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PubMed2001

Insight into the chemistry of flavin reduction and oxidation in Escherichia coli dihydroorotate dehydrogenase obtained by rapid reaction studies.

Dihydroorotate dehydrogenase (DHOD) oxidizes dihydroorotate (DHO) to orotate in the only redox reaction of pyrimidine biosynthesis. The enzyme from Escherichia coli is a membrane-bound FMN-containing enzyme that is thought to use ubiquinone as the oxidizing substrate. The chemistry of the reduction of the flavin in DHOD from E. coli by the substrate dihydroorotate (DHO) was studied at 4 degrees C in anaerobic stopped-flow experiments conducted over a broad range of pH values. A Michaelis complex that was characterized by a approximately 20 nm red-shift of the oxidized flavin absorbance formed within the dead-time of the stopped-flow instrument ( approximately 1 ms) upon mixing with DHO. The flavin of the intermediate was reduced by DHO, forming a reduced flavin-orotate charge-transfer complex. The rate constant for the flavin reduction reaction increased with pH, from a value of 1 s(-1) at pH 6.5 to approximately 360 s(-1) at pH values greater than an observed pK(a) of 9.5 which was ascribed to Ser175, the active-site base. At all pH values, the reduced flavin-orotate charge-transfer complex dissociated too slowly to be catalytically relevant. Therefore, the oxidizing quinone substrate must bind to the reduced enzyme-orotate complex at a site distinct from the substrate binding site, in agreement with steady-state kinetic studies [Björnberg, O., Grüner, A.-C., Roepstorff, P., and Jensen, K. F. (1999) Biochemistry 38, 2899-2908]. Menadione was used as a model quinone substrate to oxidize dithionite-reduced DHOD. The reduced enzyme-orotate complex reacted rapidly with menadione (180 s(-1)), demonstrating that the reduced enzyme-orotate complex is a catalytically competent intermediate.

Anaerobiosis↗
PubMed1972

Photochemical electron transport in photosynthetic reaction centers from Rhodopseudomonas spheroides. I. Kinetics of the oxidation and reduction of P-870 as affected by external factors.

Photosynthetic reaction centers from Rhodopseudomonas spheroides were prepared with the detergent lauryl dimethylamine oxide (LDAO). In contrast to reaction centers made with Triton X-100, these contained no cytochromes and little or no ubiquinone (UQ). The reduction of P-870, after its photochemical oxidation, was studied in these materials with the following results. In reaction centers made with Triton X-100, slow kinetic components (seconds to minutes) could be attributed to secondary electron acceptors or traps. In reaction centers made with LDAO the kinetics were predominantly fast (half-times, 100 msec or less); slower components could be introduced by adding UQ. Added UQ appeared to become bound to reaction centers made with LDAO, but the binding might have meant only that both components were trapped within detergent micelles. Ferricyanide could retard the reduction of oxidized P-870, apparently by capturing electrons from the reducing side of the photochemical system. Under conditions in which the participation of secondary electron acceptors seemed to have been eliminated, the recovery of P-870 was mainly by a first-order process with a half-time of about 60 msec at room temperature and 20-30 msec at about -80 degrees C and below. The transition with decreasing temperature suggested the presence of a mixed population, exhibiting both the 60 and 20 msec components, but variations in the absorption spectra with temperature did not suggest the presence of a mixed population. Absorption difference spectra in the ultraviolet were compatible with the idea that UQ added to reaction centers became reduced in the light.

Detergents↗
PubMed1990

Characterization of oxidative and reductive metabolism in vitro of nitrofluoranthenes by rat liver enzymes.

Nitrofluoranthenes (NFs) are mutagenic and carcinogenic environmental pollutants found in incomplete combustion products and urban air particulate. We have studied both oxidative and reductive metabolism in vitro of different NF isomers mediated by subcellular rat liver fractions. Under aerobic conditions only ring hydroxylation of NFs by rat liver microsomes occurred and the isomeric position of the nitro group affected both the amount and the type of phenolic metabolites formed. Liver microsomes from 3-methylcholanthrene-induced rats were most effective in giving ring hydroxylated 7- and 8-nitrofluoranthene, whereas liver microsomes from phenobarbital-pretreated rats were the most active in metabolizing 1- and 3-nitrofluoranthene. Under anaerobic conditions, only reduction of NFs mediated by both cytosolic and microsomal rat liver enzymes occurred. Cofactor requirements and inhibition experiments indicated that the reductase activity in rat liver cytosolic fractions could be ascribed to DT-diaphorase, aldehyde oxidase and/or other unknown enzymes. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and n-octylamine, and slightly by cytochrome c; flavin mononucleotide greatly enhanced this activity. 3-Nitrofluoranthene microsomal nitroreductase activity was increased by phenobarbital rat pretreatment and this increment correlated well with the content of cytochrome P450. These results indicate a participation of cytochrome P450 in the reductive metabolism of NFs by rat liver microsomes.

Animals↗
PubMed1986

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide.

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.

Adrenocorticotropic Hormone↗
PubMed2004

Ineffectiveness of allopurinol in reduction of oxidative stress in diabetic patients; a randomized, double-blind placebo-controlled clinical trial.

The objective of this randomized, double-blind placebo-controlled clinical trial was to evaluate the value of allopurinol treatment on reduction of oxidative stress in patients with diabetes type II patients. Forty-one diabetic type II subjects were randomly assigned to two groups. One group (n = 20) received 100 mg allopurinol three times a day for 14 days and the other group (n = 21) received a placebo. Blood and saliva samples were collected before and after intervention for analysis of lipid peroxidation level and total antioxidant power as indices of oxidative stress. At the beginning of the study, the groups were similar based upon age, duration of diabetes, fasting glucose, and HbA1c. Both allopurinol and placebo were effective in reduction of lipid peroxidation and total antioxidant power whether in saliva or plasma in a similar extent. HbA1c and FBS levels did not change through the study neither in case or placebo group. It is concluded that allopurinol therapy is not more effective than placebo in reduction of oxidative stress in diabetic patients. The same trend of changes in blood and saliva shown for oxidative stress indices was interesting and suggests a chance for saliva to be valuable in diagnosis of oxidative stress. However, to elaborate the exact role of allopurinol in diabetes, further large randomized clinical trials are needed.

Allopurinol↗
PubMed1994

Reductive and oxidative half-reactions of glutathione reductase from Escherichia coli.

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH and has a redox active disulfide and an FAD cofactor in each monomer. In the reductive half-reaction, FAD is reduced by NADPH and electrons pass from the reduced flavin to the redox active disulfide. The oxidative half-reaction is dithiol-disulfide interchange between the enzyme dithiol and glutathione disulfide. We have investigated the reductive and oxidative half-reactions using wild-type glutathione reductase from Escherichia coli and in an altered form of the enzyme in which the active site acid-base catalyst, His439, has been changed to an alanine residue (H439A). H439A has 0.3% activity in the NADPH/GSSG assay. The replacement affects both the oxidative half-reaction, as expected, and the reductive half-reaction--specifically, the passage of electrons from reduced flavin to the disulfide. Reduction of H439A by NADPH allows direct observation of flavin reduction. The NADPH-FAD charge transfer complex is formed in the dead time. Reduction of FAD, at a limiting rate of 250 s-1, is observed as a decrease at 460 nm and an increase at 670 nm (FADH(-)-NADP+ charge transfer). Subsequent passage of electrons from FADH- to the disulfide (increase at 460 nm and a decrease at 670 nm) is very slow (6-7 s-1) and concentration independent in H439A. The monophasic oxidative half-reaction is very slow, as expected for reduced H439A.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1975

Intermolecular complexes between N-methyl-1,4-dihydronicotinamide and flavines. The influence of steric and electronic factors on complex formation and the rate of flavine-dependent dihydronicotinamide dehydrogenation.

The reaction of N-methyldihydronicotinamide (NMNH) with flavine analogs saturates at high dihydronicotinamide concentrations. Complex formation between the reactants depends mainly on steric but not on electronic factors. Thus flavine analogs that differ up to 243 mV in their oxidation-reduction potential vary only between 0.09 and 0.17 M in Kd. When the flavine plane becomes blocked by bulky substituents, however, complex stability decreases by more than an order of magnitude. NMNH-flavine complexes show long wave optical absorption. The energy of the long wave transition decreases with increasing oxidation-reduction potential of the flavine as expected for charge transfer complexes. The first-order rate constants of flavine-dependent dihydronicotinamide dehydrogenation increase with increasing oxidation-reduction potential of the flavine but they are almost independent of Kd. The reaction is not subject to general acid-base catalysis. Thus flavine-dependent dihydronicotinamide dehydrogenation may be interpreted to proceed via a charge transfer complex between oxidized flavine and reduced nicotinamide. In the rate-limiting conversion of the charge transfer complex into products hydrogen is transferred directly, the rate being governed by the difference in oxidation-reduction potential between flavine and dihydronicotinamide. An alternative mechanism where the observed charge transfer complex is not on the reaction pathway appears to be improbable but cannot be eliminated.

Binding Sites↗
PubMed1999

Bactericidal effect of electrolyzed neutral water on bacteria isolated from infected root canals.

OBJECTIVE: The purposes of this study were to examine the time-related changes in pH, oxidation-reduction potential, and concentration of chlorine of electrolyzed neutral water and to evaluate the bactericidal effect of electrolyzed neutral water against bacteria from infected root canals. STUDY DESIGN: Various properties of electrolyzed neutral water--pH value, oxidation-reduction potential, and concentration of chlorine--were measured at different times after storage of the water in the open state, the closed state, or the closed-and-dark state. The bactericidal effect of the various electrolyzed neutral water samples was then tested against 17 strains of bacteria, including 15 strains isolated from infected canals, as well as against 1 strain of fungus. Each bacterial or fungal suspension was mixed with electrolyzed neutral water, and the 2 substances were reacted together for 1 minute. After incubation for 1 to 7 days, the bactericidal effect of the electrolyzed neutral water was determined. RESULTS: The pH value and oxidation-reduction potential of electrolyzed neutral water remained almost unchanged when the water was stored in a dark, closed container. However, the concentration of chlorine decreased from 18.4 ppm to 10.6 ppm. Electrolyzed neutral water showed a bactericidal or growth-inhibitory effect against the bacteria. CONCLUSIONS: The results indicate that electrolyzed neutral water maintains a constant pH and oxidation-reduction potential when kept in a closed container without light and that it exhibits a bacteriostatic/bactericidal action against isolates obtained from infected root canals.

Anti-Infective Agents, Local↗
PubMed1987

Determination and metabolism of dithiol-chelating agents: electrolytic and chemical reduction of oxidized dithiols in urine.

The presence of oxidized species of the dithiol-chelating agents, meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1-sulfonic acid (DMPS), in human urine was determined by chemical and electrolytic reduction methods. Urine from a human given either DMSA or DMPS was treated with electrolysis, dithiothreitol, or sodium tetrahydridoborate (NaBH4). The SH groups were derivatized with monobromobimane for the determination of unaltered dithiols. Total dithiol (unaltered and oxidized) was determined by reduction followed by derivatization with monobromobimane. The bimane derivatives were identified and quantified by HPLC and fluorescence. Although all three reduction methods gave similar results, electrolytic reduction of oxidized DMSA and chemical reduction with NaBH4 of oxidized DMPS are recommended based upon both day to day reproducibility and recovery of standards. After reduction a 4-fold increase in DMSA and a 20-fold increase in DMPS were found in urine by 12 h after an oral dose of DMSA or DMPS. These new methods for the determination of dithiols and their oxidized forms should lead to a better understanding of the metabolic properties of these increasingly important orally effective chelating agents.

Borohydrides↗
PubMed2000

Formation of W(3)A(1) electron-transferring flavoprotein (ETF) hydroquinone in the trimethylamine dehydrogenase x ETF protein complex.

The electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)) exhibits unusual oxidation-reduction properties and can only be reduced to the level of the semiquinone under most circumstances (including turnover with its physiological reductant, trimethylamine dehydrogenase (TMADH), or reaction with strong reducing reagents such as sodium dithionite). In the present study, we demonstrate that ETF can be reduced fully to its hydroquinone form both enzymatically and chemically when it is in complex with TMADH. Quantitative titration of the TMADH x ETF protein complex with sodium dithionite shows that a total of five electrons are taken up by the system, indicating that full reduction of ETF occurs within the complex. The results indicate that the oxidation-reduction properties of ETF are perturbed upon binding to TMADH, a conclusion further supported by the observation of a spectral change upon formation of the TMADH x ETF complex that is due to a change in the environment of the FAD of ETF. The results are discussed in the context of ETF undergoing a conformational change during formation of the TMADH x ETF electron transfer complex, which modulates the spectral and oxidation-reduction properties of ETF such that full reduction of the protein can take place.

Bacterial Proteins↗
PubMed1976

Oxidation and reduction of membrane-bound cytochrome c in Hemophilus parainfluenzae. Reaction with oxygen, hydrogen peroxide and nitrate.

Cytochromes of the a-, b-, c- and d-type become reduced when intact cells of Hemophilus parainfluenzae have become anaerobic following respiration with substrates such as formate or succinate, as shown previously (J. Biol. Chem. (1970) 254, 5096-5100). In the presence of formate after depletion of O2, there is an unusual two-step time course of reduction of the membrane-bound cytochrome c. The proportion of the cytochrome c which is reduced during the second stage is oxidizable by either nitrate or H2O2 and is reduced again when the nitrate or H2O2 have been depleted. We conclude that the observed two-stage reduction of cytochrome c results from the presence of an oxidant, probably H2O2, produced by reaction of formate dehydrogenase with O2. This was shown by the effects of cyanide, catalase and O2. In addition, no evidence for the production of the oxidant is seen when succinate is the substrate oxidized. Although measurements of absorption spectra indicated only one species of cytochrome c, kinetic evidence is presented for some separation of the cytochrome c into more than one electron transport pathway.

Binding Sites↗
PubMed1998

A reductant-induced oxidation mechanism for complex I.

A model for energy conversion in Complex I is proposed that is a conservative expansion of Mitchell's Q-cycle using a simple mechanistic variation of that already established experimentally for Complex III. The model accommodates the following proposals. (1) The large number of flavin and iron-sulfur redox cofactors integral to Complex I form a simple but long electron transfer chain guiding submillisecond electron transfer from substrate NADH in the matrix to the [4Fe-4S] cluster N2 close to the matrix-membrane interface. (2) The reduced N2 cluster injects a single electron into a ubiquinone (Q) drawn from the membrane pool into a nearby Qnz site, generating an unstable transition state semiquinone (SQ). The generation of a SQ species is the primary step in the energy conversion process in Complex I, as in Complex III. In Complex III, the SQ at the Qo site near the cytosolic side acts as a strong reductant to drive electronic charge across the membrane profile via two hemes B to a Qi site near the matrix side. We propose that in Complex I, the SQ at the Qnz site near the matrix side acts as a strong oxidant to pull electronic charge across the membrane profile via a quinone (Qny site) from a Qnx site near the cytosolic side. The opposing locations of matrix side Qnz and cytosolic side Qo, together with the opposite action of Qnz as an oxidant rather than a reductant, renders the Complex I and III processes vectorially and energetically complementary. The redox properties of the Qnz and Qo site occupants can be identical. (3) The intervening Qny site of Complex I acts as a proton pumping element (akin to the proton pump of Complex IV), rather than the simple electron guiding hemes B of Complex III. Thus the transmembrane action of Complex I doubles to four (or more) the number of protons and charges translocated per NADH oxidized and Q reduced. The Qny site does not exchange with the pool and may even be covalently bound. (4) The Qnx site on the cytosol side of Complex I is complementary to the Qi site on the matrix side of Complex III and can have the same redox properties. The Qnx site draws QH2 from the membrane pool to be oxidized in two single electron steps. Besides explaining earlier observations and making testable predictions, this Complex I model re-establishes a uniformity in the mechanisms of respiratory energy conversion by using engineering principles common to Complexes III and IV: (1) all the primary energy coupling reactions in the different complexes use oxygen chemistry in the guise of dioxygen or ubiquinone, (2) these reactions are highly localized structurally, utilizing closely placed catalytic redox cofactors, (3) these reactions are also highly localized energetically, since virtually all the free energy defined by substrates is conserved in the form of transition state that initiates the transmembrane action and (4) all complexes possess apparently supernumerary oxidation-reduction cofactors which form classical electron transfer chains that operate with high directional specificity to guide electron at near zero free energies to and from the sites of localized coupling.

Electron Transport↗
PubMed1989

The role of reductive and oxidative metabolism in the toxicity of mitoxantrone, adriamycin and menadione in human liver derived Hep G2 hepatoma cells.

The cytotoxic properties of quinones, such as menadione, are mediated through one electron reduction to yield semi-quinone radicals which can subsequently enter redox cycles with molecular oxygen leading to the formation of reactive oxygen radicals. In this study the role of reduction and oxidation in the toxicity of mitoxantrone was studied and its toxicity compared with that of adriamycin and menadione. The acute toxicity of mitoxantrone was not mediated through one-electron reduction, since inhibition of the enzymes glutathione reductase and catalase, responsible for protecting the cells against oxidative damage, did not affect its toxicity. Adriamycin was the most potent inhibitor of protein and RNA synthesis of the three quinones. Menadione, at concentrations up to 25 microM, did not inhibit either protein or RNA synthesis unless dicoumarol, an inhibitor of DT-diaphorase, was also present. The two-electron reduction of menadione by DT-diaphorase is therefore a protective mechanism in the cell. This enzyme also protected against the toxicity of high concentrations (100 microM) of mitoxantrone. The inhibitory effect of mitoxantrone, but not of menadione or adriamycin, on cell growth was prevented by inhibiting the activity of cytochrome P450-dependent mixed function oxidase (MFO) system using metyrapone. This suggests that mitoxantrone is oxidised to a toxic intermediate by the MFO system.

Carcinoma, Hepatocellular↗