Kenneth R. Olson

Harsh acid oxidation of activated charcoal transforms an insoluble carbon-rich source into water-soluble, disc structures of graphene decorated with multiple oxygen-containing functionalities. We term these pleiotropic nano-enzymes as “pleozymes”. A broad redox potential spans many crucial redox reactions including the oxidation of hydrogen sulfide (H₂S) to polysulfides and thiosulfate, dismutation of the superoxide radical (O₂⁻*), and oxidation of NADH to NAD⁺. The oxidation of H₂S is predicted to enhance protein persulfidation—the attachment of sulfur to cysteine residues. Persulfidated proteins act as redox intermediates, and persulfidation protects proteins from irreversible oxidation and ubiquitination, providing an important means of signaling. Protein persulfidation is believed to decline in several neurological disorders and aging. Importantly, and consistent with the role of persulfidation in signaling, the master antioxidant transcription factor Nrf2 is regulated by Keap1’s persulfidation. Here, we demonstrate that pleozymes increased overall protein persulfidation in cells from apparently healthy individuals and from individuals with the mitochondrial protein mutation responsible for Friedreich’s ataxia. We further find that pleozymes specifically enhanced Keap1 persulfidation, with subsequent increased accumulation of Nrf2 and Nrf2’s antioxidant targets.

Down syndrome (DS) is a multisystemic disorder that includes accelerated aging caused by trisomy 21. In particular, overexpression of cystathionine-β-synthase (CBS) is linked to excess intracellular hydrogen sulfide (H₂S), a mitochondrial toxin at higher concentrations, which impairs cellular viability. Concurrent overexpression of superoxide dismutase 1 (SOD1) may increase oxidative stress by generating excess hydrogen peroxide (H₂O₂) while also mitigating the toxic H₂S burden via a non-canonical sulfide-oxidizing mechanism. We investigated the phenotypic variability in basal H₂S levels in relation to DS B lymphocyte cell health and SOD1 in H₂S detoxification. The H₂S levels were negatively correlated with the DS B lymphocyte growth rates but not with CBS protein. Pharmacological inhibition of SOD1 using LCS-1 significantly increased the H₂S levels to a greater extent in DS cells while also decreasing the polysulfide products of H₂S oxidation. However, DS cells exhibited elevated H₂O₂ and lipid peroxidation, representing potential toxic consequences of SOD1 overexpression. Treatment of DS cells with a pleiotropic carbon nanozyme (pleozymes) decreased the total oxidative stress and reduced the levels of the H₂S-generating enzymes CBS and 3-mercaptopyruvate sulfurtransferase (MPST). Our results indicate that pleozymes may bridge the protective and deleterious effects of DS SOD1 overexpression on H₂S metabolism and oxidative stress, respectively, with cytoprotective benefits.

LCS-1, a putative selective inhibitor of SOD1, is a substituted pyridazinone with rudimentary similarity to quinones and naphthoquinones. As quinones catalytically oxidize H₂S to biologically active reactive sulfur species (RSS), we hypothesized LCS-1 might have similar attributes. Here, we examine LCS-1 reactions with H₂S and SOD1 using thiol-specific fluorophores, liquid chromatography–mass spectrometry, electron paramagnetic resonance (EPR), UV–vis spectrometry, and oxygen consumption. We show that LCS-1 catalytically oxidizes H₂S in buffer solutions to form RSS, namely per- and polyhydrosulfides (H₂S_n, n = 2–6). These reactions consume oxygen and produce hydrogen peroxide, but they do not have an EPR signature, nor do they affect the UV–vis spectrum. Surprisingly, LCS-1 synergizes with SOD1, but not SOD2, to oxidize H₂S to H₂S_3-6. LCS-1 forms monothiol adducts with H₂S, glutathione (GSH), and cysteine (Cys), but not with oxidized glutathione or cystine; both thiol adducts inhibit LCS-1-SOD1 synergism. We propose that LCS-1 forms an adduct with SOD1 that disrupts the intramolecular Cys⁵⁷-Cys¹⁴⁶ disulfide bond and transforms SOD1 from a dismutase to an oxidase. This would increase cellular ROS and polysulfides, the latter potentially affecting cellular signaling and/or cytoprotection.

1,4-naphthoquinones (NQs) catalytically oxidize H₂S to per- and polysufides and sulfoxides, reduce oxygen to superoxide and hydrogen peroxide, and can form NQ-SH adducts through Michael addition. Here, we measured oxygen consumption and used sulfur-specific fluorophores, liquid chromatography tandem mass spectrometry (LC-MS/MS), and UV-Vis spectrometry to examine H₂S oxidation by NQs with various substituent groups. In general, the order of H₂S oxidization was DCNQ ~ juglone > 1,4-NQ > plumbagin >DMNQ ~ 2-MNQ > menadione, although this order varied somewhat depending on the experimental conditions. DMNQ does not form adducts with GSH or cysteine (Cys), yet it readily oxidizes H₂S to polysulfides and sulfoxides. This suggests that H₂S oxidation occurs at the carbonyl moiety and not at the quinoid 2 or 3 carbons, although the latter cannot be ruled out. We found little evidence from oxygen consumption studies or LC-MS/MS that NQs directly oxidize H₂S_2–4, and we propose that apparent reactions of NQs with inorganic polysulfides are due to H₂S impurities in the polysulfides or an equilibrium between H₂S and H₂S_n. Collectively, NQ oxidation of H₂S forms a variety of products that include hydropersulfides, hydropolysulfides, sulfenylpolysulfides, sulfite, and thiosulfate, and some of these reactions may proceed until an insoluble S₈ colloid is formed.

Cardiovascular regulation of tissue oxygenation is generally viewed as an anti-drop process that prevents tissue oxygen concentration from falling below some minimum. I propose that cardiovascular regulation is predominately an anti-rise process designed to downregulate oxygen delivery. This maintains an evolutionarily conserved, reduced intracellular environment to prevent oxidation of redox-sensitive regulatory protein thiols. A number of points support this hypothesis. First, oxygen is the only nutrient with a positive, fourfold diffusion gradient from the environment to systemic tissues, minimizing the likelihood that oxygen delivery is limited. Second, hemoglobin (Hb) retains oxygen unless offloading is absolutely necessary. The allosteric properties of Hb keep oxygen tightly bound until absolutely needed, and the Bohr shift, which favors offloading, is only transient and lost when metabolism is restored. Third, a myoglobin-like Hb (xHb) would offload all of its oxygen and could easily have evolved, but it did not. Fourth, oxygen-sensitive vasoconstrictors and hyperoxic-rarefaction prevent acute and chronic over perfusion. Fifth, Fåhraeus and Fåhraeus–Lindqvist effects reduce capillary hematocrit to minimize microcirculatory oxygen content. Sixth, venous blood remains 75% saturated, wasting 75% of cardiac output were an oxygen reserve not needed. Finally, xHb-containing red blood cells could be considerably smaller and thereby decrease Fåhraeus and Fåhraeus–Lindqvist effects and cardiac load. In summary, the capacity of the cardiovascular system to deliver oxygen to the tissues generally exceeds demand, and although maintenance of an oxygen delivery reserve is important, it is more important to prevent excess oxygen delivery.

Hydrogen sulfide (H₂S) is a noxious, potentially poisonous, but necessary gas produced from sulfur metabolism in humans. In Down Syndrome (DS), the production of H₂S is elevated and associated with degraded mitochondrial function. Therefore, removing H₂S from the body as a stable oxide could be an approach to reducing the deleterious effects of H₂S in DS. In this report we describe the catalytic oxidation of hydrogen sulfide (H₂S) to polysulfides (HS_2+n⁻) and thiosulfate (S₂O₃²⁻) by poly(ethylene glycol) hydrophilic carbon clusters (PEG‐HCCs) and poly(ethylene glycol) oxidized activated charcoal (PEG‐OACs), examples of oxidized carbon nanozymes (OCNs). We show that OCNs oxidize H₂S to polysulfides and S₂O₃²⁻ in a dose‐dependent manner. The reaction is dependent on O₂ and the presence of quinone groups on the OCNs. In DS donor lymphocytes we found that OCNs increased polysulfide production, proliferation, and afforded protection against additional toxic levels of H₂S compared to untreated DS lymphocytes. Finally, in Dp16 and Ts65DN murine models of DS, we found that OCNs restored osteoclast differentiation. This new action suggests potential facile translation into the clinic for conditions involving excess H₂S exemplified by DS.

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H₂S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H₂S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H₂S to both inorganic and organic hydroper-/hydropolysulfides (R₂S_n, R=H, Cys, GSH; n = 2–4) and organic sulfoxides (GS_nOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H₂S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H₂S.

Significance: Nutraceuticals are ingested for health benefits, in addition to their general nutritional value. These dietary supplements have become increasingly popular since the late 20th century and they are a rapidly expanding global industry approaching a half-trillion U.S. dollars annually. Many nutraceuticals are promulgated as potent antioxidants. Recent Advances: Experimental support for the efficacy of nutraceuticals has lagged behind anecdotal exuberance. However, accumulating epidemiological evidence and recent, well-controlled clinical trials are beginning to support earlier animal and in vitro studies. Although still somewhat limited, encouraging results have been suggested in essentially all organ systems and against a wide range of pathophysiological conditions. Critical Issues: Health benefits of “antioxidant” nutraceuticals are largely attributed to their ability to scavenge oxidants. This has been criticized based on several factors, including limited bioavailability, short tissue retention time, and the preponderance of endogenous antioxidants. Recent attention has turned to nutraceutical activation of downstream antioxidant systems, especially the Keap1/Nrf2 (Kelch like ECH associated protein 1/nuclear factor erythroid 2-related factor 2) axis. The question now becomes, how do nutraceuticals activate this axis? Future Directions: Reactive sulfur species (RSS), including hydrogen sulfide (H₂S) and its metabolites, are potent activators of the Keap1/Nrf2 axis and avid scavengers of reactive oxygen species. Evidence is beginning to accumulate that a variety of nutraceuticals increase cellular RSS by directly providing RSS in the diet, or through a number of catalytic mechanisms that increase endogenous RSS production. We propose that nutraceutical-specific targeting of RSS metabolism will lead to the design and development of even more efficacious antioxidant therapeutic strategies. Antioxid. Redox Signal. 38, 68–94.

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H₂S) using RSS-specific fluorophores, liquid chromatography-mass spectrometry, UV-Vis absorption spectrometry, oxygen-sensitive optodes, thiosulfate-specific nanoparticles, HPLC-monobromobimane derivatization, and ion chromatographic assays. We show that NQs, catalytically oxidize H₂S to per- and polysulfides (H₂S_n, n = 2–6), thiosulfate, sulfite and sulfate in reactions that consume oxygen and are accelerated by superoxide dismutase (SOD) and inhibited by catalase. The approximate efficacy of NQs (in decreasing order) is, 1,4-NQ ≈ juglone ≈ plumbagin > 2-methoxy-1,4-NQ ≈ menadione >> phylloquinone ≈ anthraquinone ≈ menaquinone ≈ lawsone. We propose that the most probable reactions are an initial two-electron oxidation of H₂S to S⁰ and reduction of NQ to NQH₂. S⁰ may react with H₂S or elongate H₂S_n in variety of reactions. Reoxidation of NQH₂ likely involves a semiquinone radical (NQ^·−) intermediate via several mechanisms involving oxygen and comproportionation to produce NQ and superoxide. Dismutation of the latter forms hydrogen peroxide which then further oxidizes RSS to sulfoxides. These findings provide the chemical background for novel sulfur-based approaches to naphthoquinone-directed therapies.

Hydrogen sulfide has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious and neuropathological diseases have been undertaken. Here we review the possible direct targets of H₂S in mammals. H₂S directly interacts with reactive oxygen/nitrogen species and is involved in redox signaling. H₂S also reacts with hemeproteins and modulates metal-containing complexes. Once being oxidized, H₂S can persulfidate proteins by adding -SSH to the amino acid cysteine. These direct modifications by H₂S have significant impact on cell structure and many cellular functions, such as tight junctions, autophagy, apoptosis, vesicle trafficking, cell signaling, epigenetics and inflammasomes. Therefore, we conclude that H₂S is involved in many important cellular and physiological processes. Compounds that donate H₂S to biological systems can be developed as therapeutics for different diseases.