Horacio Botti

Factors affecting protein thiol reactivity and specificity in peroxide reduction.

Protein thiol reactivity generally involves the nucleophilic attack of the thiolate on an electrophile. A low pK(a) means higher availability of the thiolate at neutral pH but often a lower nucleophilicity. Protein structural factors contribute to increasing the reactivity of the thiol in very specific reactions, but these factors do not provide an indiscriminate augmentation in general reactivity. Notably, reduction of hydroperoxides by the catalytic cysteine of peroxiredoxins can achieve extraordinary reaction rates relative to free cysteine. The discussion of this catalytic efficiency has centered in the stabilization of the thiolate as a way to increase nucleophilicity. Such stabilization originates from electrostatic and polar interactions of the catalytic cysteine with the protein environment. We propose that the set of interactions is better described as a means of stabilizing the anionic transition state of the reaction. The enhanced acidity of the critical cysteine is concurrent but not the cause of catalytic efficiency. Protein stabilization of the transition state is achieved by (a) a relatively static charge distribution around the cysteine that includes a conserved arginine and the N-terminus of an α-helix providing a cationic environment that stabilizes the reacting thiolate, the transition state, and also the anionic leaving group; (b) a dynamic set of polar interactions that stabilize the thiolate in the resting enzyme and contribute to restraining its reactivity in the absence of substrate; but upon peroxide binding these active/binding site groups switch interactions from thiolate to peroxide oxygens, simultaneously increasing the nucleophilicity of the attacking sulfur and facilitating the correct positioning of the substrate. The switching of polar interaction provides further acceleration and, importantly, confers specificity to the thiol reactivity. The extraordinary thiol reactivity and specificity toward H(2)O(2) combined with their ubiquity and abundance place peroxiredoxins, along with glutathione peroxidases, as obligate hydroperoxide cellular sensors.

Sulfenic acid in human serum albumin

Summary.Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.

Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogens antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhpE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Oxidation of the albumin thiol to sulfenic acid and its implications in the intravascular compartment

Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80% of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25% of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.

Septic diaphragmatic dysfunction is prevented by Mn(III)porphyrin therapy and inducible nitric oxide synthase inhibition

ObjectiveDecreased diaphragmatic contractility and organ failure observed during sepsis is mediated by an overproduction of nitric oxide (.NO)-derived species, mitochondria being a major target of oxidative and nitrative stress. We tested the potential protective effects of (a) a novel synthetic antioxidant, the manganese(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP5+) and (b) the inducible .NO synthase inhibitor aminoguanidine (AG) on a rat model of sepsis.SettingUniversity research laboratories.Subjects and interventionsSepsis was induced by cecal ligation and perforation in rats.Measurements and resultsSystemic hemodynamics, pulmonary gas exchange, in vitro diaphragmatic function and mitochondrial respiration were evaluated. Moreover, plasma and mitochondrial oxidative and nitrative stress parameters were investigated. Sepsis determined diaphragmatic dysfunction and a significant decrease in mitochondrial coupling and respiration. Oxidative stress was evidenced by decreased plasma antioxidants and increased lipid oxidation. Tyrosine nitration was increased in the plasma and mitochondria of the septic animals. These alterations were ameliorated or prevented by either MnTE-2-PyP5+ or AG.ConclusionsOur results demonstrate that overproduction of .NO and .NO-derived reactive species play a critical role in mitochondrial impairment and diaphragmatic function during sepsis. More importantly, AG but mainly the novel metalloporphyrin MnTE-2-PyP5+ were able to ameliorate diaphragmatic and mitochondrial dysfunction and could contribute to preventing organ failure during severe sepsis.

Interactions of nitric oxide and peroxynitrite with low-density lipoprotein.

Abstract Nitric oxide (NO) is a free radical species that diffuses and concentrates in the hydrophobic core of lowdensity lipoprotein (LDL) to serve as a potent inhibitor of lipid oxidation processes. Peroxynitrite (PN), the product of the diffusionlimited reaction between NO and superoxide (O2), represents a relevant mediator of oxidative modifications in LDL. The focus of this review is the analysis of interactions between NO and PN and its secondary reactions with oxygen radicals on LDL oxidation, which are relevant in the development of the early steps as well as progression of atherosclerosis. We propose that the balance between rates of PN and NO production, which greatly depends on oxidative stress processes within the vascular wall, will critically determine the final extent of oxidative LDL modifications leading or not to scavenger receptormediated LDL uptake and foam cell formation.

Discovery of novel inhibitors of Trypanosoma cruzi trans-sialidase from in silico screening

trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC(50) values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.

Sulfenic acid—A key intermediate in albumin thiol oxidation

The single thiol of human serum albumin (HSA-SH) is the predominant plasma thiol. Both circulating albumin and pharmaceutical preparations are heterogeneous regarding the thiol redox status, as revealed by anion-exchange-hydrophobic interaction chromatography. Sulfenic acid (HSA-SOH) is an intermediate in HSA-SH oxidation processes that was detected through different techniques including mass spectrometry. Recently, quantitative data led to the determination of rate constants. The preferred fate of HSA-SOH is the formation of mixed disulfides. Alternatively, HSA-SOH can be further oxidized to sulfinic and sulfonic acids. Oxidized forms increase under disease conditions, underscoring the importance of HSA-SH as a plasma scavenger of intravascular oxidants. We here provide a critical review of the oxidation of HSA-SH in the context of the intravascular compartment, with emphasis in the methodological approaches of mass spectrometry and chromatography for the analysis of albumin thiol redox states.

Structural and enzymatic insights into the ATP-binding and autophosphorylation mechanism of a sensor histidine kinase

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.

Reactivity of peroxynitrite and nitric oxide with LDL.

Low density lipoprotein (LDL) oxidation by peroxynitrite is a complex process, finely modulated by control of peroxynitrite formation, LDL availability and free‐radical scavenging by nitric oxide (·NO), ascorbate and α‐tocopherol (α‐TOH). In the presence of CO2, lipid targets are spared at the expense of surface constituents. Since surface damage may lead to oxidation‐induced LDL aggregation and particle recognition by scavenger receptors, CO2 cannot be considered an inhibitor of peroxynitrite‐dependent LDL modifications. Chromanols, urate and ascorbate cannot scavenge peroxynitrite in the vasculature, although intermediates of urate oxidation and high ascorbate concentrations may do so in vitro. Most if not all of the protection against peroxynitrite‐induced LDL oxidation afforded by urate, ascorbate, chromanols and also ·NO should be considered to depend on their free radical scavenging abilities, including inactivation of lipid peroxyl radicals (LOO), ·NO2, and CO3·‐; as well as their capacity to reduce high oxidation states of metal centers. Peroxynitrite direct interception by reduced manganese (II) porphyrins is possibly the most powerful although unspecific strategy to inhibit peroxynitrite reactions. In light of the recent demonstration of nitrated bioactive lipids in vivo, renewed interest in the mechanisms of peroxynitrite‐ and nitric oxide‐mediated lipid nitration and nitrosation is guaranteed. IUBMB Life, 57: 407‐412, 2005