The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

Adjélé Wilson,Pierre Dorlet,Jérôme Santolini,Laura Henry,Albane Brunel

doi:10.1074/jbc.m110.195446

Abstract

Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions.

Highlights

Nitrogen monoxide (NO) is a well described radical molecule [1] that has been shown to exert major physiological functions in mammals, ranging from signaling processes to cytotoxic activities [2,3,4,5]
The natural pterin is presumably not synthesized by these bacteria, and BH4 and tetrahydrofolate increase the rate of decay of the FeIIO2 complex of several bacterial nitric-oxide synthase (NOS)-like proteins (bacNOSs) [16, 29, 30], no pterin radical has been identified in bacNOSs catalysis so far
We reported that the proximal Fe–S bond was stronger for bsNOS than for mammalian NOSs (mNOSs), suggesting a modulation of the electron donation exerted by the thiolate ligand [43]

Summary

Introduction

Nitrogen monoxide (NO) is a well described radical molecule [1] that has been shown to exert major physiological functions in mammals, ranging from signaling processes to cytotoxic activities [2,3,4,5]. The isomerization of peroxynitrite by bsNOS, in contrast with iNOS-mediated peroxynitrite activation, might reflect modification of the heme distal pocket that leads to a transient capture of NO or any other reactive nitrogen species within the catalytic site, which in turn could lead to additional chemical reactions Considering these points, one could legitimately question the role of bacNOSs as authentic NO-releasing oxygenase enzymes. We reported that the proximal Fe–S bond was stronger for bsNOS than for mNOSs, suggesting a modulation of the electron donation exerted by the thiolate ligand [43] This regulation pattern seems to be inherent to mNOSs. Unlike P450s, the sulfur atom of NOS-proximal thiolate is naturally engaged in a strong H-bond interaction with the nitrogen proton of the indole ring of the vicinal tryptophan (Scheme 1).

Results

Discussion

Conclusion