Mechanism of reaction of a suggested superoxide-dismutase mimic, Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The interaction of a recently developed intracellular superoxide dismutase analogue, Fe(II)-N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (Fe(II)-TPEN), with reactive oxygen species was investigated under in vitro conditions. The complex catalyzed the dismutation of enzyme- or radiolysis-generated superoxide with the production of H2O2; under steady-state conditions the equilibrium was strongly shifted toward Fe(III)-TPEN. Fe(II)-TPEN reacted with H2O2 to generate hydroxyl radicals in a Fenton reaction. The oxidized Fe(III)-TPEN was readily reduced by ascorbate or glutathione. Given the capacity to produce hydroxyl radicals and the reaction with cellular reductants it seems unlikely that Fe-TPEN may find widespread use as an intracellular superoxide dismutase substitute.     

The antioxidant action of taurine, hypotaurine and their metabolic precursors.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.     

Cytochrome P450 3A4-catalyzed testosterone 6beta-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps

Testosterone 6beta-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-(2)H(5); (ii) 6,6-(2)H(2); (iii) 6alpha-(2)H; (iv) 6beta-(2)H; and (v) 6beta-(3)H testosterone. Only the 6beta-hydrogen was removed by P450 3A4 and not the 6alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6beta-(1)H-, 6beta-(2)H-, and 6beta-(3)H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K(m) in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6beta-(2)H substitution. The high (D)k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high (D)k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high (D)k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.     

Stoichiometric production of hydrogen peroxide and parallel formation of ferric multimers through decay of the diferric-peroxo complex,the first detectable intermediate in ferritin mineralization

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F(peroxo), formed within 25 ms, which then leads to the release of H(2)O(2) and formation of ferric mineral precursors. The stoichiometric relationship between F(peroxo), H(2)O(2), and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H(2)O(2). By rapidly mixing apo M ferritin from frog, Fe(2+), and O(2) and allowing the reaction to proceed for 70 ms when F(peroxo) has reached its maximum accumulation, followed by spraying the reaction mixture into the H(2)O(2) assay solution, we were able to quantitatively determine the amount of H(2)O(2) produced during the decay of F(peroxo). The correlation between the amount of H(2)O(2) released with the amount of F(peroxo) accumulated at 70 ms determined by M?ssbauer spectroscopy showed that F(peroxo) decays into H(2)O(2) with a stoichiometry of 1 F(peroxo):H(2)O(2). When the decay of F(peroxo) was monitored by rapid freeze-quench M?ssbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F(peroxo). This observed parallel formation of multiple products (H(2)O(2), diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F(peroxo)) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.     

Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance

To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5.     

Stereochemistry of meso-alpha,epsilon-diaminopimelate decarboxylase reaction: the first evidence for pyridoxal 5'-phosphate dependent decarboxylation with inversion of configuration

The stereochemistry of the decarboxylation of meso-alpha,epsilon-diaminopimelate catalyzed by meso-alpha,epsilon-diaminopimelate decarboxylase (EC 4.1.1.20) of Bacillus sphaericus was determined by stereochemical analyses of [6-2H]-L-lysine produced by the reaction in D2O. The product [6-2H]-L-lysine was converted to levorotatory methyl 5-phthalimido[5-2H]valerate by the reactions not affecting the absolute configuration of the asymmetric carbon atom. By contrast, methyl 5-phthalimido[5-2H]valerate derived from [2,6-2H2]-L-lysine, which was produced from [2,6-2H2]diaminopimelate by decarboxylation in H2O, was dextrorotatory. The authentic methyl (R)-5-phthalimido[5-2H]valerate prepared from L-glutamate with glutamate decarboxylase was levorotatory. These results indicate that the meso-alpha,epsilon-diaminopimelate decarboxylase reaction proceeds in an inversion mode. The deuterium label in [6-2H]-L-lysine was fully conserved during the conversion into pelletierine through [1-2H]cadaverine by the stereospecific diamine oxidase reaction. Thus, the enzymatic decarboxylation of meso-alpha,epsilon-diaminopimelate occurs with inversion of configuration in contrast to the other amino acid decarboxylase reported so far.     

Coordination compounds derived from the interaction of streptomycin and cobalt, nickel, copper, and calcium salts characterized by 13C NMR and spectroscopic studies. Structure and bonding properties of the streptidine fraction

The coordination compounds of streptomycin (St), Co2(St)Cl4.13H2O (2), Co2(St)(NO3)4.7H2O (3), Ni2(St)Cl4.14H2O (4), Ni2(St)(NO3)4.14H2O (5), Cu2(St)Cl4.6H2O (6), and Ca(St)Cl2.8H2O (7) have been synthesized by the reaction of streptomycin sulfate (1) with three equivalents of the corresponding inorganic salt. The compounds (2)-(7) were characterized by electronic spectroscopy (in the solid state and in solution) by conductivity measurements and by 13C NMR in solution. The reaction of streptomycin with CuCl2 in water hydrolyzed the molecule giving the copper complex of the streptidine fraction (Std), Cu(Std)Cl.H2O (8). This compound was characterized by the same techniques. Detailed x-ray diffraction and 13C NMR studies of streptidine sulfate (9) were carried out.     

Stopped-flow kinetic study of vitamin E regeneration reaction with biological hydroquinones (reduced forms of ubiquinone, vitamin K, and tocopherolquinone) in solution.

A kinetic study of the regeneration reaction of vitamin E (tocopherol) with eight biological hydroquinones (HQs) (ubiquinol-10 (Q10H2 1); ubiquinol-0 (Q0H2 2); vitamin K1 HQ (VK1H2 3); vitamin K3 HQ (VK3H2 4); alpha-, beta-, and gamma-tocopherol-HQs (alpha-, beta-, and gamma-TQH2 5-7); and 2,3,5-trimethyl-1,4-HQ (TMQH2 8)) in solution was performed. The second-order rate constants (k4) for the reaction of HQs 1-8 with alpha-tocopheroxyl and 5,7-diisopropyltocopheroxyl radicals in ethanol, benzene, and isopropyl alcohol/water (5:1, v/v) solutions were measured with a stopped-flow spectrophotometer. The order of magnitude of k4 values obtained for HQs is VK1H2 > VK3H2 > alpha-TQH2 > beta-TQH2 approximately gamma-TQH2 approximately TMQH2 > Q10H2 > Q0H2, being independent of the kinds of tocopheroxyl radicals and the polarity of the solvents. The log of the k4 values obtained for HQs was found to correlate with their peak oxidation potentials. Comparing the k2 value (2.68 x 10(6) M-1 s-1 obtained for the reaction of alpha-tocopheroxyl with vitamin C (sodium ascorbate) with those (k4 = 2.54 x 10(5) and 8.15 x 10(5) M-1 s-1) obtained for the reaction of alpha-tocopheroxyl with Q10H2 and alpha-TQH2 in isopropyl alcohol/water mixtures, the former is approximately 11 and 3 times as reactive as the latter, respectively. On the other hand, the k2 value obtained for sodium ascorbate is smaller than the k4 values obtained for VK1H2 and VK3H2. These results suggest that mixtures of vitamin E and these HQs (as well as those of vitamins E and C) may function synergistically as antioxidants in various tissues and mitochondria.     

Browning of furfural and amino acids, and a novel yellow compound, furpipate, formed from lysine and furfural

Furfural is an important intermediate compound of the Maillard reaction of pentose or ascorbic acid. We examined the browning of furfural and lysine by heating and found a yellow compound, called furpipate, (E)-3-(2-furylmethylidene)-3H, 4H, 5H, 6H-pyridine-2-carboxylic acid. Furpipate is a novel pipecolic acid derivative and shows absorption maxima at 375 nm and 310 nm under acidic and alkaline conditions, respectively. This compound was the major colored compound of the heated solution containing lysine and furfural.     

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.     

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Role of histone pairs H2A,H2B and H3,H4 in the self-assembly of nucleosome core particles

The role of the histone pairs H2A,H2B and H3,H4 in the kinetics of core particle formation was investigated by using N-(1-pyrene)maleimide-labeled histone H3. The excimer emission intensity of a DNA-core histone complex prepared by direct mixing of DNA and histones in 0.2 m-NaCl is reduced by half when H2A,H2B is omitted. Fluorescence quenching studies and lifetime measurements indicate that the emission differences are probably due to static quenching. In a correctly folded nucleosome or a DNA-(H3,H4) complex, the two pyrene rings are buried and are held very close. DNA-(H3,H4) can interact with additional copies of H3,H4, but only when two dimers of H2A,H2B are correctly bound is there a specific twofold increase in excimer emission.The kinetics of the reaction of H3,H4 with DNA in 0.2 m-NaCl were followed by measuring the increase in 460 nm fluorescence. The apparent rate constant of the dominant kinetic component is ~ 2 × 10^?1 s^?1. If histones H2A,H2B are added immediately after the preparation of the DNA-(H3,H4) complex, an increase in excimer fluorescence is observed, with an apparent rate constant of ~ 6 × 10^?3 s^?1. However, if histones H2A,H2B are added one hour after DNA-(H3,H4) complex formation, there is no increase in excimer fluorescence. These results suggest that an intermediate involving the H3,H4 tetramer is formed first in nucleosome assembly. In the presence of H2A,H2B, this intermediate evolves to the final folded nucleosome, but in the absence of H2A,H2B it rearranges to an unmaturable dead-end complex. Additional experiments show that a very fast transfer of histone pairs (probably H2A,H2B) can take place between partially reconstituted nucleosomes.     

The contribution of the nuclear reaction 1H(n, gamma)2D to the yield of DNA single strand breaks in cultured mammalian cells irradiated by thermal neutrons

The yield of single strand breaks (ssb) in DNA of the HeLa S-3 cells after thermal neutron irradiation was examined using the alkaline sucrose gradient method. The contribution of the 1H(n, gamma)2D reaction to the yield of ssb was determined by substituting D2O for H2O in the irradiated medium. Calculation shows that when cells are irradiated in the H2O medium, the per cent contribution of the contaminating gamma-rays, the nuclear reaction 1H(n, gamma)2D and the other nuclear reactions is 31, 44 and 25 per cent respectively assuming additivity of effects. The estimated number of ssb induced by the nuclear reaction 1H(n, gamma)2D was at least 4.4 times greater than that by 60Co gamma-rays at the same absorbed dose. Two possible interpretations are discussed to explain the high efficiency of the 1H(n, gamma)2D reaction for ssb induction.     

Intrinsic activity of guanosine 3',5'-monophosphate-dependent protein kinase similar to adenosine 3',5'-monophosphate-dependent protein kinase. I. Phosphorylation of histone fractions.

Guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinase partially purified from silkworm pupae reacts preferentially with H1, H2A, and H2B histones but not with H3 AND H4 histones. However, the latter can serve as substrates in the presence of a stimulatory modulator as described by Kuo and Kuo (J. Biol. Chem. 251, 4283-4286 (1976)). With H2B histone as substrate high Mg2+ concentrations (50-100 mM) are necessary for the maximum rate of reaction. Although effects of the modulator and Mg2+ vary significantly with the histone fractions employed, analysis on the phosphorylation of histone fractions provides evidence that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of adenosine 3',5'-monophosphate-dependent protein kinase.     

Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.     

H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.     

Redox intermediates in the catalase cycle of catalase-peroxidases from Synechocystis PCC 6803, Burkholderia pseudomallei, and Mycobacterium tuberculosis

Monofunctional catalases (EC 1.11.1.6) and catalase-peroxidases (KatGs, EC 1.11.1.7) have neither sequence nor structural homology, but both catalyze the dismutation of hydrogen peroxide (2H2O2 --> 2H2O + O2). In monofunctional catalases, the catalatic mechanism is well-characterized with conventional compound I [oxoiron(IV) porphyrin pi-cation radical intermediate] being responsible for hydrogen peroxide oxidation. The reaction pathway in KatGs is not as clearly defined, and a comprehensive rapid kinetic and spectral analysis of the reactions of KatGs from three different sources (Synechocystis PCC 6803, Burkholderia pseudomallei, and Mycobacterium tuberculosis) with peroxoacetic acid and hydrogen peroxide has focused on the pathway. Independent of KatG, but dependent on pH, two low-spin forms dominated in the catalase cycle with absorbance maxima at 415, 545, and 580 nm at low pH and 418 and 520 nm at high pH. By contrast, oxidation of KatGs with peroxoacetic acid resulted in intermediates with different spectral features that also differed among the three KatGs. Following the rate of H2O2 degradation by stopped-flow allowed the linking of reaction intermediate species with substrate availability to confirm which species were actually present during the catalase cycle. Possible reaction intermediates involved in H2O2 dismutation by KatG are discussed.     

Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase.

The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate.     

Biophotonic hydrogen peroxide production by antibodies, T cells, and T-cell membranes

Rapidly accumulating evidence indicates that inflammatory T cells sensitively respond to their redox environment by activating signal transduction pathways. The hypothesis that T-cell receptors have the potential to catalytically transform singlet oxygen into H(2)O(2) attracted our attention since the biophysical regulation of this process would provide a new tool for therapeutically directing T cells down a preferred signaling pathway. Light-dependent production of H(2)O(2) was first described in antibodies, and we reproduced these findings. Using a real-time H(2)O(2) sensor we extended them by showing that the reaction proceeds in a biphasic way with a short-lived phase that is fast compared to the slow second phase of the reaction. We then showed that Jurkat T cells biophotonically produce about 30nM H(2)O(2)/min/mg protein when pretreated with NaN(3). This activity was concentrated 4 to 5 times in T-cell membrane preparations. The implications of these observations for the development of new therapeutic tools for inflammatory diseases are discussed.