Impact of the Pre-a Motif on Truncated Hemoglobin N Activity

Tuberculosis (TB) remains the leading cause of death by an infectious agent and therefore a global health crisis, according to the most recent report by the World Health Organization. This is due, in part, to Mycobacterium tuberculosis’ impressive defensive mechanisms against immune response, as well as the rise of Multi-Drug Resistant strains that have recently developed. Towards the turn of the century, a small heme protein called truncated hemoglobin N (trHbN) was discovered to protect the bacteria against reactive nitrogen species by converting nitric oxide (NO) to nitrate at rates far exceeding those of myoglobin and closer to those of the well-known NO dioxygenase flavohemoglobin. Ferrous oxygenated trHbN (oxy-trHbN) first converts NO to nitrate, which leaves the protein in a ferric state (met-trHbN). Met-trHbN is re-reduced to give a 5-coordinate ferrous species (red-trHbN), which is then re-oxygenated to oxy-trHbN. Recently, a unique 12-amino acid motif at the trHbN N-terminus was identified, the so-called pre-A tail, that appears to enhance the organism’s ability to convert NO to nitrate. The results presented herein show that the pre-A tail of trHbN affects every step of the putative NO dioxygenation catalytic cycle, but it affects the rate of met-trHbN re-reduction most profoundly. In a variant that lacks the pre-A tail (trHbNdelN), met-trHbNdelN was reduced about 40 times more slowly than met-trHbNWT by the non-specific reductant RuII. By comparison, the reactions of oxy-trHbN or red-trHbN with NO were only 2x – 4x slower in the trHbNdelN variant than in the wild type (the reaction of red-trHbN with NO is a good surrogate for the reaction of red-trHbN with O2). Importantly, the effect of the pre-A tail is completely lost in variants that lack distal site residues Tyr33 and Gln 58. These residues help to hold O2 firmly on the heme in oxy-trHbN, and a water molecule on the heme of met-trHbN. They also anchor a non-coordinated water molecule in the distal site of red-trHbN that blocks access by incoming diatomic gases. In a variant that lacks Tyr33 and Gln 58 (trHbNDM), met-trHbNDM is reduced 5x more rapidly by RuII than is met- trHbNWT because the distal site is now either vacant or occupied by weakly bound water, so rate-limiting water loss upon heme reduction is accelerated. A variant that lacks Tyr33, Gln 58, and the pre-A tail (met-trHbNTM), is reduced by RuII at the same rate as is met-trHbNDM, showing that tail loss does not affect the reduction rate if the distal site amino acids are absent. This is strong evidence that the pre-A tail’s primary function is to facilitate release of the distal water molecule from met-trHbN, a function that is less important in met-trHbNDM and met-trHbNTM than it is in trHbNWT

A Mechanistic Investigation of Cytochrome C Nitrite Reductase Catalyzed Reduction of Nitrite to Ammonia: The Search for Catalytic Intermediates

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) and the 2-electron reduced form of indigo trisulfonate. Experiments were done with the wild type enzyme (wtccNiR) as well as with R103Q, Y206F, and H257Q variants.A UV/Vis stopped-flow investigation of the reaction between nitrite-loaded wtccNiR and TMPD revealed for the first time that the reaction proceeds via a transient 1-electron reduced intermediate. Generation of this species is pH-independent, whereas its decay to a previously characterized 2-electron reduced intermediate is fastest at pH 6.8 and significantly slower at higher pH. The pH dependence is ascribed to the rate-limiting cleavage of the nitrite N – O bond, which requires a prior di-protonation predicted to be slow in earlier computational studies. Under steady-state conditions, S. oneidensis ccNiR catalyzed the slow 1-electron reduction of nitrite to nitric oxide, which was monitored by tracking the concomitant appearance of the colored TMPD+ radical product. The rate of TMPD+ formation was found to be directly proportional to the concentration of TMPD+ at low TMPD concentrations, providing important insights about the mechanism of NO˙ release. The steady-state studies also showed that nitrite is a substrate inhibitor of NO˙ release when TMPD is the electron source, probably because it blocks exit by NO˙ through the nitrite entry channel. The H257Q variant of ccNiR was found to have 1/400th of the wild type enzyme’s nitrite reductase activity in the standard assay in which methyl viologen monocation radical is the electron source, but nearly normal hydroxylamine reductase activity. This demonstrated that H257 is essential for nitrite reduction but not for reduction of hydroxylamine, a putative intermediate in the catalytic process. UV/Vis spectropotentiometry showed that the nitrite-loaded active site of H257Q still reduced at fairly high applied potential, but the reduction was by one electron, whereas the wild type is reduced in a concerted 2-electron step. The experiments with the variant, coupled with the pH-dependent stopped flow experiments with wtccNiR, confirm H257’s importance in facilitating nitrite reduction, but suggest that its role is to modulate the pKa values of one or more of the waters that form a complex hydrogen bonding network in the active site. This view is more conservative than earlier theoretical predictions of direct histidine involvement in nitrite diprotonation leading to N – O bond cleavage

13 reasons why the brain is susceptible to oxidative stress

The human brain consumes 20% of the total basal oxygen (O2) budget to support ATP intensive neuronal activity. Without sufficient O2 to support ATP demands, neuronal activity fails, such that, even transient ischemia is neurodegenerative. While the essentiality of O2 to brain function is clear, how oxidative stress causes neurodegeneration is ambiguous. Ambiguity exists because many of the reasons why the brain is susceptible to oxidative stress remain obscure. Many are erroneously understood as the deleterious result of adventitious O2 derived free radical and non-radical species generation. To understand how many reasons underpin oxidative stress, one must first re-cast free radical and non-radical species in a positive light because their deliberate generation enables the brain to achieve critical functions (e.g. synaptic plasticity) through redox signalling (i.e. positive functionality). Using free radicals and non-radical derivatives to signal sensitises the brain to oxidative stress when redox signalling goes awry (i.e. negative functionality). To advance mechanistic understanding, we rationalise 13 reasons why the brain is susceptible to oxidative stress. Key reasons include inter alia unsaturated lipid enrichment, mitochondria, calcium, glutamate, modest antioxidant defence, redox active transition metals and neurotransmitter auto-oxidation. We review RNA oxidation as an underappreciated cause of oxidative stress. The complex interplay between each reason dictates neuronal susceptibility to oxidative stress in a dynamic context and neural identity dependent manner. Our discourse sets the stage for investigators to interrogate the biochemical basis of oxidative stress in the brain in health and disease

Structure-function relationship in S-nitrosoglutathione reductase and the development of fluorogenic pseudo-substrates

S-nitrosation is the attachment of a nitric oxide moiety to the thiol side chain of cysteine. S-nitrosoglutathione (GSNO) acts as a bioactive reservoir for NO to maintain an equilibrium in the concentration of NO in the body. Due to this, the study of the enzyme S-nitrosoglutathione reductase has of great interest because of its ability to metabolize GSNO. S-nitrosoglutathione reductase’s activity has been linked to a number of human diseases. Chapter 1 of this thesis presents a proposed allosteric binding domain on GSNOR. Positive cooperativity (sigmoidal deviation) was observed from steady state analysis of GSNOR which indicated an affinity for the binding of GSNO at this site. The presence of such a site was further supported by Molecular docking simulations and HDX-MS which showed that the amino acids Gly321, Lys323, Asn185 and Lys188 interact with molecules bound at this site. Chapter two introduces four reagents that can function as probes or pseudo-substrates for the monitoring of enzymatic activity as well as measuring concentrations of free thiols in vitro and live cells. These reagents are N,N-di(thioamido-fluoresceinyl)-cystine (DTFCys2), N,N-di(thioamido-fluoresceinyl)-homocystine (DTFHCys2), N-amido-O-aminobenzoyl-S-nitrosoglutathione (AOASNOG), and N-thioamido-fluoresceinyl-S-nitroso-glutathione (TFSNOG). They are easy to prepare and purity and can be used in various applications

Hemoglobin-Mediated Nitric Oxide Signaling

The rate that hemoglobin reacts with nitric oxide (NO) is limited by how fast NO can diffuse into the heme pocket. The reaction is as fast as any ligand/protein reaction can be and the result, when hemoglobin is in its oxygenated form, is formation of nitrate in what is known as the dioxygenation reaction. As nitrate, at the concentrations made through the deoxygenation reaction, is biologically inert, the only role hemoglobin was once thought to play in NO signaling was to inhibit it. However, there are now several mechanisms that have been discovered by which hemoglobin may preserve, control, and even create NO activity. These mechanisms involve compartmentalization of reacting species and conversion of NO from or into other species such as nitros othiols or nitrite which could transport NO activity. Despite the tremendous amount of work devoted to this field, major questions concerning precise mechanisms of NO activity preservation as well as if and how Hb creates NO activity remain unanswered

Unravelling ties in the nitrogen network: Polyamines and nitric oxide emerging as essential players in signalling roadway

Nitrogen (N) is a central mineral nutrient essential for plant development and growth. It is usually scarcely found in soils, so the knowledge of the overall plant N metabolism deserves substantial attention. Polyamines (PAs) are N-containing low-molecular-weight compounds of polycationic nature involved in essential processes all throughout the life of plants whereas nitric oxide (NO) is a gaseous free radical involved in signalling cascades related to many physiological events. PAs and NO share signalling functions and interact with each other in several biological functions, mainly in stress responses. Biosynthesis pathways of PAs and NO are overlapped; PAs induce NO formation, but it is still not completely defined whether PAs act as substrates, cofactors, or signals for promoting NO synthesis and also, which are the mechanisms involved in NO regulation of PAs metabolism. Polyamine levels are of vital importance in the regulation of the network of N-metabolising pathways in plants, as they are components of the core of the overall N metabolism. In light of the importance of improving the efficiency of N uptake and distribution, it is time to elucidate the intricate relationship among N as a nutrient with PAs and NO as emerging signalling molecules. The close cooperation among these players in the whole N metabolism is an interesting target for the development of biotechnological tools for sustainable agriculture.Fil: Recalde, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gómez Mansur, Nabila María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Cabrera, Andrea Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Matayoshi, Carolina Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gallego, Susana Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Groppa, María Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Benavides, Maria Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O_2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O_2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O_2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important iron–oxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs

Oxidative Stress Modulators and Functional Foods

This book “Oxidative Stress Modulators and Functional Foods” is focused on the antioxidant role of natural products, involving their ability to modulate oxidative stress and/or reverse disease studied both in vitro and in animal models. Additionally, the molecular mechanisms of these actions and the modulation of signalling pathways related to inflammation, apoptosis, and survival response in the redox system by natural products are included

Recent developments in effective antioxidants : the structure and antioxidant properties

Since the last few years, the growing interest in the use of natural and synthetic antioxidants as functional food ingredients and dietary supplements, is observed. The imbalance between the number of antioxidants and free radicals is the cause of oxidative damages of proteins, lipids, and DNA. The aim of the study was the review of recent developments in antioxidants. One of the crucial issues in food technology, medicine, and biotechnology is the excess free radicals reduction to obtain healthy food. The major problem is receiving more effective antioxidants. The study aimed to analyze the properties of efficient antioxidants and a better understanding of the molecular mechanism of antioxidant processes. Our researches and sparing literature data prove that the ligand antioxidant properties complexed by selected metals may significantly affect the free radical neutralization. According to our preliminary observation, this efficiency is improved mainly by the metals of high ion potential, e.g., Fe(III), Cr(III), Ln(III), Y(III). The complexes of delocalized electronic charge are better antioxidants. Experimental literature results of antioxidant assays, such as diphenylpicrylhydrazyl (DPPH) and ferric reducing activity power assay (FRAP), were compared to thermodynamic parameters obtained with computational methods. The mechanisms of free radicals creation were described based on the experimental literature data. Changes in HOMO energy distribution in phenolic acids with an increasing number of hydroxyl groups were observed. The antioxidant properties of flavonoids are strongly dependent on the hydroxyl group position and the catechol moiety. The number of methoxy groups in the phenolic acid molecules influences antioxidant activity. The use of synchrotron techniques in the antioxidants electronic structure analysis was proposed