Neuroglobin (Ngb) is a globin expressed in the nervous system of humans and other organisms that is involved in the protection of the brain from ischemic damage. Despite considerable interest, however, the in vivo function of Ngb is still a conundrum. In this paper we report a number of kinetic experiments with O ₂ and NO that we have interpreted on the basis of the 3D structure of Ngb, now available for human and murine metNgb and murine NgbCO. The reaction of reduced deoxyNgb with O ₂ and NO is slow ( t _1/2 ≈ 2 s) and ligand concentration-independent, because exogenous ligand binding can only occur upon dissociation of the distal His-64, which is coordinated to the ferrous heme iron. By contrast, NgbO ₂ reacts very rapidly with NO, yielding metNgb and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{NO}}_{3}^{-}\end{equation*} by means of a heme-bound peroxynitrite intermediate. Steady-state amperometric experiments show that Ngb is devoid of O ₂ reductase and NO reductase activities. To achieve this result, we have set up a protocol for efficient reduction of metNgb using a mixture of FMN and NADH under bright illumination. The results are discussed with reference to a global scheme inspired by the 3D structures of metNgb and NgbCO. Based on the ligand-linked conformational changes discovered by crystallography, the pathways of the reactions with O ₂ and NO provide a framework that may account for the involvement of Ngb in controlling the activation of a protective signaling mechanism.

Escherichia coli flavorubredoxin is a member of the family of the A-type flavoproteins, which are built by two core domains: a metallo-β-lactamase-like domain, at the N-terminal region, harboring a non-heme di-iron site, and a flavodoxin-like domain, containing one FMN moiety. The enzyme fromE. coli has an extra module at the C terminus, containing a rubredoxin-like center. The A-type flavoproteins are widespread among strict and facultative anaerobes, as deduced from the analysis of the complete prokaryotic genomes. In this report we showed that the recombinant enzyme purified from E. coli has nitric-oxide reductase activity with a turnover number of ∼15 mol of NO·mol enzyme⁻¹·s⁻¹, which was well within the range of those determined for the canonical hemeb₃ -Fe_B containing nitric-oxide reductases (e.g. ∼10–50 mol NO·mol enzyme⁻¹·s⁻¹ for the Paracoccus denitrificans NOR). Furthermore, it was shown that the activity was due to the A-type flavoprotein core, as the rubredoxin domain alone exhibited no activity. Thus, a novel family of prokaryotic NO reductases, with a non-heme di-iron site as the catalytic center, was established.

Hydrogen sulfide (H₂S) impairs mitochondrial respiration by potently inhibiting the heme-copper cytochrome c oxidase. Since many prokaryotes, including Escherichia (E.) coli, generate H₂S and encounter high H₂S levels particularly in the human gut, herein we tested whether bacteria can sustain sulfide-resistant O₂-dependent respiration. E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo₃ enzyme and two bd oxidases much less sensitive to cyanide. Working on the isolated enzymes, we found that, whereas the bo₃ oxidase is inhibited by sulfide with half-maximal inhibitory concentration IC₅₀ = 1.1 ± 0.1 μM, under identical experimental conditions both bd oxidases are insensitive to sulfide up to 58 μM. In E. coli respiratory mutants, both O₂-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo₃ oxidase alone, but unaffected by ≤200 μM sulfide when either bd enzyme acted as the only terminal oxidase. Accordingly, wild-type E. coli showed sulfide-insensitive respiration and growth under conditions favouring the expression of bd oxidases. In all tested conditions, cyanide mimicked the functional effect of sulfide on bacterial respiration. We conclude that bd oxidases promote sulfide-resistant O₂-consumption and growth in E. coli and possibly other bacteria. The impact of this discovery is discussed.

Hydrogen sulfide (H₂S) has emerged as a relevant signaling molecule in physiology, taking its seat as a bona fide gasotransmitter akin to nitric oxide (NO) and carbon monoxide (CO). After being merely regarded as a toxic poisonous molecule, it is now recognized that mammalian cells are equipped with sophisticated enzymatic systems for H₂S production and breakdown. The signaling role of H₂S is mainly related to its ability to modify different protein targets, particularly by promoting persulfidation of protein cysteine residues and by interacting with metal centers, mostly hemes. H₂S has been shown to regulate a myriad of cellular processes with multiple physiological consequences. As such, dysfunctional H₂S metabolism is increasingly implicated in different pathologies, from cardiovascular and neurodegenerative diseases to cancer. As a highly diffusible reactive species, the intra‐ and extracellular levels of H₂S have to be kept under tight control and, accordingly, regulation of H₂S metabolism occurs at different levels. Interestingly, even though H₂S, NO, and CO have similar modes of action and parallel regulatory targets or precisely because of that, there is increasing evidence of a crosstalk between the three gasotransmitters. Herein are reviewed the biochemistry, metabolism, and signaling function of hydrogen sulfide, as well as its interplay with the other gasotransmitters, NO and CO.