Alfred Hausladen

A metabolic enzyme for S -nitrosothiol conserved from bacteria to humans

Considerable evidence indicates that NO biology involves a family of NO-related molecules and that S-nitrosothiols (SNOs) are central to signal transduction and host defence. It is unknown, however, how cells switch off the signals or protect themselves from the SNOs produced for defence purposes. Here we have purified a single activity from Escherichia coli, Saccharomyces cerevisiae and mouse macrophages that metabolizes S-nitrosoglutathione (GSNO), and show that it is the glutathione-dependent formaldehyde dehydrogenase. Although the enzyme is highly specific for GSNO, it controls intracellular levels of both GSNO and S-nitrosylated proteins. Such ‘GSNO reductase’ activity is widely distributed in mammals. Deleting the reductase gene in yeast and mice abolishes the GSNO-consuming activity, and increases the cellular quantity of both GSNO and protein SNO. Furthermore, mutant yeast cells show increased susceptibility to a nitrosative challenge, whereas their resistance to oxidative stress is unimpaired. We conclude that GSNO reductase is evolutionarily conserved from bacteria to humans, is critical for SNO homeostasis, and protects against nitrosative stress.

Flavohemoglobin denitrosylase catalyzes the reaction of a nitroxyl equivalent with molecular oxygen

We have previously reported that bacterial flavohemoglobin (HMP) catalyzes both a rapid reaction of heme-bound O2 with nitric oxide (NO) to form nitrate [HMP-Fe(II)O2 + NO → HMP-Fe(III) + NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}] and, under anaerobic conditions, a slower reduction of heme-bound NO to an NO− equivalent (followed by the formation of N2O), thereby protecting against nitrosative stress under both aerobic and anaerobic conditions, and rationalizing our finding that NO is rapidly consumed across a wide range of O2 concentrations. It has been alternatively suggested that HMP activity is inhibited at low pO2 because the enzyme is then in the relatively inactive nitrosyl form [koff/kon for NO (0.000008 μM) ≪ koff/kon for O2 (0.012 μM) and KM for O2 = 30–100 μM]. To resolve this discrepancy, we have directly measured heme-ligand turnover and NADH consumption under various O2/NO concentrations. We find that, at biologically relevant O2 concentrations, HMP preferentially binds NO (not O2), which it then reacts with oxygen to form nitrate (in essence NO− + O2 → NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}). During steady-state turnover, the enzyme can be found in the ferric (FeIII) state. The formation of a heme-bound nitroxyl equivalent and its subsequent oxidation is a novel enzymatic function, and one that dominates the oxygenase activity under biologically relevant conditions. These data unify the mechanism of HMP/NO interaction with those recently described for the nematode Ascaris and mammalian hemoglobins, and more generally suggest that the peroxidase (FeIII)-like properties of globins have evolved for handling of NO.

Endogenous protein S-nitrosylation in E. coli: regulation by OxyR

Handling Nitrosative Stress S-nitrosylation of proteins is a principal mechanism of cellular signaling in eukaryotes but has not been reported in microbes. The transcription factor OxyR serves to protect bacterial cells from reactive oxygen species produced by cell metabolism in the presence of oxygen. Seth et al. (p. 470) found that when cells grew in the presence of nitrate, OxyR was modified by S-nitrosylation of the same cysteine residue that gets oxidized in cells grown aerobically. However, the nitrosylated OxyR activated a different set of genes—some of which appeared to protect the cell from excessive S-nitrosylation. Bacteria adapting to growth in the presence of nitrate activate a transcriptional regulator also involved in oxidative stress. Endogenous S-nitrosylation of proteins, a principal mechanism of cellular signaling in eukaryotes, has not been observed in microbes. We report that protein S-nitrosylation is an obligate concomitant of anaerobic respiration on nitrate in Escherichia coli. Endogenous S-nitrosylation during anaerobic respiration is controlled by the transcription factor OxyR, previously thought to operate only under aerobic conditions. Deletion of OxyR resulted in large increases in protein S-nitrosylation, and S-nitrosylation of OxyR induced transcription from a regulon that is distinct from the regulon induced by OxyR oxidation. Furthermore, products unique to the anaerobic regulon protected against S-nitrosothiols, and anaerobic growth of E. coli lacking OxyR was impaired on nitrate. Thus, OxyR serves as a master regulator of S-nitrosylation, and alternative posttranslational modifications of OxyR control distinct transcriptional responses.

Assessment of nitric oxide signals by triiodide chemiluminescence

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures.

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

Thioredoxin-interacting Protein (Txnip) Is a Feedback Regulator of S-Nitrosylation

Nitric oxide exerts a plethora of biological effects via protein S-nitrosylation, a redox-based reaction that converts a protein Cys thiol to a S-nitrosothiol. However, although the regulation of protein S-nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus-coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin-interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin-mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S-nitrosylation.

Hemoglobin βCys93 is essential for cardiovascular function and integrated response to hypoxia

Significance Oxygen delivery by RBC Hb is essential for life. Just three amino acids in Hb are conserved in all mammals and birds, but only two of those are required to carry oxygen. The third, a Cys within the β-chain, βCys93, has been assigned a role in carrying nitric oxide, which mediates vasodilation. However, the physiological importance of RBC-mediated vasoregulation is unknown. We show that blood flow and tissue oxygenation are markedly impaired in mice with a βCys93Ala mutation. The βCys93Ala mutation also results in myocardial ischemia, cardiac decompensation, and enhanced mortality. These findings support a new view of the respiratory cycle wherein, remarkably, RBCs regulate blood flow and (βCys93NO)-Hb is necessary for adequate tissue oxygenation and normal cardiovascular function. Oxygen delivery by Hb is essential for vertebrate life. Three amino acids in Hb are strictly conserved in all mammals and birds, but only two of those, a His and a Phe that stabilize the heme moiety, are needed to carry O2. The third conserved residue is a Cys within the β-chain (βCys93) that has been assigned a role in S-nitrosothiol (SNO)-based hypoxic vasodilation by RBCs. Under this model, the delivery of SNO-based NO bioactivity by Hb redefines the respiratory cycle as a triune system (NO/O2/CO2). However, the physiological ramifications of RBC-mediated vasodilation are unknown, and the apparently essential nature of βCys93 remains unclear. Here we report that mice with a βCys93Ala mutation are deficient in hypoxic vasodilation that governs blood flow autoregulation, the classic physiological mechanism that controls tissue oxygenation but whose molecular basis has been a longstanding mystery. Peripheral blood flow and tissue oxygenation are decreased at baseline in mutant animals and decline excessively during hypoxia. In addition, βCys93Ala mutation results in myocardial ischemia under basal normoxic conditions and in acute cardiac decompensation and enhanced mortality during transient hypoxia. Fetal viability is diminished also. Thus, βCys93-derived SNO bioactivity is essential for tissue oxygenation by RBCs within the respiratory cycle that is required for both normal cardiovascular function and circulatory adaptation to hypoxia.

Identification of S-nitroso-CoA reductases that regulate protein S-nitrosylation

Significance Coenzyme A (CoA) is a small-molecular-weight thiol that plays a central role in cellular metabolism. We have discovered a novel, phylogenetically conserved class of enzymes that reduce S-nitroso-CoA (SNO-CoA) and thereby regulate protein S-nitrosylation. These denitrosylases, identified as alcohol dehydrogenase 6 (Adh6) in yeast and aldo-keto reductase 1A1 in mammals, may be analogized to deacetylases, which regulate CoA-mediated protein acetylation. In yeast, Adh6 (previously without ascribed cellular function) regulates endogenous protein S-nitrosylation (heretofore unknown) including function-altering S-nitrosylation that impacts CoA-related metabolism. Thus, our findings establish a novel role for CoA in protein S-nitrosylation (operating through SNO-CoA), which is governed by specific enzymes. This mechanism may regulate the influence of nitric oxide on cellular metabolism in health and disease. Coenzyme A (CoA) mediates thiol-based acyl-group transfer (acetylation and palmitoylation). However, a role for CoA in the thiol-based transfer of NO groups (S-nitrosylation) has not been considered. Here we describe protein S-nitrosylation in yeast (heretofore unknown) that is mediated by S-nitroso-CoA (SNO-CoA). We identify a specific SNO-CoA reductase encoded by the alcohol dehydrogenase 6 (ADH6) gene and show that deletion of ADH6 increases cellular S-nitrosylation and alters CoA metabolism. Further, we report that Adh6, acting as a selective SNO-CoA reductase, protects acetoacetyl–CoA thiolase from inhibitory S-nitrosylation and thereby affects sterol biosynthesis. Thus, Adh6-regulated, SNO-CoA–mediated protein S-nitrosylation provides a regulatory mechanism paralleling protein acetylation. We also find that SNO-CoA reductases are present from bacteria to mammals, and we identify aldo-keto reductase 1A1 as the mammalian functional analog of Adh6. Our studies reveal a novel functional class of enzymes that regulate protein S-nitrosylation from yeast to mammals and suggest that SNO-CoA–mediated S-nitrosylation may subserve metabolic regulation.

Is the flavohemoglobin a nitric oxide dioxygenase

Forrester and Foster [1] review potential mechanisms of nitric oxide (NO) catabolism by the flavohemoglobin HMP, a major source of NO-detoxifying activity in prokaryotes and simple eukaryotes including yeast: dioxygenation of NO to NO3 (NO dioxygenase), nitroxylation of oxygen to NO3 (denitrosylase), and reduction of NO to N2O in the absence of O2 (NO reductase). They argue for NO dioxygenation as the principal, physiologically relevant mechanism, based on rates of NO consumption (faster in the presence of O2) and the finding that HMP activity can be inactivated by a mutation (Tyr29Phe) that disrupts binding of O2 without affecting the binding of NO. We disagree with this assessment, however, as it reflects a misapprehension of the biochemistry of the enzyme and of the criteria for physiological relevance. Multiple lines of evidence favor the denitrosylase mechanism (nitroxylation of oxygen) as primary and the importance of the NO reductase mechanism in specific physiological situations. The dioxygenase activity may be a fortuitous consequence of laboratory conditions. Simply put, the question is whether the flavohemoglobin binds NO or O2 during turnover in situ. Bacteria express flavohemoglobin in either aerobic or anaerobic environments, but they only express flavohemoglobin aerobically in the presence of NO. As pointed out by Forrester and Foster [1], the relative affinities of HMP strongly favor NO binding over O2. KM values are 0.28 mM for NO versus 100 mM for O2 and KD values favor NO by a factor of 1500 [2]. Moreover, O2 limitation is typical of growing microorganisms, in which respiratory activity can result in levels of dissolved O2 of less than 5% (o10 mM) [3], and pathogenic bacteria will encounter ‘‘high’’ NO concentrations (antimicrobial concentrations of NO are *0.28 mM) in microaerobic or anaerobic environments [4–8]. HMP will thus bind NO (not O2) in physiological situations. The ferrous (Fe2þ)–NO complex in HMP is turned over very rapidly across a wide range of O2 concentrations (9–125 mM O2) [9]. Rates of denitrosylation thus compare favorably (within a factor of 2) to rates of NO dioxygenation [9]. Reanalysis of several published works reveals that the majority of HMP is in the nitrosyl form during turnover, including work by Gardner et al. [2] using 200 mM O2 and 2 mM NO (Fig. 5 in Ref. [2]; NO-ligated HMP is in 15-fold excess over O2-ligated HMP based on KD’s); by Mills et al. [10] using 200 mM O2 and 35 mM NO (Fig. 4 in Ref. [10]; a 260-fold excess of Fe–NO over Fe–O2); by the same authors [10] with repeated additions of 35 mM NO (1000-fold excess of Fe–NO); and by Hausladen et al. [9] using 10 mM NO and 12.5 mM O2 (1200-fold excess of Fe–NO) (Fig. 3 in Ref. [9]). It is important to note that absorbance spectra recorded during turnover reflect entirely the nitrosyl (ferrous or ferric) forms. As O2 concentrations increase, the spectra change from ferrous-nitrosyl to increasingly ferric-nitrosyl character, while no evidence of the ferrous-oxy complex (i.e., the dioxygenase mechanism) is seen [9].

S-Nitrosoglutathione Reductase Deficiency Confers Improved Survival and Neurological Outcome in Experimental Cerebral Malaria

ABSTRACT Artesunate remains the mainstay of treatment for cerebral malaria, but it is less effective in later stages of disease when the host inflammatory response and blood-brain barrier integrity dictate clinical outcomes. Nitric oxide (NO) is an important regulator of inflammation and microvascular integrity, and impaired NO bioactivity is associated with fatal outcomes in malaria. Endogenous NO bioactivity in mammals is largely mediated by S-nitrosothiols (SNOs). Based on these observations, we hypothesized that animals deficient in the SNO-metabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), which exhibit enhanced S-nitrosylation, would have improved outcomes in a preclinical model of cerebral malaria. GSNOR knockout (KO) mice infected with Plasmodium berghei ANKA had significantly delayed mortality compared to WT animals (P < 0.0001), despite higher parasite burdens (P < 0.01), and displayed markedly enhanced survival versus the wild type (WT) when treated with the antimalarial drug artesunate (77% versus 38%; P < 0.001). Improved survival was associated with higher levels of protein-bound NO, decreased levels of CD4+ and CD8+ T cells in the brain, improved blood-brain barrier integrity, and improved coma scores, as well as higher levels of gamma interferon. GSNOR KO animals receiving WT bone marrow had significantly reduced survival following P. berghei ANKA infection compared to those receiving KO bone barrow (P < 0.001). Reciprocal transplants established that survival benefits of GSNOR deletion were attributable primarily to the T cell compartment. These data indicate a role for GSNOR in the host response to malaria infection and suggest that strategies to disrupt its activity will improve clinical outcomes by enhancing microvascular integrity and modulating T cell tissue tropism.