Jon M. Fukuto

Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling

Significance Reactive sulfur-containing compounds, such as l-cysteine hydropersulfide (CysSSH), reportedly form in mammals. However, the biological relevance of these reactive sulfur species remains unclear. We determined that CysSSH was synthesized from cystine by cystathionine β-synthase and cystathionine γ-lyase, which in turn may contribute to high levels of glutathione hydropersulfide (>100 μM) and other CysSSH derivatives of peptides/proteins formed in cells, tissues, and plasma from mice and humans. Compared with glutathione and hydrogen sulfide, CysSSH derivatives were superior nucleophiles and reductants and capable of regulating electrophilic cell signaling mediated by 8-nitroguanosine 3′,5′-cyclic monophosphate. Altogether, it is proposed that reactive Cys persulfides and S-polythiolation have critical regulatory functions in redox cell signaling. Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine β-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 μM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H2O2 and a recently described biologically generated electrophile 8-nitroguanosine 3′,5′-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H2S can be generated from persulfide degradation, much of the reported biological activity associated with H2S may actually be that of persulfides. That is, H2S may act primarily as a marker for the biologically active of persulfide species.

Small molecule signaling agents: the integrated chemistry and biochemistry of nitrogen oxides, oxides of carbon, dioxygen, hydrogen sulfide, and their derived species.

Several small molecule species formally known primarily as toxic gases have, over the past 20 years, been shown to be endogenously generated signaling molecules. The biological signaling associated with the small molecules NO, CO, H₂S (and the nonendogenously generated O₂), and their derived species have become a topic of extreme interest. It has become increasingly clear that these small molecule signaling agents form an integrated signaling web that affects/regulates numerous physiological processes. The chemical interactions between these species and each other or biological targets is an important factor in their roles as signaling agents. Thus, a fundamental understanding of the chemistry of these molecules is essential to understanding their biological/physiological utility. This review focuses on this chemistry and attempts to establish the chemical basis for their signaling functions.

Thiol Chemistry in Peroxidase Catalysis and Redox Signaling

The oxidation chemistry of thiols and disulfides of biologic relevance is described. The review focuses on the interaction and kinetics of hydrogen peroxide with low-molecular-weight thiols and protein thiols and, in particular, on sulfenic acid groups, which are recognized as key intermediates in several thiol oxidation processes. In particular, sulfenic and selenenic acids are formed during the catalytic cycle of peroxiredoxins and glutathione peroxidases, respectively. In turn, these enzymes are in close redox communication with the thioredoxin and glutathione systems, which are the major controllers of the thiol redox state. Oxidants formed in the cell originate from several different sources, but the major producers are NADPH oxidases and mitochondria. However, a different role of the oxygen species produced by these sources is apparent as oxidants derived from NADPH oxidase are involved mainly in signaling processes, whereas those produced by mitochondria induce cell death in pathways including also the thioredoxin system, presently considered an important target for cancer chemotherapy.

Redox chemistry and chemical biology of H2S, hydropersulfides, and derived species: implications of their possible biological activity and utility.

Hydrogen sulfide (H2S) is an endogenously generated and putative signaling/effector molecule. Despite its numerous reported functions, the chemistry by which it elicits its functions is not understood. Moreover, recent studies allude to the existence of other sulfur species besides H2S that may play critical physiological roles. Herein, the basic chemical biology of H2S as well as other related or derived species is discussed and reviewed. This review particularly focuses on the per- and polysulfides which are likely in equilibrium with free H2S and which may be important biological effectors themselves.

The reaction of H2S with oxidized thiols: Generation of persulfides and implications to H2S biology

Hydrogen sulfide is an endogenously generated molecule with many reported physiological functions. Although several biological targets have been proposed, the biochemical mechanisms by which it elicits activity are not established. Thus, in an effort to begin to delineate the fundamental biological chemistry of H(2)S, we have examined the reaction of H(2)S with oxidized thiols and thiol proteins in order to determine whether persulfide formation occurs, is stable and how this may affect protein function. We have found that persulfides are easily generated, relatively stable and can alter enzyme activity. Moreover, we have begun to develop methodology for in situ generation of persulfides to facilitate further study of this potentially important species.

NG-hydroxy-l-arginine and nitric oxide inhibit Caco-2 tumor cell proliferation by distinct mechanisms

The objective of this study was to elucidate the role and mechanism of nitric oxide (NO) synthase (NOS) in modulating the growth of the Caco-2 human colon carcinoma cell line. The two novel observations reported here are, first, that N G-hydroxy-l-arginine (NOHA) inhibits Caco-2 tumor cell proliferation, likely by inhibiting arginase activity, and, second, that NO causes cytostasis by mechanisms that might involve inhibition of ornithine decarboxylase (ODC) activity. Both arginase and ODC are enzymes involved in the conversion of arginine to polyamines required for cell proliferation. Cell growth was monitored by cell count, cell protein analysis, and DNA synthesis. NOHA (1-30 μM) and NO in the form of DETA/NO (1-30 μM) inhibited cell proliferation by 30-85%. The cytostatic effect of NOHA was prevented by addition of excess ornithine, putrescine, spermidine, or spermine to cell cultures, whereas the cytostatic effect of NO (DETA/NO) and α-difluoromethylornithine (ODC inhibitor) was unaffected by ornithine but was prevented by putrescine, spermidine, or spermine. The cytostatic effect of NOHA appeared to be independent of its conversion to NO, and the effect of NO appeared to be independent of cGMP. NOHA inhibited urea production by Caco-2 cells and inhibited arginase catalytic activity (85% at 3 μM), whereas NO (DEA/NO and SNAP) inhibited ODC activity (≥60% at 30 μM) without affecting arginase activity. Coculture of Caco-2 cells with lipopolysaccharide/cytokine-activated rat aortic endothelial cells markedly slowed Caco-2 cell proliferation, and this was blocked by NOS inhibitors. These observations that NOHA and NO may inhibit sequential steps in the arginine-polyamine pathway suggest a novel biological role for NOS in the inhibition of cell proliferation of certain tumor cells and possibly other cell types.

Nitric oxide reaction with red blood cells and hemoglobin under heterogeneous conditions

Understanding the interaction of nitric oxide (NO) with red blood cells (RBCs) is vital to elucidating the metabolic fate of NO in the vasculature. Because hemoglobin (Hb) is the most abundant intraerythrocytic protein and reacts rapidly with NO, the interaction of NO with Hb has been studied extensively. We and others have shown the NO reaction with RBCs is nearly 1,000-fold slower than the reaction with cell-free Hb. Because the reaction rate of NO with cell-free Hb and RBCs is quite different, we hypothesize that different reaction products evolve under locally high NO concentrations, which can be generated by bolus NO addition or NO inhalation. Here we use electron paramagnetic resonance to show that bolus NO addition to cell-free Hb solutions results in nitrosylhemoglobin [HbFe(II)NO] formation as a minor product through a MetHb-dependent pathway. Further, the RBC is shown to be more prone to form HbFe(II)NO under this heterogeneous condition compared with an equivalent free-Hb solution. In both cases, trapping MetHb with cyanide blocked the formation of HbFe(II)NO. We conclude that the formation of HbFe(II)NO is a heterogeneous phenomenon involving three successive reactions of NO with the same heme molecule. These results were supported further by mathematically modeling NO–Hb reactions and diffusion.

Biological nitric oxide signalling: chemistry and terminology.

Biological nitrogen oxide signalling and stress is an area of extreme clinical, pharmacological, toxicological, biochemical and chemical research interest. The utility of nitric oxide and derived species as signalling agents is due to their novel and vast chemical interactions with a variety of biological targets. Herein, the chemistry associated with the interaction of the biologically relevant nitrogen oxide species with fundamental biochemical targets is discussed. Specifically, the chemical interactions of nitrogen oxides with nucleophiles (e.g. thiols), metals (e.g. hemeproteins) and paramagnetic species (e.g. dioxygen and superoxide) are addressed. Importantly, the terms associated with the mechanisms by which NO (and derived species) react with their respective biological targets have been defined by numerous past chemical studies. Thus, in order to assist researchers in referring to chemical processes associated with nitrogen oxide biology, the vernacular associated with these chemical interactions is addressed.

The specificity of nitroxyl chemistry is unique among nitrogen oxides in biological systems

The importance of nitric oxide in mammalian physiology has been known for nearly 30 years. Similar attention for other nitrogen oxides such as nitroxyl (HNO) has been more recent. While there has been speculation as to the biosynthesis of HNO, its pharmacological benefits have been demonstrated in several pathophysiological settings such as cardiovascular disorders, cancer, and alcoholism. The chemical biology of HNO has been identified as related to, but unique from, that of its redox congener nitric oxide. A summary of these findings as well as a discussion of possible endogenous sources of HNO is presented in this review.

A comparison of the chemistry associated with the biological signaling and actions of nitroxyl (HNO) and nitric oxide (NO)

Nitric oxide (NO) and nitroxyl (HNO) are reported to have numerous biological activities with significant therapeutic potential. Many of these activities are overlapping. The chemistry by which these two species act is likely to be distinct in spite of their apparent close structural similarities. Discussed in this review is the chemistry of NO and HNO with their likely biological targets - thiolproteins, metalloproteins (more specifically iron heme proteins) and free radical processes. Based on the chemistry discussed, it can be concluded that the biological actions of NO are likely due primarily to its interactions with metal centers and reaction with radical species. The likely biological targets for HNO are, similarly, metal centers and radical species (albeit with different chemistry compared to NO). HNO is also particularly good at directly modifying thiols while NO-mediated thiol modification requires other reactants to be present and is not as facile. Thus, a fundamental difference between NO and HNO that likely distinguishes them with regards to their biological activity is the greater propensity for HNO to react with thiols compared to NO.