370 Radical-Scavenging Reactions of Ascorbic Acid and Its Analogue in the Presence of a Redox-Inactive Metal Ion Ikuo Nakanishi1, Keiko Inami2, Shogo Nomura2, Kei Ohkubo3,4, Tomonori Kawashima1, Masataka Mochizuki2, Shunichi Fukuzumi3,4, Toshihiko Ozawa5, and Ken-ichiro Matsumoto1 1National Institute of Radiological Sciences, Japan, 2Tokyo University of Science,Japan, 3Osaka University, Japan, 4ALCA - Japan Science and Technology Agency, Japan, 5Yokohama College of Pharmacy, Japan Ascorbic acid (AscH2) is an important naturally occurring, water-soluble antioxidant. However, the detailed radical-scavenging mechanism of AscH2 has yet to be fully clarified. In this study, we investigated the radical-scavenging kinetics of AscH2 and 5,6-isopropylidene ascorbic acid (iAscH2) in the absence and presence of magnesium ion. AscH2 efficiently scavenged galvinoxyl and 2,2-diphenyl-1-picrylhydrazy radicals in deaerated methanol at 25 °C. The pseudo-first-order rate constants (kobs) determined for these reactions were constant with the change in the AscH2 concentration. The kobs values were significantly decreased in the presence of 0.1 M Mg(ClO4)2. Similar results were obtained for the reactions of iAscH2 with these radicals in deaerated acetonitrile. The detailed radical-scavenging mechanisms will be discussed based on the kinetic data obtained in this study.

371 Specific Residues in Peroxiredoxins Promote Peroxide Reactivity Through Effects on Cysteine pKa, Transition State Stabilization and Oligomerization Kimberly J Nelson1, Derek Parsonage1, Amanda ED Van Swearingen2, Ye Yuan3, Freddie R Salsbury3, Andrea Hall4, P Andrew Karplus4, and Leslie B Poole1 1Wake Forest School of Medicine, 2Duke University, 3Wake Forest University, 4Oregon State University Reversible thiol modification is a major component of the ROS-linked modulation of cell signaling pathways. In healthy cells, ROS levels will be controlled and peroxides that are released during signaling events will preferentially oxidize highly reactive cysteine residues within proteins. Oxidation at such sites can alter

protein structure and/or function, leading to modulation of redox-sensitive pathways. The development of methods to predict redox sensitive sites is limited by the lack of knowledge about the structural characteristics that promote sensitivity of particular cysteines toward peroxide-mediated oxidation. To better understand the structural features and residues present in the peroxiredoxin (Prx) family members that allow them to react rapidly (at kcat/Km ~107 or 108 M-1 s-1) with peroxides, we describe here the creation and characterization of select variants of the Salmonella typhimurium peroxiredoxin AhpC. Despite the fact that Thr residues are not usually regarded as titratable, mutation of a conserved Thr residue in the active site of AhpC causes an increase in pKa that is as dramatic as that observed with mutation of the conserved nearby Arg. Mutations to residues that were previously proposed to stabilize the transition state also significantly decrease the rate of reaction with peroxide, supporting the importance of substrate activation in AhpC. Finally, we show that oligomerization to dimers and decamers increases the peroxide reactivity of this protein and helps stabilize the thiolate at the active site, at least in part by promoting favorable residue conformations within the fully folded AhpC active site structure.

372 Singlet Molecular Oxygen Generation by Oleic Acid Hydroperoxides with Nitronium Ion Alexsandra Cristina Scalfo1, Fernanda Manso Prado1, Sayuri Miyamoto1, Marisa Helena Gennari de Medeiros1, and Paolo Di Mascio1 1Universidade de São Paulo, Brazil Singlet molecular oxygen [O2 (1Δg)] is an electronically excited reactive species, being involved in different physiological and pathological processes. Relevant examples of potential biological sources of O2 (1Δg) are decomposition of lipid hydroperoxides into peroxyl radicals and enzymatic process catalyzed by peroxidases or oxygenases. Recently nitrated fatty acids have been extensively studied, since they represent an important class of signaling mediators in inflammatory process. These compounds are able to promote post-translational modification of proteins due to their electrophilic nature. Regarding the chemical characteristics of unsaturated fatty acid hydroperoxides and their behavior towards nitronium ion, the aim of this study is to investigate the generation of O2 (1Δg) through nitrated oleic acid hydroperoxide and/or its derivatives. Direct evidences of O2 (1Δg) production by the reaction of oleic acid hydroperoxide and nitronium tetrafluoroborate (NO2BF4) arose from measurement of monomol light emission in near infrared region ( = 1270 nm). Additional proof for its production came from the use of sodium azide, a physical quencher of O2 (1Δg). Moreover, mass spectrometry analysis of products formed in this reaction demonstrated the presence of nitrated oleic acid hydroperoxide with m/z 359. Thus, the results obtained until now indicated that nitrated oleic acid hydroperoxides can be an additional source of O2 (1Δg), even in biological systems. Keywords: singlet oxygen, fatty acids, oleic acid hydroperoxides, nitronium ion, mass spectrometry Acknowledgements: CAPES, CNPq, Pró Reitoria de Pesquisa-USP INCT de Processos Redox em Biomedicina – Redoxoma and FAPESP.

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