Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Oxidation of 3-hydroxyanthranilic acid to the phenoxazinone cinnabarinic acid by peroxyl radicals and by compound I of peroxidases or catalase

Since 3-hydroxyanthranilic acid (3HAA), an oxidation product of tryptophan metabolism, is a powerful radical scavenger [Christen, S., Peterhans, E., ; Stocker, R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2506], its reaction with peroxyl radicals was investigated further. Exposure to aqueous peroxyl radicals generated at constant rate under air from the thermolabile radical initiator 2,2'-azobis[2-amid-inopropane] hydrochloride (AAPH) resulted in rapid consumption of 3HAA with initial accumulation of its cyclic dimer, cinnabarinic acid (CA). The initial rate of formation of the phenoxazinone CA accounted for approximately 75% of the initial rate of oxidation of 3HAA, taking into account that 2 mol of 3HAA are required to form 1 mol of CA. Consumption of 3HAA under anaerobic conditions (where alkyl radicals are produced from AAPH) was considerably slower and did not result in detectable formation of CA. Addition of superoxide dismutase enhanced autoxidation of 3HAA as well as the initial rates of peroxyl radical-induced oxidation of 3HAA and formation of CA by approximately 40-50%, whereas inclusion of xanthine/xanthine oxidase decreased the rate of oxidation of 3HAA by approximately 50% and inhibited formation of CA almost completely, suggesting that superoxide anion radical (O2.-) was formed and reacted with reaction intermediate(s) to curtail formation of CA. Formation of CA was also observed when 3HAA was added to performed compound I of horseradish peroxidase (HRPO) or catalytic amounts of either HRPO, myeloperoxidase, or bovine liver catalase together with glucose/glucose oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of valine and isoleucine in plants I. Formation of α-acetolactate in Phaseolus radiatus

1. 1. The presence of an enzyme system in plants catalyzing the formation of α-acetolactate from pyruvate has been demonstrated; the system in green gram (Phaseolus radiatus) has been partially purified and its characteristics have been studied.2. Free acetaldehyde is formed as a product of the reaction and so the reaction is mainly diverted towards the formation of acetoin. 3. The system requires thiamine pyrophosphate and a divalent metal ion (Mn2+ or Mg2+) for maximum activity. The optimum pH is around 6.0 and the optimum temperature is 60°. 4. The system is very labile in absence of pyruvate, Mn2+ and DPT. 5. The Km values for pyruvate, Mn2+, Mg2+ and DPT are 3·10−2 M. 5·10−5 M, 2·10−5 M, and e·10−6 M respectively. The activation energy is 3540 cal/mole. 6. The enzyme is strongly inhibited by p-chloromercuribenzoate and the inhibition can be reversed partially by 2-mercaptoethanol, BAL or cysteine. Heavy metals, such as Hg2+ and Ag+, are inhibitory but l-valine does not inhibit the reaction.

A regional chemostratigraphically-defined correlation framework for the late Triassic TAG-I Formation in Blocks 402 and 405a, Algeria

The Triassic Argilo-Gréseux Inférieur Formation (TAG-I) is one of the principal hydrocarbon reservoirs in the Berkine Basin of Algeria. Sedimentological studies have shown that it exhibits marked spatial and temporal facies variations on both a local field scale and a regional basinal scale. This variability, combined with a lack of diagnostic flora and fauna, makes regional correlation within the unit difficult. In turn, the lack of a consistent regional stratigraphic framework hampers the comparison of the various correlation schemes devised by operators in the basin. Contrasting the TAG-I in Blocks 402 and 405a exemplifies the problems encountered when attempting regionally to define a correlation framework for the interval. Between these two blocks, a distance of approximately 200 km, there are marked changes in the style of deposition from sand-dominated, proximal fluvial systems in the SW (Block 405a, MLN, MLC, KMD and MLNW fields) to a more distal, more clay-prone system in the NE (Block 402, ROD/BRSE/BSFN, SFNE and BSF fields). A chemostratigraphic study of the TAG-I in these two blocks has allowed a four-fold correlation framework to be defined, where each chemostratigraphic package has distinctive geochemical features. Chemostratigraphic Package 10, the oldest unit, lies above the Hercynian Unconformity, but beneath a geochemically identifiable hiatal surface. Chemostratigraphic Package 20 lies above the hiatal surface but is separated from the overlying packages by a mineralogical change identifiable in both claystone and sandstone geochemistry. Chemostratigraphic Packages 30 and 40 are chemically somewhat similar, but are separated by a regional event interpreted as a period of dolocrete and lacustrine development. By combining the geochemical differentiation of the units and recognition of their stratal boundaries, it is possible to define a correlation for the TAG-I between Blocks 402 and 405a. The proposed correlation between the two blocks suggests that the northern parts of Block 405a may have been occupied by a spur or subsidiary channel from the main SW–NE-trending fluvial system, resulting in one of the chemically defined packages being demonstrably absent in the MLNW, MLN, KMD and MLC fields when compared with the other areas of the study.

Revisiting Asian monsoon formation and change associated with Tibetan Plateau forcing: I. Formation

Numerical experiments with different idealized land and mountain distributions are carried out to study the formation of the Asian monsoon and related coupling processes. Results demonstrate that when there is only extratropical continent located between 0 and 120°E and between 20/30°N and the North Pole, a rather weak monsoon rainband appears along the southern border of the continent, coexisting with an intense intertropical convergence zone (ITCZ). The continuous ITCZ surrounds the whole globe, prohibits the development of near-surface cross-equatorial flow, and collects water vapor from tropical oceans, resulting in very weak monsoon rainfall. When tropical lands are integrated, the ITCZ over the longitude domain where the extratropical continent exists disappears as a consequence of the development of a strong surface cross-equatorial flow from the winter hemisphere to the summer hemisphere. In addition, an intense interaction between the two hemispheres develops, tropical water vapor is transported to the subtropics by the enhanced poleward flow, and a prototype of the Asian monsoon appears. The Tibetan Plateau acts to enhance the coupling between the lower and upper tropospheric circulations and between the subtropical and tropical monsoon circulations, resulting in an intensification of the East Asian summer monsoon and a weakening of the South Asian summer monsoon. Linking the Iranian Plateau to the Tibetan Plateau substantially reduces the precipitation over Africa and increases the precipitation over the Arabian Sea and the northern Indian subcontinent, effectively contributing to the development of the South Asian summer monsoon.

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85 angstrom. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. (C) 2009 Elsevier Inc. All rights reserved.

A Reactive Intermediate, Ni-5(C6H4N3)(6)(CO)(4)], in the Formation of Nonameric Clusters of Nickel, Ni-9(C6H4N3)(12)(CO)(6)] and Ni-9(C6H4N3)(12)(CO)(6)].2(C3H7NO)

Three new molecular compounds, Ni-5(bta)(6)(CO)(4)], I, Ni-9(bta)(12)(CO)(6)], II, Ni-9(bta)(12)(CO)(6)]. 2(C3H7NO), III, (bta = benzotriazole) were prepared employing solvothermal reactions. Of these, I have pentanuclear nickel, whereas II and III have nonanuclear nickel species. The structures are formed by the connectivity between the nickel and benzotriazole giving rise to the 5- and 9-membered nickel clusters. The structures are stabilised by extensive pi aEuro broken vertical bar pi and C-H... pi interactions. Compound II and III are solvotamorphs as they have the same 9-membered nickel clusters and have different solvent molecules. To the best of our knowledge, the compounds I-III represent the first examples of the same transition element existing in two distinct coordination environment in this class of compounds. The studies reveal that compound I is reactive and could be an intermediate in the preparation of II and III. Thermal studies indicate that the compounds are stable upto 350(a similar to)C and at higher temperatures (similar to 800(a similar to)C) the compounds decompose into NiO. Magnetic studies reveal that II is anti-ferromagnetic.

O6-carboxymethylguanine DNA adduct formation and lipid peroxidation upon in vitro gastrointestinal digestion of haem-rich meat

Scope Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have been put forward to explain this causal relationship, i.e. N-nitroso compound (NOC) formation and lipid peroxidation (LPO). Methods and Results In this study, the NOC-derived DNA adduct O6-carboxymethylguanine (O6-CMG) and the LPO product malondialdehyde (MDA) were measured in individual in vitro gastrointestinal digestions of meat types varying in haem content (beef, pork, chicken). While MDA formation peaked during the in vitro small intestinal digestion, alkylation and concomitant DNA adduct formation was observed in seven (out of 15) individual colonic digestions using separate faecal inocula. From those, two haem-rich meat digestions demonstrated a significantly higher O6-CMG formation (p < 0.05). MDA concentrations proved to be positively correlated (p < 0.0004) with haem content of digested meat. The addition of myoglobin, a haem-containing protein, to the digestive simulation showed a dose–response association with O6-CMG (p = 0.004) and MDA (p = 0.008) formation. Conclusion The results suggest the haem-iron involvement for both the LPO and NOC pathway during meat digestion. Moreover, results unambiguously demonstrate that DNA adduct formation is very prone to inter-individual variation, suggesting a person-dependent susceptibility to colorectal cancer development following haem-rich meat consumption.

Proton-transfer versus nontransfer in compounds of the diazo-dye precursor 4-(phenyldiazenyl) aniline (aniline yellow) with strong organic acids: the 5-sulfosalicylate and the dichroic benzenesulfonate salts, and the 1:2 adduct with 3,5-dinitrobenzoic

The structures of two 1:1 proton-transfer red-black dye compounds formed by reaction of aniline yellow [4-(phenyldiazenyl)aniline] with 5-sulfosalicylic acid and benzenesulfonic acid, and a 1:2 nontransfer adduct compound with 3,5-dinitrobenzoic acid have been determined at either 130 or 200 K. The compounds are 2-(4-aminophenyl)-1-phenylhydrazin-1-ium 3-carboxy-4-hydroxybenzenesulfonate methanol solvate, C12H12N3+.C7H5O6S-.CH3OH (I), 2-(4-aminophenyl)-1-hydrazin-1-ium 4-(phenydiazinyl)anilinium bis(benzenesulfonate), 2C12H12N3+.2C6H5O3S-, (II) and 4-(phenyldiazenyl)aniline-3,5-dinitrobenzoic acid (1/2) C12H11N3.2C~7~H~4~N~2~O~6~, (III). In compound (I) the diaxenyl rather than the aniline group of aniline yellow is protonated and this group subsequently akes part in a primary hydrogen-bonding interaction with a sulfonate O-atom acceptor, producing overall a three-dimensional framework structure. A feature of the hydrogen bonding in (I) is a peripheral edge-on cation-anion association involving aromatic C--H...O hydrogen bonds, giving a conjoint R1/2(6)R1/2(7)R2/1(4)motif. In the dichroic crystals of (II), one of the two aniline yellow species in the asymmetric unit is diazenyl-group protonated while in the other the aniline group is protonated. Both of these groups form hydrogen bonds with sulfonate O-atom acceptors and thee, together with other associations give a one-dimensional chain structure. In compound (III), rather than proton-transfer, there is a preferential formation of a classic R2/2(8) cyclic head-to-head hydrogen-bonded carboxylic acid homodimer between the two 3,5-dinitrobenzoic acid molecules, which in association with the aniline yellow molecule that is disordered across a crystallographic inversion centre, result in an overall two-dimensional ribbon structure. This work has shown the correlation between structure and observed colour in crystalline aniline yellow compounds, illustrated graphically in the dichroic benzenesulfonate compound.

Hybrid framework iron(II) phosphate-oxalates

Two inorganic-organic hybrid framework iron phosphate-oxalates, I, [N2C4H12](0.5)[Fe-2(HPO4)(C2O4)(1.5)] and II, [Fe-2(OH2)PO4(C2O4)(0.5)] have been synthesized by hydrothermal means and the structures determined by X-ray crystallography. Crystal Data: compound I, monoclinic, spacegroup = P2(1)/c (No. 14), a=7.569(2) Angstrom, b=7.821(2) Angstrom, c=18.033(4) Angstrom, beta=98.8(1)degrees, V=1055.0(4) Angstrom(3), Z=4, M=382.8, D-calc=2.41 g cm(-3) MoK alpha, R-F=0.02; compound II, monoclinic, spacegroup=P2(1)/c (No. 14), a=10.240(1) b=6.375(3) Angstrom, 9.955(1) Angstrom, beta=117.3(1)degrees, V=577.4(1) Angstrom(3), Z=4, M=268.7, D-calc=3.09 g cm(-3) MoK alpha, R-F=0.03. These materials contain a high proportion of three-coordinated oxygens and [Fe2O9] dimeric units, besides other interesting structural features. The connectivity of Fe2O9 is entirely different in the two materials resulting in the formation of a continuous chain of Fe-O-Fe in II. The phosphate-oxalate containing the amine, I, forms well-defined channels. Magnetic susceptibility measurements show Fen to be in the high-spin state (t(2g)(4)e(g)(2)) in II, and in the intermediate-spin state (t(2g)(5)e(g)(1)) in I.

Time- and Temperature-Dependent Study in the Three-Component Zinc-Triazolate-Oxybis(benzoate) System: Stabilization of New Topologies

Three new three-dimensional zinc-triazolate-oxybis(benzoate) compounds. [{Zn-3(H2O)(2)}{C12H8O(COO)(2)}(2)-{C2H2N3}(2)]center dot 2H(2)O(I), [Zn-7{C12H8O(COO)(2)}(4){C2H2N3}(6)]center dot H2O, (II), and[{Zn-5(OH)(2)}{C12H8O(COO)(2)}(3){C2H2N3}(2)] (III), synthesized by a hydrothermal reaction of a mixture of Zn(OAc)(2)center dot 2H(2)O, 4,4'-oxybis(benzoic acid), 1,2,4-triazole, NaOH, and water. Compound I has an interpenetrated diamond structure and II and III have pillared-layer related structures. The formation of a hydrated phase (I) at low temperature and a completely dehydrated phase (III) at high temperature suggests the importance of thermodynamic factors in the formation of three compounds. Transformation studies of I in the presence of water shows the formation of a simple Zn-OBA compound, [Zn(OBA)(H2O)] (IV), at 150 and 180 degrees C and compound III at 200 degrees C. The compounds have been characterized by single-crystal X-ray diffraction, powder X-ray diffraction. thermogravimetric analysis, IR, and photoluminescence studies.

Synthesis, Structure, and Transformation Studies in a Family of Inorganic-Organic Hybrid Framework Structures Based on Indium

Eight new open-framework inorganic-organic hybrid compounds based on indium have been synthesized employing hydrothermal methods. All of the compounds have InO6, C2O4, and HPO3/HPO4/SO4 units connected to form structures of different dimensionality Thus, the compounds have zero- (I), two- (II, III, IV, V, VII, and VIII), and three-dimensionally (VI) extended networks. The formation of the first zero-dimensional hybrid compound is noteworthy In addition, concomitant polymorphic structures have been observed in the present study. The molecular compound, I, was found to be reactive, and the transformation studies in the presence of a base (pyridine) give rise to the polymorphic structures of II and III, while the addition of an acid (H3PO3) gives rise to a new indium phosphite with a pillared layer structure (T1). Preliminary density functional theory calculations suggest that the stabilities of the polymorphs are different, with one of the forms (II) being preferred over the other, which is consistent with the observed experimental behavior. The oxalate units perform more than one role in the present structures. Thus, the oxalate units connect two In centers to satisfy the coordination requirements as well as to achieve charge balance in compounds II, IV, and VI. The terminal oxalate units observed in compounds I, IV, and V suggest the possibility of intermediate structures. Both in-plane and out-of-plane connectivity of the oxalate units were observed in compound VI. The 31 compounds have been characterized by powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and P-31 NMR studies.

I*(P-2(1/2)) production from CH3I photodissociation at 236 nm

The quantum yield of I*((2)p(1/2)) production from CH3I photolysis at 236 nm in the gas phase has been measured as 0.69 +/- 0.03. The implication is that direct excitation to the (1)Q(1) excited state is significant at this wavelength. The dynamics of I* formation at other excitation energies covering the entire A-band of absorption of CH3I has been discussed in the light of this measurement.

Crystal and molecular structure of synthetic cholesterol compounds vitamin D3-analogues (I) 24(S)-hydroxy coprastan-3-one & (II) 24(R)-hydroxy coprastan-3-one

The title compound I (24-(S)-Hydroxy Coprastan-3-one) crystallises in orthorhombic space group P2(1)2(1)2(1) with Z = 4. The unit cell dimensions are a = 6.701(2)Angstrom, b = 11.506(8)Angstrom, c = 32.183(4)Angstrom, V = 2481(2)Angstrom (3), D-cal = 1.077 Mg/m(3). The tide compound II (24-(R)-Hydroxy Coprastan-3-one) crystallises in orthorhombic space group P212121 with two molecules per assymetric unit and with Z = 8. The Unit cell dimensions are a = 10.954(2)Angstrom, b = 21.757(6)Angstrom, c = 21.130(7)Angstrom, V = 5035.0(2)Angstrom (3), D-cal = 1.062 Mg/m(3). In compound I and in both the molecules of compound II, the rings A, B & C are in chair conformation and the five membered ring D is in envelope conformation. The priority sequence attached to the chiral carbon C24 has "S" designation in compound I and "R" designation in compound II. The structures are stabilized by C-H . . .O and O-H---O hydrogen bonds.

Electron flow through cytochrome P450

The cytochromes P450 (P450s) are a remarkable class of heme enzymes that catalyze the metabolism of xenobiotics and the biosynthesis of signaling molecules. Controlled electron flow into the thiolate-ligated heme active site allows P450s to activate molecular oxygen and hydroxylate aliphatic C–H bonds via the formation of high-valent metal-oxo intermediates (compounds I and II). Due to the reactive nature and short lifetimes of these intermediates, many of the fundamental steps in catalysis have not been observed directly. The Gray group and others have developed photochemical methods, known as “flash-quench,” for triggering electron transfer (ET) and generating redox intermediates in proteins in the absence of native ET partners. Photo-triggering affords a high degree of temporal precision for the gating of an ET event; the initial ET and subsequent reactions can be monitored on the nanosecond-to-second timescale using transient absorption (TA) spectroscopies. Chapter 1 catalogues critical aspects of P450 structure and mechanism, including the native pathway for formation of compound I, and outlines the development of photochemical processes that can be used to artificially trigger ET in proteins. Chapters 2 and 3 describe the development of these photochemical methods to establish electronic communication between a photosensitizer and the buried P450 heme. Chapter 2 describes the design and characterization of a Ru-P450-BM3 conjugate containing a ruthenium photosensitizer covalently tethered to the P450 surface, and nanosecond-to-second kinetics of the photo-triggered ET event are presented. By analyzing data at multiple wavelengths, we have identified the formation of multiple ET intermediates, including the catalytically relevant compound II; this intermediate is generated by oxidation of a bound water molecule in the ferric resting state enzyme. The work in Chapter 3 probes the role of a tryptophan residue situated between the photosensitizer and heme in the aforementioned Ru-P450 BM3 conjugate. Replacement of this tryptophan with histidine does not perturb the P450 structure, yet it completely eliminates the ET reactivity described in Chapter 2. The presence of an analogous tryptophan in Ru-P450 CYP119 conjugates also is necessary for observing oxidative ET, but the yield of heme oxidation is lower. Chapter 4 offers a basic description of the theoretical underpinnings required to analyze ET. Single-step ET theory is first presented, followed by extensions to multistep ET: electron “hopping.” The generation of “hopping maps” and use of a hopping map program to analyze the rate advantage of hopping over single-step ET is described, beginning with an established rhenium-tryptophan-azurin hopping system. This ET analysis is then applied to the Ru-tryptophan-P450 systems described in Chapter 2; this strongly supports the presence of hopping in Ru-P450 conjugates. Chapter 5 explores the implementation of flash-quench and other phototriggered methods to examine the native reductive ET and gas binding events that activate molecular oxygen. In particular, TA kinetics that demonstrate heme reduction on the microsecond timescale for four Ru-P450 conjugates are presented. In addition, we implement laser flash-photolysis of P450 ferrous–CO to study the rates of CO rebinding in the thermophilic P450 CYP119 at variable temperature. Chapter 6 describes the development and implementation of air-sensitive potentiometric redox titrations to determine the solution reduction potentials of a series of P450 BM3 mutants, which were designed for non-native cyclopropanation of styrene in vivo. An important conclusion from this work is that substitution of the axial cysteine for serine shifts the wild type reduction potential positive by 130 mV, facilitating reduction by biological redox cofactors in the presence of poorly-bound substrates. While this mutation abolishes oxygenation activity, these mutants are capable of catalyzing the cyclopropanation of styrene, even within the confines of an E. coli cell. Four appendices are also provided, including photochemical heme oxidation in ruthenium-modified nitric oxide synthase (Appendix A), general protocols (Appendix B), Chapter-specific notes (Appendix C) and Matlab scripts used for data analysis (Appendix D).