Reactions of Recombinant Neuronal Nitric Oxide Synthase with Redox Cycling Xenobiotics: A Mechanistic Study

Mindaugas Lesanavičius,Narimantas Čėnas,Jean-Luc Boucher

doi:10.3390/ijms23020980

Abstract

Neuronal nitric oxide synthase (nNOS) catalyzes single-electron reduction of quinones (Q), nitroaromatic compounds (ArNO2) and aromatic N-oxides (ArN → O), and is partly responsible for their oxidative stress-type cytotoxicity. In order to expand a limited knowledge on the enzymatic mechanisms of these processes, we aimed to disclose the specific features of nNOS in the reduction of such xenobiotics. In the absence or presence of calmodulin (CAM), the reactivity of Q and ArN → O increases with their single-electron reduction midpoint potential (E17). ArNO2 form a series with lower reactivity. The calculations according to an “outer-sphere” electron transfer model show that the binding of CAM decreases the electron transfer distance from FMNH2 to quinone by 1–2 Å. The effects of ionic strength point to the interaction of oxidants with a negatively charged protein domain close to FMN, and to an increase in accessibility of the active center induced by high ionic strength. The multiple turnover experiments of nNOS show that, in parallel with reduced FAD-FMN, duroquinone reoxidizes the reduced heme, in particular its Fe2+-NO form. This finding may help to design the heme-targeted bioreductively activated agents and contribute to the understanding of the role of P-450-type heme proteins in the bioreduction of quinones and other prooxidant xenobiotics.

Highlights

Nitric oxide (NO) is produced physiologically to perform a range of signaling functions and as an immune response agent [1]
Fe previous studies [22], we examined the reactions of Neuronal nitric oxide synthase (nNOS) with a number
The lower reactivity of ArNO2 was attributed to their k22 ~ 106 M−1 s−1 [29], which is much lower than that of quinones, 108 M−1 s−1 [30]

Summary

Introduction

Nitric oxide (NO) is produced physiologically to perform a range of signaling functions and as an immune response agent [1]. The mammalian NO synthases (NOS, EC 1.14.13.39). Are dimeric flavohemoproteins that catalyze the conversion of L-arginine to NO· and citrulline at the expense of NADPH. The monomer of NOS consists of a heme domain with a tetrahydrobiopterin (H4 B) bound at its N-terminus, and a FAD- and FMN-containing reductase domain at its C-terminus. The reductase and oxygenase domains are linked by a calmodulin (CAM)-binding sequence ([2,3], and references therein). The FAD-FMN domain of NOS is highly similar to that of microsomal NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) [4,5,6]. The redox equivalents are transferred along the pathway NADPH → FAD

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