Thwarted Belonging and Perceived Burdensomeness During Middle and Older Adulthood: The Role of Generativity.

Using a sample of middle-aged and older adults, this research explores associations between generativity and two key risk factors for suicide: thwarted belonging (T.B.) and perceived burdensomeness (P.B.). These variables are typically studied as predictors of suicide; the current study is unique in examining their psychosocial correlates. Erikson described, generativity as a psychosocial construct that characterizes adult well-being in mid-life, conceptualized as the sense one has successfully guided and contributed to the younger generation through mentoring. Using the Midlife in the United States Survey (MIDUS), the current analyses indicate that generativity is associated with lower levels of P.B. and T.B., even after accounting for measures of hopelessness, depressive symptoms, financial stability, perceived neighborhood quality, chronic health conditions, and respondent's demographic characteristics including gender and age. Results are discussed in terms of applications for suicide-risk prevention, and with regard to the promotion of positive psychosocial development across the lifespan.

Developmental Changes in the Relation between Youth Disclosure and Parenting Behavior: A Cohort-sequential Analysis.

To address a gap in the literature regarding the development of youth disclosure across the transition to adolescence, the current research uses a cohort-sequential approach to study youth disclosure from middle childhood through adolescence. Longitudinal data from three cohorts of parents were utilized (N = 1359; children at T1 were in grades 2 [M = 8.00 years, SD = 0.57 years, 45% female], 4 [M = 10.12 years, SD = 0.60 years, 45% female], and 9 [M = 15.19 years, SD = 0.57 years, 48% female]). Parents were assessed annually over a 3-year time period. The focal analyses explored contemporaneous associations between characteristics of the parent-youth relationship (specifically, parental rejection and parental consistent discipline) and youth disclosure after accounting for person-specific trajectories of disclosure. Associations of gender, age, and socioeconomic status with disclosure were also assessed. Regarding trajectories of youth disclosure, results indicate that youth disclose less information to their parents about their daily lives as they get older; this trend was consistent across gender and socioeconomic status. In terms of associations with youth disclosure, when parents provided more consistent discipline or engaged in less rejection of their child, youth disclosure increased, even after accounting for their own trajectory of disclosure across time. In addition, the association of consistent discipline with youth disclosure became stronger with increased youth age. Results are discussed in terms of implications for understanding youth autonomy development, and the dyadic and developmental impact of parenting behaviors over time.

"People Think It's Easy Because I Smile, But It's Not Easy": The Lived Experiences of Six African American Single Mothers.

Grounded in Resilience Theory (Masten et al., 1990; Walsh, 1996; Walsh, 2002; Walsh, 2003) with a specific focus on parental resilience (Gavidia-Payne et al., 2015), this qualitative phenomenological study explored the lived experiences of low-income Female adult single parent (FASP) families. Each family had at least one adolescent aged 11 through 21 enrolled in special education classes and engaging in risk behaviors. Two central research questions were addressed. The first focused on the lived experiences of FASP and the second focused on their perspectives on how schools can enhance their support. Data were collected through face-to-face, in-depth, semi-structured interviews. Three major themes emerged: Life adjustment, The child is the priority, and Perseverance revealing their experience to be one of resilience. Participants also shared and provided insight on their perspectives regarding how schools can better support families. Findings revealed limited parent-school collaboration and highlight the need for further research with this population in the context of resilience. Suggestions for schools and communities working with such families are discussed.

The nitroxide/antioxidant 3-carbamoyl proxyl attenuates disease severity in murine models of severe asthma.

Asthma is characterized by airway inflammation, hyper-responsiveness, symptoms of dyspnea, wheezing and coughing. In most patients, asthma is well controlled using inhaled corticosteroids and bronchodilators. A minority of patients with asthma develop severe disease, which is frequently only partially responsive or even resistant to treatment with corticosteroids. Severe refractory asthma is associated with structural changes in the airways, termed "airway remodeling", and/or with neutrophilic rather than eosinophilic airway inflammation. While oxidative stress plays an important role in the pathophysiology of asthma, cyclic nitroxide stable radicals, which are unique and efficient catalytic antioxidants, effectively protect against oxidative injury. We have demonstrated that the nitroxide 3-carbamoyl proxyl (3-CP) attenuates airway inflammation and hyperresponsiveness in allergic asthma as well as bleomycin-induced fibrosis both using murine models, most probably through modulation of oxidative stress. The present study evaluates the effect of 3-CP on airway inflammation and remodeling using two murine models of severe asthma where mice are sensitized and challenged either by ovalbumin (OVA) or by house dust mite (HDM). 3-CP was orally administered during the entire period of the experiment or during the challenge period alone where its effect was compared to that of dexamethasone. The induced increase by OVA and by HDM of BALf cell counts, airway hyperresponsiveness, fibrosis, transforming growth factor-beta (TGF-ß) levels in BALf and protein nitration levels of the lung tissue was significantly reduced by 3-CP. The effect of 3-CP, using two different murine models of severe asthma, is associated at least partially with attenuation of oxidative stress and with TGF-ß expression in the lungs. The results of this study suggest a potential use of 3-CP as a novel therapeutic agent in different forms of severe asthma.

3-Carbamoyl-proxyl nitroxide radicals attenuate bleomycin-induced pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with a poor prognosis and limited treatment options. Oxidative and nitrosative stress is implicated as one of the main pathogenic pathways in IPF. The rationale for the use of antioxidants to treat lung fibrosis is appealing, however to date a consistent beneficial effect for such an approach has not been observed. We have recently demonstrated that nitroxides, particularly 3-carbamoyl-proxyl (3-CP), markedly reduce airway inflammation, airway hyper-responsiveness, and protein nitration of the lung tissue in a mouse model of ovalbumin-induced acute asthma, thus prompting its use for the treatment of IPF. The present study investigates the effect of 3-CP on the development of lung fibrosis using the murine intratracheal bleomycin model. 3-CP was administered either intranasally or orally during the entire experiment or starting 7 days after induction of the lung injury. 3-CP was found to be both a preventive and a therapeutic drug reducing the lung fibrosis (histological score), the increase in collagen content, protein nitration, TGF-ß levels, the degree of weight loss as well as inhibiting the impairment of lung function. Nitroxides are catalytic antioxidants that preferentially detoxify radicals, and therefore the effect of 3-CP on the severity of the disease supports the involvement of reactive oxygen and nitrogen species in the disease pathology.

Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited.

There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO-) and superoxide (HO2•/O2•-). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely •OH and •NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO-. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH and •NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.

Sibling Adjustment to Childhood Chronic Illness: An Integrative Review.

Siblings of children with a chronic illness are among the close family members impacted by childhood chronic illness. Family roles, routines, and functioning are adjusted to fit the needs of the child and family throughout the course of the illness. Empirical articles regarding adjustment of healthy siblings of children with chronic illnesses ages 0 to 17 years, published in 2000 to 2019 were searched on PsycInfo, PubMed, and Medline using the keywords "sibling adjustment," "sibling chronic," and "sibling illness." Through thematic synthesis, four broad topics emerged concerning sibling adjustment to chronic illness: (a) experiences, (b) psychosocial adjustment, (c) coping, and (d) communication. Implications for family nursing and family health practitioners and future research directions are presented.

Hydroxylamines inhibit tyrosine oxidation and nitration: The role of their respective nitroxide radicals.

In vivo, nitroxide antioxidants distribute within minutes throughout all tissues, but are reduced to their respective hydroxylamines due to the cellular reducing environment, which apparently limits their application. To distinguish their antioxidative activity from that of their respective nitroxides, the kinetics and mechanism of their inhibitory effect on the enzymatic oxidation and nitration of tyrosine have been studied. The inhibitory effect of the hydroxylamines on the oxidation and nitration of tyrosine induced by HRP/H2O2 and HRP/H2O2/nitrite was investigated by following the kinetics of the formation of their respective nitroxides, H2O2 decomposition, release of O2 and accumulation of tyrosine oxidation and nitration products. The distinction between the antioxidative activities of nitroxides and of their respective hydroxylamines is hindered due to oxidation of hydroxylamines to nitroxides, which catalytically inhibit tyrosine oxidation and nitration. The results demonstrate that (i) hydroxylamines inhibit tyrosine oxidation and nitration and their inhibitory effect increases as the reduction potential of their respective nitroxides decreases; (ii) the 6-membered ring hydroxylamines are more effective antioxidants than the 5-membered hydroxylamine derived from 3-carbamoyl proxyl and (iii) the 6-membered ring hydroxylamines are as effective antioxidants as their respective nitroxides, whereas the 3-carbamoyl proxyl is even a weaker antioxidant than its respective hydroxylamine. In general, cyclic hydroxylamines are more effective antioxidants than common antioxidants such as ascorbic and uric acids, which are depleted giving rise to secondary radicals that, might be toxic. In the case of hydroxylamines, the secondary radicals are their respective nitroxides, which are efficient catalytic antioxidants.

Sources of social support and gender in perceived stress and individual adjustment among Latina/o college-attending emerging adults.

OBJECTIVES: We explored the role of particular sources of social support (friends, romantic partners, family) as moderators and mediators in the associations between perceived stress and individual well-being (loneliness, depressive symptoms, and self-rated physical health). We also tested the possible moderating effect of gender to ascertain whether women and men are differentially impacted by social support's diverse sources. METHOD: Participants were 163 Latina/o emerging adults attending college (85% women; Mage = 20.2 years, range: 18-25). RESULTS: Holding perceived stress constant, friend support was negatively associated with loneliness, romantic partner support was negatively related to depressive symptoms, and family support was positively associated with self-rated physical health. Friend or romantic partner support moderated the relationships between perceived stress and loneliness, and self-rated physical health, but not depressive symptoms. Perceived stress and loneliness were indirectly and positively associated through lower friends and romantic partner supports, perceived stress and depressive symptoms were indirectly and positively related through lower romantic partner support, and perceived stress and self-rated physical health were related indirectly and negatively through lower family support. Gender moderated the relationships between family and friend support and self-rated physical health, and between friend support and depressive symptoms. Particular sources of support mediated the associations of perceived stress with well-being. CONCLUSIONS: Results highlight how social support helps Latina/o youth cope with stress and mitigate challenges associated with their college transition. Social support implications for physical and psychological health differ for male and female Latina/o college-attending emerging adults. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

The Impact of Widely Publicized Suicides on Search Trends: Using Google Trends to Test the Werther and Papageno Effects.

The objective of this study was to examine the impact of widely publicized suicides on the Werther and Papageno Effects using internet search trends. A list of widely publicized suicides from 2010 through 2018 was compiled along with dates of death for each of these individuals. Google.com/trends data were then collected for searches for "how to suicide" and "suicide prevention" for 14 days prior to a widely publicized suicide/14 days after a widely publicized suicide and 7 days prior to a widely publicized suicide/7 days after a widely publicized suicide. Comparisons were then made between these time periods for "how to suicide" and "suicide prevention." Some celebrities, such as Robin Williams (2014) and Aaron Hernandez (2017) were associated with increased searches. However, for many there was no increase in search trends. Limited support was found for the impact of widely publicized suicides on internet search trends with one case supporting a Werther Effect and one case supporting a Papageno Effect. The finding that only some celebrities were associated with increased searches may be a byproduct of the impact of celebrity status on these effects, with more prominent celebrities having the greatest impact.

Mechanistic insight into the catalytic inhibition by nitroxides of tyrosine oxidation and nitration.

BACKGROUND: Nitroxide antioxidants (RNO•) protect from injuries associated with oxidative stress. Tyrosine residues in proteins are major targets for oxidizing species giving rise to irreversible cross-linking and protein nitration, but the mechanisms underlying the protective activity of RNO• on these processes are not sufficiently clear. METHODS: Tyrosine oxidation by the oxoammonium cation (RN+=O) was studied by following the kinetics of RNO• formation using EPR spectroscopy. Tyrosine oxidation and nitration were investigated using the peroxidase/H2O2 system without and with nitrite. The inhibitory effect of RNO• on these processes was studied by following the kinetics of the evolved O2 and accumulation of tyrosine oxidation and nitration products. RESULTS: Tyrosine ion is readily oxidized by RN+=O, and the equilibrium constant of this reaction depends on RNO• structure and reduction potential. RNO• catalytically inhibits tyrosine oxidation and nitration since it scavenges both tyrosyl and •NO2 radicals while recycling through RN+=O reduction by H2O2, tyrosine and nitrite. The inhibitory effect of nitroxide on tyrosine oxidation and nitration increases as its reduction potential decreases where the 6-membered ring nitroxides are better catalysts than the 5-membered ones. CONCLUSIONS: Nitroxides catalytically inhibit tyrosine oxidation and nitration. The proposed reaction mechanism adequately fits the results explaining the dependence of the nitroxide inhibitory effect on its reduction potential and on the concentrations of the reducing species present in the system. GENERAL SIGNIFICANCE: Nitroxides protect against both oxidative and nitrative damage. The proposed reaction mechanism further emphasizes the role of the reducing environment to the efficacy of these catalysts.

Carbon dioxide-catalyzed peroxynitrite reactivity - The resilience of the radical mechanism after two decades of research.

Peroxynitrite, ONOO-, formed in tissues that are simultaneously generating NO• and O2•-, is widely regarded as a major contributor to oxidative stress. Many of the reactions involved are catalyzed by CO2 via formation of an unstable adduct, ONOOC(O)O-, that undergoes O-O bond homolysis to produce NO2• and CO3•- radicals, whose yields are equal at about 0.33 with respect to the ONOO- reactant. Since its inception two decades ago, this radical-based mechanism has been frequently but unsuccessfully challenged. The most recent among these [Serrano-Luginbuehl et al. Chem. Res. Toxicol.31:721-730; 2018] claims that ONOOC(O)O- is stable, predicts a yield of NO2•/CO3•- of less than 0.01 under physiological conditions and, contrary to widely accepted viewpoints, suggests that radical generation is inconsequential to peroxynitrite-induced oxidative damage. Here we review the experimental and theoretical evidence that support the radical model and show this recently proposed alternative mechanism to be incorrect.

Direct Observation of Acyl Nitroso Compounds in Aqueous Solution and the Kinetics of Their Reactions with Amines, Thiols, and Hydroxamic Acids.

Acyl nitroso compounds or nitrosocarhonyls (RC(O)N═O) are reactive short-lived electrophiles, and their hydrolysis and reactions with nucleophiles produce HNO. Previously, direct detection of acyl nitroso species in nonaqueous media has been provided by time-resolved infrared spectroscopy demonstrating that its half-life is about 1 ms. In the present study hydroxamic acids (RC(O)NHOH) are oxidized electrochemically in buffered aqueous solutions (pH 5.9-10.2) yielding transient species characterized by their maximal absorption at 314-330 nm. These transient species decompose via a first-order reaction yielding mainly HNO and the respective carboxylic acid and therefore are ascribed to RC(O)N═O. The sufficiently long half-life of RC(O)N═O in aqueous solution allows for the first time the study of the kinetics of its reactions with various nucleophiles demonstrating that the nucleophilic reactivity follows the order thiolate > hydroxamate > amine. Metal chelates of CH3C(O)NHOH catalyze the hydrolysis of CH3C(O)N═O at the efficacy order of CuII > ZnII > NiII > CoII where only CuII catalyzes the hydrolysis also in the absence of the hydroxamate. Finally, oxidation of hydroxamic acids generates HNO, and the rate of this process is determined by the half-life of the respective acyl nitroso compound.

Design, Synthesis, and Biological Evaluation of Allosteric Effectors That Enhance CO Release from Carboxyhemoglobin.

Carbon monoxide (CO) poisoning causes between 5,000-6,000 deaths per year in the US alone. The development of small molecule allosteric effectors of CO binding to hemoglobin (Hb) represents an important step toward making effective therapies for CO poisoning. To that end, we have found that the synthetic peptide IRL 2500 enhances CO release from COHb in air, but with concomitant hemolytic activity. We describe herein the design, synthesis, and biological evaluation of analogs of IRL 2500 that enhance the release of CO from COHb without hemolysis. These novel structures show improved aqueous solubility and reduced hemolytic activity and could lead the way to the development of small molecule therapeutics for the treatment of CO poisoning.

Nitrogen Dioxide Reaction with Nitroxide Radical Derived from Hydroxamic Acids: The Intermediacy of Acyl Nitroso and Nitroxyl (HNO).

Hydroxamic acids (RC(O)NHOH) form a class of compounds that display interesting chemical and biological properties The chemistry of RC(O)NHOH) is associated with one- and two-electron oxidations forming the respective nitroxide radical (RC(O)NHO•) and acyl nitroso (RC(O)N═O), respectively, which are relatively unstable species. In the present study, the kinetics and mechanism of the •NO2 reaction with nitroxide radicals derived from acetohydroxamic acid, suberohydroxamic acid, benzohydroxamic acid, and suberoylanilide hydroxamic acid have been studied in alkaline solutions. Ionizing radiation was used to generate about equal yields of these radicals, demonstrating that the oxidation of the transient nitroxide radical by •NO2 produces HNO and nitrite at about equal yields. The rate constant of •NO2 reaction with the nitroxide radical derived from acetohydroxamic acid has been determined to be (2.5 ± 0.5) × 109 M-1 s-1. This reaction forms a transient intermediate absorbing at 314 nm, which decays via a first-order reaction whose rate increases upon increasing the pH or the hydroxamic acid concentration. Transient intermediates absorbing around 314 nm are also formed during the oxidation of hydroxamic acids by H2O2 catalyzed by horseradish peroxidase. It is shown that HNO is formed during the decomposition of these intermediates, and therefore, they are assigned to acyl nitroso compounds. This study provides for the first time a direct spectrophotometric detection of acyl nitroso compounds in aqueous solutions allowing the study of their chemistry and reaction kinetics.

A longitudinal examination of social connectedness and suicidal thoughts and behaviors among adolescents.

BACKGROUND: This study examines the relationship between three different types of social connectivity and suicidal thoughts and behaviors. METHODS: Using the Add Health dataset, three domains of social connection were explored: parental connection, school connection, and social integration. Logistic regression was used to examine whether changes over time in connectedness predicted suicidal thoughts and behavior. RESULTS: Youth whose difference scores on social integration and parental connectedness increased were less likely to experience suicidal ideation. Increases in difference scores for perceived school connectedness protected youth who reported ideation from engaging in a suicide attempt. CONCLUSIONS: Perceptions of social connection are key factors in understanding adolescent suicidal thoughts and behaviors. It is important to consider social connection across different relationship contexts.

Mechanism of HRP-catalyzed nitrite oxidation by H2O2 revisited: Effect of nitroxides on enzyme inactivation and its catalytic activity.

The peroxidative activity of horseradish peroxidase (HRP) undergoes progressive inactivation while catalyzing the oxidation of nitrite by H2O2. The extent of inactivation increases as the pH increases, [nitrite] decreases or [H2O2] increases, and is accompanied by a loss of the Soret peak of HRP along with yellow-greenish coloration of the solution. HRP-catalyzed nitrite oxidation by H2O2 involves not only the formation of compounds I and II as transient heme species, but also compound III, all of which in turn, oxidize nitrite yielding •NO2. The rate constant of nitrite oxidation by compound III is at least 10-fold higher than that by compound II, which is also reducible by •NO2 where its reduction by nitrite is the rate-determining step of the catalytic cycle. The extent of the loss of the Soret peak of HRP is lower than the loss of its peroxidative activity implying that deterioration of the heme moiety leading to iron release only partially contributes toward heme inactivation. Cyclic stable nitroxide radicals, such as 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO), 4-OH-TPO and 4-NH2-TPO at µM concentrations detoxify •NO2 thus protecting HRP against inactivation mediated by this radical. Hence, HRP inactivation proceeds via nitration of the porphyrin ring most probably through compound I reaction with •NO2, which partially leads to deterioration of the heme moiety. The nitroxide acts catalytically since its oxidation by •NO2 yields the respective oxoammonium cation, which is readily reduced back to the nitroxide by H2O2, superoxide ion radical, and nitrite. In addition, the nitroxide catalytically inhibits tyrosine nitration mediated by HRP/H2O2/nitrite reactions system as it efficiently competes with tyrosyl radical for •NO2. The inhibition by nitroxides of tyrosine nitration is demonstrated also in the case of microperoxidase (MP-11) and cytochrome c revealing an additional role played by nitroxide antioxidants.

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity.

BACKGROUND: Horseradish peroxidase (HRP) catalyzes H2O2 dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H2O2-induced inactivation, have been investigated. METHODS: HRP reaction with H2O2 was studied by following H2O2 depletion, O2 evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow. RESULTS: Nitroxide protects HRP against H2O2-induced inactivation. The rate of H2O2 dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H2O2. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO)>4-OH-TPO>3-carbamoyl proxyl>4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III. CONCLUSIONS: Nitroxide catalytically protects HRP against inactivation induced by H2O2 while modulating its catalase-like activity. The protective role of nitroxide at µM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex. GENERAL SIGNIFICANCE: Nitroxides catalytically protect heme proteins against inactivation induced by H2O2 revealing an additional role played by nitroxide antioxidants in vivo.

Nitroxides catalytically inhibit nitrite oxidation and heme inactivation induced by H2O2, nitrite and metmyoglobin or methemoglobin.

Stable nitroxide radicals have multiple biological effects, although the mechanisms underlying them are not fully understood. Their protective effect against oxidative damage has been mainly attributed to scavenging deleterious radicals, oxidizing reduced metal ions and reducing oxyferryl centers of heme proteins. Yet, the potential of nitroxides to protect heme proteins against inactivation while suppressing or enhancing their catalytic activities has been largely overlooked. We have studied the effect of nitroxides, including TPO (2,2,6,6-tetramethylpiperidin-N-oxyl), 4-OH-TPO, 4-oxo-TPO and 3-carbamoyl proxyl, on the peroxidase-like activity of metmyoglobin (MbFeIII) and methemoglobin (HbFeIII) using nitrite as an electron donor by following heme absorption, H2O2 consumption, O2 evolution and nitrite oxidation. The results demonstrate that the peroxidase-like activity is accompanied by a progressive heme inactivation where MbFeIII is far more resistant than HbFeIII. Nitroxides convert the peroxidase-like activity into catalase-like activity while inhibiting heme inactivation and nitrite oxidation in a dose-dependent manner. The nitroxide facilitates H2O2 dismutation, yet none of its reactions with any of the intermediates formed in these systems is rate-determining, and therefore its effect on the rate of the catalysis is hardly dependent on the kind of the nitroxide derivative and its concentration. The nitroxide at µM concentrations range catalytically inhibits nitrite oxidation, and consequently prevents tyrosine nitration induced by heme protein/H2O2/nitrite due to its fast oxidation by •NO2 forming the respective oxoammonium cation, which is reduced back to the nitroxide by H2O2 and by superoxide radical. The nitroxides are superior over common antioxidants, which their reaction with •NO2 always yields secondary radicals leading eventually to consumption of the antioxidant. A mechanism is proposed, and the kinetic simulations fit very well the experimental data in the case of MbFeIII where most of the rate constants of the reactions involved are independently known.

Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and "suicide inactivation" of metmyoglobin.

BACKGROUND: Metmyoglobin (MbFe(III)) reaction with H(2)O(2) has been a subject of study over many years. H(2)O(2) alone promotes heme destruction frequently denoted "suicide inactivation," yet the mechanism underlying H(2)O(2) dismutation associated with MbFe(III) inactivation remains obscure. METHODS: MbFe(III) reaction with excess H(2)O(2) in the absence and presence of the nitroxide was studied at pH 5.3-8.1 and 25°C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H(2)O(2) depletion, O(2) evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy. RESULTS: The rates of both H(2)O(2) dismutation and heme inactivation processes depend on [MbFe(III)], [H(2)O(2)] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe(III) molecule catalyzes the dismutation of 50±10 H(2)O(2) molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe(III) while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe(IV)O by H(2)O(2) and by nitroxide, respectively. CONCLUSIONS: The nitroxide effects on H(2)O(2) dismutation catalyzed by MbFe(III) demonstrate that MbFe(IV)O reduction by H(2)O(2) is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I-H(2)O(2) adduct, which decomposes to a MbFe(IV)O and an inactivated heme at a ratio of 25:1. GENERAL SIGNIFICANCE: The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.