Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine P-Synthase

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Silvina Bartesaghi1, Nicolás Campolo1, Federico Issoglio2, Ari Zeida2, Tilman Grune3, Darío Estrín2, and Rafael Radi1 1 Universidad de la República, Uruguay, 2Universidad de Buenos Aires, Argentina, 3German Institute of Human Nutrition, Germany Glutamine synthetase (GS) is a key metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonia. At the central nervous system, it is located mainly in the cytosol of astrocytes, playing an important role in ammonia detoxification and prevention of glutamatedependent excitotoxicity. Alterations in GS activity may lead to astroglial dysfunction, affecting thus neuronal function and survival. Several in vitro and in vivo studies have shown that GS activity is highly susceptible to biologically-relevant reactive oxygen and nitrogen species, in particular to peroxynitrite (ONOO), formed by the diffusion-controlled reaction between superoxide radical (O2Ɣ-) and nitric oxide (ƔNO). Peroxynitrite-derived radicals, promote protein tyrosine nitration, a post-translational modification that can seriously affect protein structure and function, yielding 3nitrotyrosine (3-NT). In this context, tyrosine nitration of GS has been identified as one of the main oxidative modifications associated to enzyme inactivation in a series of pathological conditions. Critical tyrosine residues participate in the enzyme active site and are involved in the binding of the substrates ATP and ammonia. This work aims to establish the molecular mechanisms involved in GS inactivation by oxidative modifications mediated by ONOO-. For this purpose, we have performed studies with the pure enzyme. Firstly, we purified recombinant human GS (rGS) expressed in Escherichia coli, exposed it to ONOO- under different conditions and evaluated enzyme function and oxidation/nitration after treatment. Bolus addition of ONOOcaused a dose-dependent decrease in GS activity (70% inactivation); concomitantly, an increase in relative 3-NT levels was observed In addition, we performed molecular dynamics simulations (MD) to examine human GS structural properties and nitration mechanisms. Further studies are underway to quantitatively establish the role of tyrosine nitration on GS inactivation and to map the tyrosine nitration sites responsible of inactivation.

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Sebastián Carballal1, Carmen Gherasim2, Pramod K. Yadav2, Ernesto Cuevasanta 1, David P. Ballou2, Beatriz Alvarez1, and Ruma Banerjee2 1 Universidad de la República, Uruguay, 2University of Michigan, USA Cystathionine E-synthase (CBS) is a key enzyme in the metabolism of sulfur amino acids in mammals. It catalyzes the

pyridoxal 5´-phosphate (PLP)-dependent condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. Human CBS contains a non-catalytic heme b cofactor, hexa coordinated to cysteine and histidine. Fe(III)-CBS is relatively stable and unreactive. It can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NOƔ) leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O2 back to Fe(III)-CBS forming superoxide radical anion (O2Ɣ-). We have recently demonstrated that Fe(II)-CBS has the ability to reduce nitrite (NO2-) to NOƔ‡ leading to Fe(II)NOƔ-CBS formation, suggesting a possible new role for CBS in NOƔ signaling. In this study, we characterized the kinetics of NO2- reduction by Fe(II)-CBS by stopped-flow spectrophotometry using low dithionite concentrations. In addition, we assessed the possible generation of nitroxyl (HNO) as the product of spontaneous decay of Fe(II)NO‡-CBS under anaerobic conditions. Reoxidation of Fe(II)NO‡-CBS by O2 showed complex kinetic behavior with a multiphasic time course. Peroxynitrite (ONOO-) formation was detected using the fluorescent probe coumarin boronic acid. Thus, in addition to being a potential source of O2Ɣ-, CBS constitutes a previously unrecognized source of peroxynitrite. This study adds important new elements to our understanding of the heme reactivity in CBS and adds to the evidence that CBS represents a point of interplay between NO2-, ONOO-, NOƔ, HNO, O2Ɣ-, CO and H2S.

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Marta T. Ignasiak1, Maryam Karimi2, David I. Pattison2, and Michael J Davies1 1 University of Copenhagen, Denmark, 2The Heart Research Institute, Sydney, Australia Controlled production of oxidants such as HOCl, HOBr, HOSCN, ONOOH and H2O2 by leukocytes is critical for the immune response and killing of invading pathogens, but uncontrolled or inappropriate generation of these species at sites of inflammation causes tissue damage with this being associated with multiple inflammatory diseases. Previous studies have reported that thiols (RSH) and thioethers 565¶  HJWKH DPLQR DFLGV &\V DQG 0HW  DUHPDMRU WDUJHWVIRU inflammatory oxidants and that these reactions are rapid. Reaction has also been reported with disulfide bonds (e.g. cystine), with these reactions occurring with lower rate constants. The effect of structure and environment on these values has not been examined. We now report rate constants for the reaction of oxidants with multiple disulfide containing compounds, ranging from model compounds to proteins, obtained using stopped-flow spectrophotometry or competition kinetic approaches. It is shown that the rate constants for these reactions at pH 7.4 are highly dependent on both the oxidant and environment of the disulfide bond. The reactivity of many disulfides is lower than for thiols, and decreases in the order HOBr > HOCl > HOSCN > ONOOH > H2O2. However some disulfides are markedly more reactive, by up to 5 orders of magnitude, than others (k 10(3) 10(8) M-1s-1 for HOCl), indicating that some disulfide bonds, including those in some proteins are much more susceptible to oxidation than others. These processes result in the formation of WKLRVXOILQDWHV>562 65¶@WKDWFDQXQGHUJRIXUWKHUUHDFWLRQV7KLV pattern of enhanced reactivity holds across a wide range of oxidants, suggesting that this is a general phenomenon, with

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