Uncoupled redox stress: how a temporal misalignment of redox-regulated processes and circadian rhythmicity exacerbates the stressed state.

Diurnal and seasonal rhythmicity, entrained by environmental and nutritional cues, is a vital part of all life on Earth operating at every level of organization; from individual cells, to multicellular organisms, whole ecosystems and societies. Redox processes are intrinsic to physiological function and circadian regulation, but how they are integrated with other regulatory processes at the whole-body level is poorly understood. Circadian misalignment triggered by a major stressor (e.g. viral infection with SARS-CoV-2) or recurring stressors of lesser magnitude such as shift work elicit a complex stress response that leads to desynchronization of metabolic processes. This in turn challenges the system's ability to achieve redox balance due to alterations in metabolic fluxes (redox rewiring). We infer that the emerging 'alternative redox states' do not always revert readily to their evolved natural states; 'Long COVID' and other complex disorders of unknown aetiology are the clinical manifestations of such rearrangements. To better support and successfully manage bodily resilience to major stress and other redox challenges needs a clear perspective on the pattern of the hysteretic response for the interaction between the redox system and the circadian clock. Characterization of this system requires repeated (ideally continuous) recording of relevant clinical measures of the stress responses and whole-body redox state (temporal redox phenotyping). The human/animal body is a complex 'system of systems' with multi-level buffering capabilities, and it requires consideration of the wider dynamic context to identify a limited number of stress-markers suitable for routine clinical decision making. Systematically mapping the patterns and dynamics of redox biomarkers along the stressor/disease trajectory will provide an operational model of whole-body redox regulation/balance that can serve as basis for the identification of effective interventions which promote health by enhancing resilience.

Systems redox biology in health and disease.

Living organisms need to be able to cope with environmental challenges and other stressors and mount adequate responses that are as varied as the spectrum of those challenges. Understanding how the multi-layered biological stress responses become integrated across and between different levels of organization within an organism can provide a different perspective on the nature and inter-relationship of complex systems in health and disease. We here compare two concepts which have been very influential in stress research: Selye's 'General Adaptation Syndrome' and Sies's 'Oxidative Stress' paradigm. We show that both can be embraced within a more general framework of 'change and response'. The 'Reactive Species Interactome' allows each of these to be considered as distinct but complementary aspects of the same system, representative of roles at different levels of organization within a functional hierarchy. The versatile chemistry of sulfur - exemplified by hydrogen sulfide, glutathione and proteinous cysteine thiols - enriched by its interactions with reactive oxygen, nitrogen and sulfur species, would seem to sit at the heart of the 'Redox Code' and underpin the ability of complex organisms to cope with stress.

Electroanalysis at a Single Giant Vesicle Generating Enzymatically a Reactive Oxygen Species.

In the framework of artificial or synthetic cell development, giant liposomes are common basic structures. Their enclosed membrane allows encapsulating proteins, DNA, reactants, etc., while its phospholipid nature allows some exchanges with the surrounding medium. Biochemical reactions induced inside giant liposomes or vesicles are often monitored or imaged by fluorescence microscopy techniques. Here, we show that electrochemistry performed with ultramicroelectrodes is perfectly suitable to monitor an enzymatic reaction occurring in a single giant unilamellar vesicle. Glucose oxidase (GOx) was microinjected inside individual vesicles containing 1 mM glucose. H2O2 was thus generated in the vesicle and progressively diffused across the membrane toward the surrounding environment. An ultramicroelectrode sensitive to H2O2 (black platinum-modified carbon surface) was placed next to the membrane and provided a direct detection of the hydrogen peroxide flux generated by the enzyme activity. Electrochemistry offered a highly sensitive (in situ detection), selective (potential applied at the electrode), time-resolved analysis (chronoamperometry) of the GOx activity over an hour duration, without modifying the internal giant unilamellar vesicles (GUV) medium. These results demonstrate that electroanalysis with microsensors is well adapted and complementary to fluorescence microscopy to sense enzymatic activities, for instance, generating reactive oxygen species, at single vesicles further used to develop artificial cells.

COVID-19: A Redox Disease-What a Stress Pandemic Can Teach Us About Resilience and What We May Learn from the Reactive Species Interactome About Its Treatment.

Significance: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 2019 (COVID-19), affects every aspect of human life by challenging bodily, socioeconomic, and political systems at unprecedented levels. As vaccines become available, their distribution, safety, and efficacy against emerging variants remain uncertain, and specific treatments are lacking. Recent Advances: Initially affecting the lungs, COVID-19 is a complex multisystems disease that disturbs the whole-body redox balance and can be long-lasting (Long-COVID). Numerous risk factors have been identified, but the reasons for variations in susceptibility to infection, disease severity, and outcome are poorly understood. The reactive species interactome (RSI) was recently introduced as a framework to conceptualize how cells and whole organisms sense, integrate, and accommodate stress. Critical Issues: We here consider COVID-19 as a redox disease, offering a holistic perspective of its effects on the human body, considering the vulnerability of complex interconnected systems with multiorgan/multilevel interdependencies. Host/viral glycan interactions underpin SARS-CoV-2's extraordinary efficiency in gaining cellular access, crossing the epithelial/endothelial barrier to spread along the vascular/lymphatic endothelium, and evading antiviral/antioxidant defences. An inflammation-driven "oxidative storm" alters the redox landscape, eliciting epithelial, endothelial, mitochondrial, metabolic, and immune dysfunction, and coagulopathy. Concomitantly reduced nitric oxide availability renders the sulfur-based redox circuitry vulnerable to oxidation, with eventual catastrophic failure in redox communication/regulation. Host nutrient limitations are crucial determinants of resilience at the individual and population level. Future Directions: While inflicting considerable damage to health and well-being, COVID-19 may provide the ultimate testing ground to improve the diagnosis and treatment of redox-related stress diseases. "Redox phenotyping" of patients to characterize whole-body RSI status as the disease progresses may inform new therapeutic approaches to regain redox balance, reduce mortality in COVID-19 and other redox diseases, and provide opportunities to tackle Long-COVID. Antioxid. Redox Signal. 35, 1226-1268.

The Redox architecture of physiological function.

The ability of organisms to accommodate variations in metabolic need and environmental conditions is essential for their survival. However, an explanation is lacking as to how the necessary accommodations in response to these challenges are organized and coordinated from (sub)cellular to higher-level physiological functions, especially in mammals. We propose that the chemistry that enables coordination and synchronization of these processes dates to the origins of Life. We offer a conceptual framework based upon the nature of electron exchange (Redox) processes that co-evolved with biological complexification, giving rise to a multi-layered system in which intra/intercellular and inter-organ exchange processes essential to sensing and adaptation stay fully synchronized. Our analysis explains why Redox is both the lingua franca and the mechanism that enable integration by connecting the various elements of regulatory processes. We here define these interactions across levels of organization as the 'Redox Interactome'. This framework provides novel insight into the chemical and biological basis of Redox signalling and may explain the recent convergence of metabolism, bioenergetics, and inflammation as well as the relationship between Redox stress and human disease.

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases.

Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr ), of dissociation of NO from the ferric heme-NO complex (kd ), and of oxidation of the ferrous heme-NO complex (kox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound FeIII-N(O)OO- intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.

The evolution of nitric oxide signalling diverges between animal and green lineages.

Nitric oxide (NO) is a ubiquitous signalling molecule with widespread distribution in prokaryotes and eukaryotes where it is involved in countless physiological processes. While the mechanisms governing nitric oxide (NO) synthesis and signalling are well established in animals, the situation is less clear in the green lineage. Recent investigations have shown that NO synthase, the major enzymatic source for NO in animals, is absent in land plants but present in a limited number of algae. The first detailed analysis highlighted that these new NO synthases are functional but display specific structural features and probably original catalytic activities. Completing this picture, analyses were undertaken in order to investigate whether major components of the prototypic NO/cyclic GMP signalling cascades mediating many physiological effects of NO in animals were also present in plants. Only a few homologues of soluble guanylate cyclases, cGMP-dependent protein kinases, cyclic nucleotide-gated channels, and cGMP-regulated phosphodiesterases were identified in some algal species and their presence did not correlate with that of NO synthases. In contrast, S-nitrosoglutathione reductase, a critical regulator of S-nitrosothiols, was recurrently found. Overall, these findings highlight that plants do not mediate NO signalling through the classical NO/cGMP signalling module and support the concept that S-nitrosation is a ubiquitous NO-dependent signalling mechanism.

What does "NO-Synthase" stand for ?

Mammalian NO-Synthases (NOSs) are the enzymatic sources of Nitric Oxide (NO°), a paradigmatic gasotransmitter involved in many (patho)-physiological processes. The increasing number of available genomes led to the identification of hundreds of new NOS proteins throughout the kingdoms of life, calling for a global investigation of this family of proteins. These new NOSs are commonly believed to share the same structure, functioning and role as mammalian NOSs. The scope of this article is to highlight the singularity of these NOSs and to describe their complex structural and functional diversity. NOS appears as a unique enzymatic machinery that exhibits a complex Structure - Activity - Function relationship. Its sophisticated redox mechanism and enzymatic regulation, coupled to the vast biological chemistry of reactive nitrogen species, leads to a specific cross-talk between NOS catalysis and its biological environment that implies a complex evolution of NOS function. This paper addresses the relationship between structure, function and evolution of NOS proteins using three NOS model families and advocates for an integrative and interdisciplinary approach that combines modelling studies, structural characterization, and in vitro/in vivo functional investigations.

The NOS-like protein from the microalgae Ostreococcus tauri is a genuine and ultrafast NO-producing enzyme.

The exponential increase of genomes' sequencing has revealed the presence of NO-Synthases (NOS) throughout the tree of life, uncovering an extraordinary diversity of genetic structure and biological functions. Although NO has been shown to be a crucial mediator in plant physiology, NOS sequences seem present solely in green algae genomes, with a first identification in the picoplankton species Ostreococcus tauri. There is no rationale so far to account for the presence of NOS in this early-diverging branch of the green lineage and its absence in land plants. To address the biological function of algae NOS, we cloned, expressed and characterized the NOS oxygenase domain from Ostreococcus tauri (OtNOSoxy). We launched a phylogenetic and structural analysis of algae NOS, and achieved a 3D model of OtNOSoxy by homology modeling. We used a combination of various spectroscopies to characterize the structural and electronic fingerprints of some OtNOSoxy reaction intermediates. The analysis of OtNOSoxy catalytic activity and kinetic efficiency was achieved by stoichiometric stopped-flow. Our results highlight the conserved and particular features of OtNOSoxy structure that might explain its ultrafast NO-producing capacity. This integrative Structure-Catalysis-Function approach could be extended to the whole NOS superfamily and used for predicting potential biological activity for any new NOS.

Revisiting the Val/Ile Mutation in Mammalian and Bacterial Nitric Oxide Synthases: A Spectroscopic and Kinetic Study.

Nitric oxide is produced in mammals by the nitric oxide synthase (NOS) isoforms at a catalytic site comprising a heme associated with a biopterin cofactor. Through genome sequencing, proteins that are highly homologous to the oxygenase domain of NOSs have been identified, in particular in bacteria. The active site is highly conserved except for a valine residue in the distal pocket that is replaced with an isoleucine in bacteria. This switch was previously reported to influence the kinetics of the reaction. We have used the V346I mutant of the mouse inducible NOS (iNOS) as well as the I224V mutant of the NOS from Bacillus subtilis (bsNOS) to study their spectroscopic signatures in solution and look for potential structural differences compared to their respective wild types. Both mutants seem destabilized in the absence of substrate and cofactor. When both substrate and cofactor are present, small differences can be detected with Nω-hydroxy-l-arginine compared to arginine, which is likely due to the differences in the hydrogen bonding network of the distal pocket. Stopped-flow experiments evidence significant changes in the kinetics of the reaction due to the mutation as was already known. We found these effects particularly marked for iNOS. On the basis of these results, we performed rapid freeze-quench experiments to trap the biopterin radical and found the same results that we had obtained for the wild types. Despite differences in kinetics, a radical could be trapped in both steps for the iNOS mutant but only for the first step in the mutant of bsNOS. This strengthens the hypothesis that mammalian and bacterial NOSs may have a different mechanism during the second catalytic step.

Nitric oxide synthase in plants: Where do we stand?

Over the past twenty years, nitric oxide (NO) has emerged as an important player in various plant physiological processes. Although many advances in the understanding of NO functions have been made, the question of how NO is produced in plants is still challenging. It is now generally accepted that the endogenous production of NO is mainly accomplished through the reduction of nitrite via both enzymatic and non-enzymatic mechanisms which remain to be fully characterized. Furthermore, experimental arguments in favour of the existence of plant nitric oxide synthase (NOS)-like enzymes have been reported. However, recent investigations revealed that land plants do not possess animal NOS-like enzymes while few algal species do. Phylogenetic and structural analyses reveals interesting features specific to algal NOS-like proteins.

Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases.

The activation of the peroxynitrite anion (PN) by hemoproteins, which leads to its detoxification or, on the contrary to the enhancement of its cytotoxic activity, is a reaction of physiological importance that is still poorly understood. It has been known for some years that the reaction of hemoproteins, notably cytochrome P450, with PN leads to the buildup of an intermediate species with a Soret band at ∼435 nm (I435). The nature of this intermediate is, however, debated. On the one hand, I435 has been presented as a compound II species that can be photoactivated to compound I. A competing alternative involves the assignment of I435 to a ferric-nitrosyl species. Similar to cytochromes P450, the buildup of I435 occurs in nitric oxide synthases (NOSs) upon their reaction with excess PN. Interestingly, the NOS isoforms vary in their capacity to detoxify/activate PN, although they all show the buildup of I435. To better understand PN activation/detoxification by heme proteins, a definitive assignment of I435 is needed. Here we used a combination of fine kinetic analysis under specific conditions (pH, PN concentrations, and PN/NOSs ratios) to probe the formation of I435. These studies revealed that I435 is not formed upon homolytic cleavage of the O-O bond of PN, but instead arises from side reactions associated with excess PN. Characterization of I435 by resonance Raman spectroscopy allowed its identification as a ferric iron-nitrosyl complex. Our study indicates that the model used so far to depict PN interactions with hemo-thiolate proteins, i.e., leading to the formation and accumulation of compound II, needs to be reconsidered.

Oxygen activation in NO synthases: evidence for a direct role of the substrate.

Nitric oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho-physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast-kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise Fe(II)-O2 reaction intermediates in the presence of L-arginine (Arg), alkyl- and aryl-guanidines. The presence of new reaction intermediates, such as ferric haem-peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H-bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (N(ω)-hydroxy-L-arginine oxidation).

Analysis of the Expression and Activity of Nitric Oxide Synthase from Marine Photosynthetic Microorganisms.

Nitric oxide (NO) functions as a signaling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of L-arginine to NO and L-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported in 2010. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage.In this chapter we describe detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis of spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of its activity or the use of fluorescent probes and Griess assay for in vivo NO determination. We further discuss the advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organisms that can generate misunderstandings in the interpretation of results.

A heme-binding domain controls regulation of ATP-dependent potassium channels.

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

Redox Control of the Human Iron-Sulfur Repair Protein MitoNEET Activity via Its Iron-Sulfur Cluster.

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, mNT has been implicated in cytosolic Fe-S repair of a key regulator of cellular iron homeostasis. Here, we aimed to decipher the mechanism by which mNT triggers its Fe-S repair capacity. By using tightly controlled reactions combined with complementary spectroscopic approaches, we have determined the differential roles played by both the redox state of the mNT cluster and dioxygen in cluster transfer and protein stability. We unambiguously demonstrated that only the oxidized state of the mNT cluster triggers cluster transfer to a generic acceptor protein and that dioxygen is neither required for the cluster transfer reaction nor does it affect the transfer rate. In the absence of apo-acceptors, a large fraction of the oxidized holo-mNT form is converted back to reduced holo-mNT under low oxygen tension. Reduced holo-mNT, which holds a [2Fe-2S](+)with a global protein fold similar to that of the oxidized form is, by contrast, resistant in losing its cluster or in transferring it. Our findings thus demonstrate that mNT uses an iron-based redox switch mechanism to regulate the transfer of its cluster. The oxidized state is the "active state," which reacts promptly to initiate Fe-S transfer independently of dioxygen, whereas the reduced state is a "dormant form." Finally, we propose that the redox-sensing function of mNT is a key component of the cellular adaptive response to help stress-sensitive Fe-S proteins recover from oxidative injury.

EPR characterisation of the ferrous nitrosyl complex formed within the oxygenase domain of NO synthase.

Nitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form.

Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.