Despite the worldwide public health impact of malaria, neither the mechanism by which the Plasmodium parasite detoxifies and sequesters haem, nor the action of current antimalarial drugs is well understood. The haem groups released from the digestion of the haemoglobin of infected red blood cells are aggregated into an insoluble material called haemozoin or malaria pigment. Synthetic beta-haematin (FeIII-protoporphyrin-IX)2 is chemically, spectroscopically and crystallographically identical to haemozoin and is believed to consist of strands of FeIII-porphyrin units, linked into a polymer by propionate oxygen-iron bonds. Here we report the crystal structure of beta-haematin determined using simulated annealing techniques to analyse powder diffraction data obtained with synchrotron radiation. The molecules are linked into dimers through reciprocal iron-carboxylate bonds to one of the propionic side chains of each porphyrin, and the dimers form chains linked by hydrogen bonds in the crystal. This result has implications for understanding the action of current antimalarial drugs and possibly for the design of new therapeutic agents.

The rapid and spontaneous interaction between superoxide (O2-.) and nitric oxide (NO) to yield the potent oxidants peroxynitrite (ONOO-) and peroxynitrous acid (ONOOH), has been suggested to represent an important pathway by which tissue may be injured during inflammation. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized ONOO-, there has been little information available quantifying the interaction between O2-. and NO in the absence or the presence of redox-active iron. Using the hypoxanthine (HX)/xanthine oxidase system to generate various fluxes of O2-. and H2O2 and the spontaneous decomposition of the spermine/NO adduct to produce various fluxes of NO, we found that in the absence of redox-active iron, the simultaneous production of equimolar fluxes of O2-. and NO increased the oxidation of dihydrorhodamine (DHR) from normally undetectable levels to approximately 15 microM, suggesting the formation of a potent oxidant. Superoxide dismutase, but not catalase, inhibited this oxidative reaction, suggesting that O2-. and not hydrogen peroxide (H2O2) interacts with NO to generate a potent oxidizing agent. Excess production of either radical virtually eliminated the oxidation of DHR. In the presence of 5 microM Fe+3-EDTA to insure optimum O2-.-driven Fenton chemistry, NO enhanced modestly HX/xanthine oxidase-induced oxidation of DHR. As expected, both superoxide dismutase and catalase inhibited this Fe-catalyzed oxidation reaction. Excess NO production with respect to O2-. flux produced only modest inhibition (33%) of DHR oxidation. In a separate series of studies, we found that equimolar fluxes of O2-. and NO in the absence of iron only modestly enhanced hydroxylation of benzoic acid from undetectable levels to 0.6 microM 2-hydroxybenzoate. In the presence of 5 microM Fe+3-EDTA, HX/xanthine oxidase-mediated hydroxylation of benzoic acid increased dramatically from undetectable levels to 4.5 microM of the hydroxylated product. Superoxide dismutase and catalase were both effective at inhibiting this classic O2-.-driven Fenton reaction. Interestingly, NO inhibited this iron-catalyzed hydroxylation reaction in a concentration-dependent manner such that fluxes of NO approximating those of O2-. and H2O2 virtually abolished the hydroxylation of benzoic acid. We conclude that in the absence of iron, equimolar fluxes of NO and O2-. interact to yield potent oxidants such as ONOO-/ONOOH, which oxidize organic compounds. Excess production of either radical remarkably inhibits these oxidative reactions. In the presence of low molecular weight redox-active iron complexes, NO may enhance or inhibit O2-.-dependent oxidation and hydroxylation reactions depending upon their relative fluxes.

Peroxynitrite, the reaction product of nitric oxide (NO) and superoxide (O 2 . ) is assumed to decompose upon protonation in a first order process via intramolecular rearrangement to NO 3 ؊ . The present study was carried out to elucidate the origin of NO 2 ؊ found in decomposed peroxynitrite solutions. As revealed by stopped-flow spectroscopy, the decay of peroxynitrite followed firstorder kinetics and exhibited a pK a of 6.8 ؎ 0.1. The reaction of peroxynitrite with NO was considered as one possible source of NO 2 ؊ , but the calculated second order rate constant of 9.1 ؋ 10 4 M ؊1 s ؊1 is probably too small to explain NO 2 ؊ formation under physiological conditions. Moreover, pure peroxynitrite decomposed to NO 2 ؊ without apparent release of NO. Determination of NO 2 ؊ and NO 3 ؊ in solutions of decomposed peroxynitrite showed that the relative amount of NO 2 ؊ increased with increasing pH, with NO 2 ؊ accounting for about 30% of decomposition products at pH 7.5 and NO 3 ؊ being the sole metabolite at pH 3.0. Formation of NO 2 ؊ was accompanied by release of stoichiometric amounts of O 2 (0.495 mol/mol of NO 2 ؊ ). The two reactions yielding NO 2 ؊ and NO 3 ؊ showed distinct temperature dependences from which a difference in E act of 26.2 ؎ 0.9 kJ mol ؊1 was calculated. The present results demonstrate that peroxynitrite decomposes with significant rates to NO 2 ؊ plus O 2 at physiological pH. Through formation of biologically active intermediates, this novel pathway of peroxynitrite decomposition may contribute to the physiology and/or cytotoxicity of NO and superoxide.The reaction between nitric oxide (NO) and superoxide anion (O 2 . ) yields peroxynitrite with a second order rate constant near the diffusion-controlled limit (k ϭ 4.3-6.7 ϫ 10 9 M Ϫ1 s Ϫ1 ) (1, 2). The reaction constitutes an important sink for O 2 . because it is about twice as fast as the maximum velocity of SOD. 1 Consequently, peroxynitrite has been implicated in many pathological conditions including stroke (3), heart disease (4), and atherosclerosis (5, 6). The potential cellular targets for peroxynitrite cytotoxicity include the antioxidants ascorbate, ␣-tocopherol, and uric acid (7-10), protein and non-protein sulfhydryls (11), DNA (12), and membrane phospholipids (13). Decomposition of peroxynitrite is complex (14, 15). The anion is rather stable in alkaline solutions but decomposes rapidly (t 1/2 ϭ 1 s at pH 7.4, 37°C) upon protonation to peroxynitrous acid (ONOOH) (pK a ϭ 6.8) (16). Two pathways of ONOOH decomposition have been proposed. Some studies have argued that ONOOH is cleaved homolytically to generate hydroxyl and NO 2 radicals. This hypothesis is based on the sensitivity to hydroxyl radical scavengers of certain peroxynitrite-induced reactions, including the formation of malondialdehyde from deoxyribose and the hydroxylation on the benzene ring of sodium benzoate, phenylalanine, and tyrosine (16, 17). Studies on decomposition of peroxynitrite by electron paramagnetic resonance spectroscopy with the spin traps 5,5-dime...

Cu-catalyzed cross-dehydrogenative coupling (CDC) methodologies were developed based on the oxidative activation of sp 3 C-H bonds adjacent to a nitrogen atom. Various sp, sp 2 , and sp 3 C-H bonds of pronucleophiles were used in the Cu-catalyzed CDC reactions. Based on these results, the mechanisms of the CDC reactions also are discussed.

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic b-hematin (bH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to bH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.

The intraerythrocytic parasite Plasmodium—the causative agent of malaria—produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1β (IL-1β). However, the mechanism regulating Hz recognition and IL-1β maturation has not been identified. Here, we show that Hz induces IL-1β production. Using knockout mice, we showed that Hz-induced IL-1β and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1β augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.

An increasing number of biological roles are ascribed to S-nitrosothiol compounds. Their inherent instability in multicomponent solutions is recognized as forming the basis for their physiological effects, such as the release of nitric oxide or the posttranslational modification of protein cysteine residues. This reactivity also contributes to the lack of fundamental physical and spectroscopic data that have been reported. We have addressed this issue through characterization of the physical and spectroscopic properties of a group of commonly used S-nitrosothiols. The S-nitrosothiol Ph3CSNO, which is readily prepared by the biphasic nitrosation of Ph3CSH, is characterized by X-ray diffraction, vibrational spectroscopy, electrochemistry, and spectroelectrochemistry. Its behavior is contrasted with that of known S-nitrosothiols derived from glutathione and N-acetyl-d,l-penicillamine, which also are demonstrated to undergo facile electrochemical and chemical denitrosylation. The structure and vibrational data are contrasted with ab initio results calculated with density functional theory, B3LYP/6-311+G*, which indicates that electron transfer populates an orbital that is strongly ON−SR antibonding in character. The bond lengths observed for Ph3CSNO (N−O 1.18 Å, S−N 1.79 Å) indicate a formal nitrogen-to-oxygen double bond and sulfur−oxygen single bond. However, theoretical calculations show a measure of delocalization over the −CSNO framework. This is supported by experimental results that show low ν(NO) vibrational frequencies (1470−1515 cm-1) and a large ΔG ⧧ (10.7 kcal/mol) for syn−anti interconversion determined by variable-temperature 15N NMR. Together these results demonstrate an important new reactivity pattern for this biologically critical class of compounds.

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