indicating that the enzyme does not display high-affinity pterin binding upon short-term exposure to BH4
Overnight incubation of ECo-nNOS with BH4 yielded an enzyme with very similar pterin-binding properties as the Sf9-expressed enzyme. This demonstrates that the expression system is not essential for high-affinity pterin binding and that BH4 need not be present during enzyme synthesis

Summary

Introduction

Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. A key role in the mechanism of NO synthesis is played by the essential cofactor tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-L-biopterin, BH4).[5−8] In the absence of BH4, the enzyme still oxidizes NADPH and reduces O2, but the oxidation of Arg is blocked, a phenomenon known as uncoupling.[3−6] Under these conditions, NOS produces O2− instead of NO, with major potential pathophysiological consequences: the bioavailability of NO decreases by diminished NO production and efficient scavenging of NO by O2−, and the reaction between O2− and NO results in the formation of the strongly oxidizing and presumably deleterious product peroxynitrite (ONOO−).

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