Christopher L. Bianco

Nitroxyl (HNO) signaling.

Nitroxyl (HNO) has become a nitrogen oxide of significant interest due to its reported biological activity. The actions of HNO in the cardiovascular system appear to make it a good candidate for therapeutic applications for cardiovascular disorders and other potentially important effects have been noted as well. Although the chemistry associated with this activity has not been firmly established, the propensity for HNO to react with thiols and metals are likely mechanisms. Herein, are described the biological activity of HNO and some of the chemistry of HNO that may be responsible for its biological effects.

The Reactive Species Interactome: Evolutionary Emergence, Biological Significance, and Opportunities for Redox Metabolomics and Personalized Medicine

Abstract Significance: Oxidative stress is thought to account for aberrant redox homeostasis and contribute to aging and disease. However, more often than not, administration of antioxidants is ineffective, suggesting that our current understanding of the underlying regulatory processes is incomplete. Recent Advances: Similar to reactive oxygen species and reactive nitrogen species, reactive sulfur species are now emerging as important signaling molecules, targeting regulatory cysteine redox switches in proteins, affecting gene regulation, ion transport, intermediary metabolism, and mitochondrial function. To rationalize the complexity of chemical interactions of reactive species with themselves and their targets and help define their role in systemic metabolic control, we here introduce a novel integrative concept defined as the reactive species interactome (RSI). The RSI is a primeval multilevel redox regulatory system whose architecture, together with the physicochemical characteristics of its constituents, allows efficient sensing and rapid adaptation to environmental changes and various other stressors to enhance fitness and resilience at the local and whole-organism level. Critical Issues: To better characterize the RSI-related processes that determine fluxes through specific pathways and enable integration, it is necessary to disentangle the chemical biology and activity of reactive species (including precursors and reaction products), their targets, communication systems, and effects on cellular, organ, and whole-organism bioenergetics using system-level/network analyses. Future Directions: Understanding the mechanisms through which the RSI operates will enable a better appreciation of the possibilities to modulate the entire biological system; moreover, unveiling molecular signatures that characterize specific environmental challenges or other forms of stress will provide new prevention/intervention opportunities for personalized medicine. Antioxid. Redox Signal. 27, 684–712.

The chemical biology of protein hydropersulfides: Studies of a possible protective function of biological hydropersulfide generation

The recent discovery of significant hydropersulfide (RSSH) levels in mammalian tissues, fluids and cells has led to numerous questions regarding their possible physiological function. Cysteine hydropersulfides have been found in free cysteine, small molecule peptides as well as in proteins. Based on their chemical properties and likely cellular conditions associated with their biosynthesis, it has been proposed that they can serve a protective function. That is, hydropersulfide formation on critical thiols may protect them from irreversible oxidative or electrophilic inactivation. As a prelude to understanding the possible roles and functions of hydropersulfides in biological systems, this study utilizes primarily chemical experiments to delineate the possible mechanistic chemistry associated with cellular protection. Thus, the ability of hydropersulfides to protect against irreversible electrophilic and oxidative modification was examined. The results herein indicate that hydropersulfides are very reactive towards oxidants and electrophiles and are modified readily. However, reduction of these oxidized/modified species is facile generating the corresponding thiol, consistent with the idea that hydropersulfides can serve a protective function for thiol proteins.

The Chemical Biology of Hydropersulfides (RSSH): Chemical Stability, Reactivity and Redox Roles

Recent reports indicate the ubiquitous prevalence of hydropersulfides (RSSH) in mammalian systems. The biological utility of these and related species is currently a matter of significant speculation. The function, lifetime and fate of hydropersulfides will be assuredly based on their chemical properties and reactivity. Thus, to serve as the basis for further mechanistic studies regarding hydropersulfide biology, some of the basic chemical properties/reactivity of hydropersulfides was studied. The nucleophilicity, electrophilicity and redox properties of hydropersulfides were examined under biological conditions. These studies indicate that hydropersulfides can be nucleophilic or electrophilic, depending on the pH (i.e. the protonation state) and can act as good one- and two-electron reductants. These diverse chemical properties in a single species make hydropersulfides chemically distinct from other, well-known sulfur containing biological species, giving them unique and potentially important biological function.

The chemical biology of the persulfide (RSSH)/perthiyl (RSS·) redox couple and possible role in biological redox signaling.

The recent finding that hydropersulfides (RSSH) are biologically prevalent in mammalian systems has prompted further investigation of their chemical properties in order to provide a basis for understanding their potential functions, if any. Hydropersulfides have been touted as hyper-reactive thiol-like species that possess increased nucleophilicity and reducing capabilities compared to their thiol counterparts. Herein, using persulfide generating model systems, the ability of RSSH species to act as one-electron reductants has been examined. Not unexpectedly, RSSH is relatively easily oxidized, compared to thiols, by weak oxidants to generate the perthiyl radical (RSS·). Somewhat surprisingly, however, RSS· was found to be stable in the presence of both O2 and NO and only appears to dimerize. Thus, the RSSH/RSS· redox couple is readily accessible under biological conditions and since dimerization of RSS· may be a rare event due to low concentrations and/or sequestration within a protein, it is speculated that the general lack of reactivity of individual RSS· species may allow this couple to be utilized as a redox component in biological systems.

Examining the reaction of NO and H2S and the possible cross-talk between the two signaling pathways

The small, low molecular weight molecules nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are all endogenously generated in mammalian systems via regulated enzymatic catalysis (1). Along with dioxygen (O2), these small molecules, as well as their derived species, constitute a unique and expanding class of signaling species with important biological function. These species play critical roles in numerous biological processes, including regulation of enzyme activity, protein structure and function, and cellular defense (1). The initial idea that low molecular weight species can be biosynthesized enzymatically for the purpose of regulating vital signaling function came from work on NO. It has been unequivocally demonstrated that NO is made via the actions of nitric oxide synthase (NOS) enzymes. Biosynthesized NO then activates the ironheme-containing enzyme soluble guanylate cyclase (sGC) via coordination to the ironheme, leading to increases in the second messenger cGMP (2). In the vascular system, the NO-sGC-cGMP pathway elicits smooth muscle relaxation and this pathway is a major mechanism for controlling vascular tone. Importantly, the discovery of NO as an endogenously generated small-molecule signaling species evolved from an initial understanding of its target, sGC, and the fact that exogenous/pharmacological addition of NO (or NO-donors) led to enzyme activation. The discovery that NO was biosynthesized for this purpose was a watershed moment in the understanding of physiological signaling because it represented a unique biological pathway whereby a small, freely diffusible, nonionic molecule, previously known mostly as a toxic species, could be synthesized, diffuse to its target, sGC, and elicit a biological response. Following this seminal work, other endogenously generated small-molecule species, such as CO and H2S, have also been shown to possess important signaling properties. However, unlike NO, whose biological target was known, the mechanisms by which these other signaling molecules elicit their … [↵][1]1To whom correspondence should be addressed. Email: jon.fukuto{at}sonoma.edu. [1]: #xref-corresp-1-1

The chemical biology of HNO signaling.

Nitroxyl (HNO) is a simple molecule with significant potential as a pharmacological agent. For example, its use in the possible treatment of heart failure has received recent attention due to its unique therapeutic properties. Recent progress has been made on the elucidation of the mechanisms associated with its biological signaling. Importantly, the biochemical mechanisms described for HNO bioactivity are consistent with its unique and novel chemical properties/reactivity. To date, much of the biology of HNO can be associated with interactions and modification of important regulatory thiol proteins. Herein will be provided a description of HNO chemistry and how this chemistry translates to some of its reported biological effects.

Biological hydropersulfides and related polysulfides – a new concept and perspective in redox biology

The chemical biology of thiols (RSH, e.g., cysteine and cysteine‐containing proteins/peptides) has been a topic of extreme interest for many decades due to their reported roles in protein structure/folding, redox signaling, metal ligation, cellular protection, and enzymology. While many of the studies on thiol/sulfur biochemistry have focused on thiols, relatively ignored have been hydropersulfides (RSSH) and higher order polysulfur species (RSSnH, RSSnR, n > 1). Recent and provocative work has alluded to the prevalence and likely physiological importance of RSSH and related RSSnH. RSSH of cysteine (Cys‐SSH) has been found to be prevalent in mammalian systems along with Cys‐SSH‐containing proteins. The RSSH functionality has not been examined to the extent of other biologically relevant sulfur derivatives (e.g., sulfenic acids, disulfides, etc.), whose roles in cell signaling are strongly indicated. The recent finding of Cys‐SSH biosynthesis and translational incorporation into proteins is an unequivocal indication of its fundamental importance and necessitates a more profound look into the physiology of RSSH. In this Review, we discuss the currently reported chemical biology of RSSH (and related species) as a prelude to discussing their possible physiological roles.

Selenols are resistant to irreversible modification by HNO.

The discovery of nitric oxide (NO) as an endogenously generated signaling species in mammalian cells has spawned a vast interest in the study of the chemical biology of nitrogen oxides. Of these, nitroxyl (azanone, HNO) has gained much attention for its potential role as a therapeutic for cardiovascular disease. Known targets of HNO include hemes/heme proteins and thiols/thiol-containing proteins. Recently, due to their roles in redox signaling and cellular defense, selenols and selenoproteins have also been speculated to be additional potential targets of HNO. Indeed, as determined in the current work, selenols are targeted by HNO. Such reactions appear to result only in formation of diselenide products, which can be easily reverted back to the free selenol. This characteristic is distinct from the reaction of HNO with thiols/thiolproteins. These findings suggest that, unlike thiolproteins, selenoproteins are resistant to irreversible oxidative modification, support that Nature may have chosen to use selenium instead of sulfur in certain biological systems for its enhanced resistance to electrophilic and oxidative modification.

The reaction of hydrogen sulfide with disulfides: formation of a stable trisulfide and implications for biological systems

The signalling associated with hydrogen sulfide (H2S) remains to be established, and recent studies have alluded to the possibility that H2S‐derived species play important roles. Of particular interest are hydropersulfides (RSSH) and related polysulfides (RSSnR, n > 1). This work elucidates the fundamental chemical relationship between these sulfur species as well as examines their biological effects.