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PubMed2003

In vivo changes of the oxidation-reduction state of NADP and of the ATP/ADP cellular ratio linked to the photosynthetic activity in Chlamydomonas reinhardtii.

The ATP/ADP and NADP/NADPH ratios have been measured in whole-cell extract of the green alga Chlamydomonas reinhardtii, to understand their availability for CO(2) assimilation by the Calvin cycle in vivo. Measurements were performed during the dark-light transition of both aerobic and anaerobic cells, under illumination with saturating or low light intensity. Two different patterns of behavior were observed: (a) In anaerobic cells, during the lag preceding O(2) evolution, ATP was synthesized without changes in the NADP/NADPH ratio, consistently with the operation of cyclic electron flow. (b) In aerobiosis, illumination increased the ATP/ADP ratio independently of the intensity used, whereas the amount of NADPH was decreased at limiting photon flux and regained the dark-adapted level under saturating photon flux. Moreover, under these conditions, the addition of low concentrations of uncouplers stimulated photosynthetic O(2) evolution. These observations suggest that the photosynthetic generation of reducing equivalents rather than the rate of ATP formation limits the photosynthetic assimilation of CO(2) in C. reinhardtii cells. This situation is peculiar to C. reinhardtii, because neither NADPH nor ATP limited this process in plant leaves, as shown by their increase upon illumination in barley (Hordeum vulgare) leaves, independent of light intensity. Experiments are presented and were designed to evaluate the contribution of different physiological processes that might increase the photosynthetic ATP/NADPH ratio-the Mehler reaction, respiratory ATP supply following the transfer of reducing equivalents via the malate/oxaloacetate shuttle, and cyclic electron flow around PSI-to this metabolic situation.

Adenosine Diphosphate↗
PubMed1995

Drosophila insulin receptor: lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation.

The glycosylation of the Drosophila insulin receptor (DIR) has been compared to that of the rat insulin receptor by examining the binding of receptors to the lectins wheat germ agglutinin, Concanavalin-A, and lentil lectin. Although rat insulin receptors bound and were specifically eluted from all three lectins, only a small fraction of the DIR (< 5%) was retained on wheat germ agglutinin. In contrast, the DIR bound strongly to Concanavalin-A and lentil lectin and was recovered from lentil lectin columns after elution with alpha-methyl-mannoside. The pattern of lectin binding indicates that glycosylation of the DIR and rat insulin receptors differs, with the DIR containing primarily high mannose-type oligosaccharides. After lectin chromatography, the DIR exhibited an elevated level of basal autophosphorylation and kinase activity, which could be restored to a low level by incubation with 0.5 mM dithiothreitol (DTT). DTT did not, however, affect ligand-stimulated kinase activity. The ability of low concentrations of DTT to deactivate the DIR kinase suggests that, like the mammalian receptor, beta-subunit thiols may be involved in regulation of conformational changes between activated and unactivated receptor states. Interestingly, DTT-induced deactivation of the DIR was blocked by preincubation with an antipeptide antibody against the carboxy-terminal domain of the DIR. This suggests that the DIR carboxyl terminus undergoes a conformational change during the activation-inactivation cycle of the kinase, which can be sterically hindered by the antibody. Conformational changes in this region of the mammalian receptor have been observed, and these data suggest that features of the insulin receptor activation mechanism have been substantially conserved during evolution.

Amino Acid Sequence↗
PubMed1995

[Study of squirrel myoglobin. Oxidation-reduction reaction between squirrel oxymyoglobin and ferricytochrome c].

Metmyoglobin (met-Mb) of Yakutian ground squirrel (Citellus undulatus) was isolated from skeletal muscles and fractionated using gel and ion-exchange chromatography. An electrophoretically homogeneous major fraction of met-Mb (pI 7.05) was obtained with a 90% yield. The rates of the redox reaction between ground squirrel oxy-Mb and horse and ground squirrel ferri-Cyt c were studied in the pH range of 5-8 at ionic strengths ranging from 0.01 to 1. The experimental results were compared to the earlier obtained data for sperm whale and pig Mbs whose three-dimensional structures and physico-chemical properties are well studied. It was shown that the properties of ground squirrel Mb are very similar to those of pig Mb but differ from those of whale Mb. The amino acid sequences of about 50 Mbs from various mammals as well as from some avian and chordate species (Protein Data Bank) were compared. It was found that rodent myoglobins can be divided into two distinct groups. Myoglobins of animals living in water are identical with those of sperm whale and other cetaceans, while myoglobins of ground rodents closely resemble pig Mb. The number of non-homologous amino acid substitutes in each of the two groups (1-2 positions) is much less compared to all rodent myoglobins (8 positions). The charge structure of the Mb contact site interacting with Cyt c in the course of the electron transfer reaction is strictly conservative for all mammalian Mbs tested in this study (but not for birds and chordates). It is assumed that under certain extreme conditions oxy-Mb can directly interact with cell organelles, such as mitochondria.

Animals↗
PubMed2006

Biological decolorization of reactive anthraquinone and phthalocyanine dyes under various oxidation-reduction conditions.

The decolorization of two anthraquinone dyes (Reactive Blue 4 [RB4] and Reactive Blue 19 [RB19]) and two phthalocyanine dyes (Reactive Blue 7 [RB7] and Reactive Blue 21 [RB21]) was investigated at an initial dye concentration of 300 mg/L using an unacclimated, enrichment culture. The culture was fed a mixture of organic compounds and maintained initially under aerobic conditions, and then progressively developed anoxic/ anaerobic conditions. Biotransformation-related decolorization of the dyes did not take place under aerobic conditions, but use of the feed organic mixture and biomass production by the enrichment culture were not affected. Complete ammonia removal occurred in the control and all dye-amended cultures. The development and extent of nitrification were much lower in the latter cultures, in which ammonia removal via air stripping was the dominant mechanism. Prolonged incubation of the culture under anoxic/anaerobic conditions with multiple carbon source additions resulted in a high decolorization extent of anthraquinone dyes (over 84%) and only partial decolorization of phthalocyanine dyes (49 to 66%). Development of significant methanogenic activity took place in the control and, to a lesser extent, in the two phthalocyanine dye-amended cultures, but the anthraquinone dyes severely inhibited the development of methanogenic activity. The RB4 and RB19 decolorization was attributed to nonreversible, microbially mediated dye transformation(s), demonstrated by the accumulation of decolorization products with absorbance maxima in the 420- to 460-nm region. The decolorization of RB4 and RB19 followed Michaelis-Menten kinetics. At an initial dye concentration of 300 mg/L, the observed maximum decolorization rate per unit biomass was 9.1 and 37.5 mg dye/mg volatile suspended solids x day for the RB4 and RB19, respectively. Thus, partial decolorization of reactive phthalocyanine dyes and extensive biological decolorization of reactive anthraquinone dyes is feasible only under anoxic/anaerobic conditions.

Anthraquinones↗
PubMed1987

On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes.

We have confirmed the propensity of fragments of cytochrome c to form complexes that reproduce the structure and, in part, the functionality, of the native protein by preparing four novel complexes. We have used trypsin under three different sets of conditions in sequence to prepare a contiguous two-fragment complex (1-55).(56-104). One of the intermediates is a stable overlapping complex (1-65).(56-104). Conditions for limited acid hydrolysis of peptide bonds in cytochrome c have been developed that optimize the yield of fragments (1-50) and (51-104). These two fragments also form a stable association, as do (1-50) and (56-104). These complexes are potentially useful for the semisynthesis of analogues modified in the region of the cleavage sites, which include a number of highly conserved amino acid residues, and are being used for studies of protein folding, interactions with oxidase, cytochrome c immunogenicity and of artificially induced spontaneous resyntheses between complexing fragments. Like other known two-fragment complexes of cytochrome c, they exhibit normal visible spectra, including the presence of the 695 nm band, indicative of a functional haem crevice. Studies of their biological activities and redox potentials lead to a number of conclusions on structure-function relationships in cytochrome c. Most significantly there is a linear relationship between the logarithm of electron-transfer rates from cytochrome c reductase and redox potential in this series of analogues, indicating that such transfer is thermodynamically controlled. This discovery contributes to our understanding of the interaction of cytochrome and reductase. Since the relationship is obeyed by other types of analogues, except for those that involve modification of the active site of cytochrome c, we have a useful diagnostic for those residues that participate directly in electron transfer.

Chromatography, Ion Exchange↗
PubMed1995

Electrostatic effects of surface acidic amino acid residues on the oxidation-reduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough).

The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor. These proteins function as low-potential one-electron transferases in bacteria. A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function. The structural basis of this phenomenon is not yet thoroughly understood. In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated. These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor. Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate. Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations. There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted. However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type. These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution. Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family, apparently through the destabilization of the flavin hydroquinone anion.

Amino Acids, Dicarboxylic↗
PubMed1994

Oxidation-reduction properties of short-chain acyl-CoA dehydrogenase: effects of substrate analogs.

Redox potentials of short-chain acyl-CoA dehydrogenase from the anaerobe, Megasphaera elsdenii, have been determined by means of uv-visible spectroelectrochemistry in the presence of substrate analogs. During redox titrations in the presence of 2-azabutyryl-CoA, up to 85% anionic FAD semiquinone was stabilized with a molar absorbance at 387 nm of 19 mM-1 cm-1. Despite a slow reduction of short-chain acyl-CoA dehydrogenase by 2-azabutyryl-CoA (< 2% reduction/h), a dissociation constant of 0.7 microM was measured and redox potentials, E1(0') and E2(0'), of -0.07 and -0.17 V, respectively, were determined at pH 7.0 for the first and second electrons in reduction of the FAD of short-chain acyl-CoA dehydrogenase. The analog, 2-azoctanoyl-CoA, did not reduce short-chain acyl-CoA dehydrogenase, bound with a dissociation constant of 2 microM, stabilized up to 47% anionic FAD semiquinone, and gave values of -0.08 and -0.11 V for E1(0') and E2(0') at pH 6.9. In contrast to 2-aza-acyl-CoA, the thioethers, butyl-CoA, octyl-CoA, and allyl-CoA, and the thioester, acetyl-CoA, did not bind strongly (Kd > or = 50 microM) and caused no significant change in the redox properties of short-chain acyl-CoA dehydrogenase. The two-electron redox potential, Em, remained at -0.08 V at pH 7.0 and there was no stabilization of FAD semiquinone in the presence of these analogs. These results show that no single feature of substrate structure, the thioester carbonyl, the presence of 2,3-unsaturation, or a fatty alkyl chain of appropriate length, can account for the 0.06-V positive change in redox potential which is observed in the presence of the substrate couple, crotonyl-CoA/butyryl-CoA (M. T. Stankovich and S. Soltysik (1987) Biochemistry 26 2627-2632). As outlined above, 2-azabutyryl-CoA or 2-azaoctanoyl-CoA did cause marked changes in the redox properties of short-chain acyl-CoA dehydrogenase, but the preferential stabilization of FAD semiquinone and negative change in Em distinguish the effects of 2-azaacyl-CoA from those of the substrate couple.

Acyl Coenzyme A↗
PubMed1975

The oxygenated complexes of the two catalytically active oxidation-reduction states of L-tryptophan-2,3-dioxygenase.

The oxygenated complexes of the two catalytically active forms of pseudomonad and rat liver L-tryptophan-2,3-dioxygenase (EC 1.13.11.11) have been studied. As was previously reported (ISHIMURA, Y., NORZAKI, M., HAYAISHI, O., TAMURA, M., AND YAMAZAK-I I. (1970) J. Biol. Chem. 245, 3593-3602), we observe that the fully reduced form of pseudomonad tryptophan oxygenase during steady state catalysis exists predominantly as the L-tryptophan ferroheme-O2 enzyme complex (lambdamax = 415 nm, 540 nm, 570 nm). However, during steady state catalysis by a half-reduced form of both the pseudomonad and hepatic enzymes, the predominant species present manifest absorption spectra indicative of ternary complexes in which all the heme exists as ferriheme (Soret, 407 nm), there being no trace of a ferroheme-O2 complex. Carbon monoxide is a competitive inhibitor with respect to molecular oxygen of catalysis by either the half-reduced or fully reduced forms of pseudomonad tryptophan oxygenase. During steady state catalysis in the presence of CO, the fully reduced form of the enzyme exists as a mixture of the oxyferroheme (Soret = 415 nm) and carboxyferroheme (Soret = 421 nm) enzyme complexes. However, if the same experiment is repeated with the half-reduced form of the pseudomonad enzyme, all of the enzyme is in the ferriheme state, even though CO is inhibiting this form of the enzyme to the same degree as it does the fully reduced form. We conclude that for the half-reduced form of pseudomonad tryptophan oxygenase the substrate, O2, and the inhibitor, CO, are not binding to the heme moieties, but are bound elsewhere, presumably to the Cu(I) moieties. Examination of the kinetic mechanisms of the half-reduced and fully reduced forms of pseudomonad tryptophan oxygenase using the inhibitors carbon monoxide and 5-fluorotryptophan confirmed that the fully reduced enzyme binds L-tryptophan before O2 (FORMAN, H., AND FEIGELSON, P. (1971) Biochemistry 10, 760-763) and that for the half-reduced enzyme O2 binds first. In the presence of 5-fluorotryptophan a relatively stable oxyferroheme enzyme complex was generated with the fully reduced form of pseudomonad tryptophan oxygenase. Thus, saturation of the catalytic site alone either with the substrate, L-tryptophan, or the competitive inhibitor, 5-fluorotryptophan, enhances binding of O2 to the ferroheme moieties of the enzyme. The resistance of this complex to photolysis indicates that the bound molecular oxygen is predominantly present as superoxide, O2-minus.

Animals↗
PubMed1976

Apparent oxidation-reduction potential of Clostridium acidi-urici ferredoxin. Effect of pH, ionic strength, and amino acid replacements.

The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes.

Amino Acids↗
PubMed1987

Nitrous oxide reduction in nodules: denitrification or n(2) fixation?

Detached cowpea nodules that contained a nitrous oxide reductase-positive (Nor) rhizobium strain (8A55) and a nitrous oxide reductase-negative (Nor) rhizobium strain (32H1) were incubated with 1% N(2)O (95 atom% N) in the following three atmospheres: (i) aerobic with C(2)H(2) (10%), (ii) aerobic without C(2)H(2), and (iii) anaerobic (argon atmosphere) without C(2)H(2). The greatest production of N(2) occurred anaerobically with 8A55, yet very little was formed with 32H1. Although acetylene reduction activity was slightly higher with 32H1, about 10 times more N(2) was produced aerobically by 8A55 than by 32H1 in the absence of acetylene. The major reductive pathway of N(2)O reduction by denitrifying rhizobium strain 8A55 is by nitrous oxide reductase rather than nitrogenase.

Journal Article↗
PubMed2002

Gene cluster of nitrous oxide reduction in the deep sea of mariana trench.

The gene of nitrous oxide reductase (nosZ) and entire region of nos gene cluster from Pseudomonas sp. strain MT-1, which is isolated from the sediment of Mariana Trench, was identified. Gene organization in nos gene cluster is similar to that of other bacteria, and nosZ was highly homologous to nosZ of other Pseudomonads. The remarkable property was the existence of a gap from Arg-302 to Ser-309 (numbered by NosZ of P. aeruginosa), which might be a feature of marine bacterial NosZ. It is interesting that nosZ of the strain MT-1 is not related so closely to P. stutzeri, although 16S rDNA of the strain MT-1 is highly homologous to that of P. stutzeri. The existence of nosZ gene in the strain MT-1 indicated the presence of a complete denitrification (nitrate to N(2)) in the Mariana Trench. This is the first report on genetic analysis of denitrification in the deep sea.

Amino Acid Sequence↗
PubMedDate not supplied

[Effect of ethyl alcohol on oxidation-reduction processes in the myocardium].

Evidence is given of disturbances of aerobic oxidative process in the myocardial mitochondria occurring in acute and chronic alcoholic intoxication. A single introduction of alcohol in a dose of 1 and 3 g/kg to rats and dogs produces "hypoxyform" changes in the myocardium, viz. a fall of PO2, a reduced activity of most NAD-independent enzymes, activation of succinate- and lactate-dehydrogenases in a moderate dissociation of oxidative phosphorylation reactions. The damaging effect of the alcohol persists for 24 hours after its one-time introduction. Following a 2-month long alcoholic intoxication there was seen a depressed respiratory activity of the mitochondria and of the study enzymes, except for cytochrome-C-oxidase. It is assumed that the cardiotoxic effect of alcohol is due to the damage of the energy-producing system of the mitochondria.

Alcoholic Intoxication↗
PubMed1977

The relationship between the activity of chloroplast photosystem II and the midpoint oxidation-reduction potential of cytochrome b-559.

The role of cytochrome b-559 in Photosystem II reactions has been investigated using hydroxylamine treatment of chloroplast membranes. Incubation of chloroplasts with hydroxylamine in darkness resulted in inhibition of water oxidation and a decrease in the amplitude of cytochrome b-559 reducible by hydroquinone. The loss of water oxidizing activity perfectly correlated with the decrease in amplitude of cytochrome b-559 reduction. Potentiometric titration of cytochrome b-559 after hydroxylamine treatment revealed a component with Em7.8 at +240 mV in addition to a lower potential species at +90 mV. This compared to control chloroplasts in which cytochrome b-559 exists in the typical high potential state, Em7.8 = +383 mV, in addition to some of the low potential (Em7.8 = +77 mV) form. Photosystem II activity could be further inhibited by incubation with hydroxylamine in the light. In these chloroplasts only low rates of photooxidation of artificial electron donors were observed compared to 'dark' chloroplasts. In addition, the hydroxylamine light treatment caused a further change in cytochrome b-559 redox properties; a single component, Em7.8 = 90 mV is seen in titration curves. The role of cytochrome b-559 in Photosystem II functioning is discussed on the basis of these observations which suggest a dependence of photooxidizing ability of Photosystem II on the redox properties of this cytochrome.

Chloroplasts↗
PubMedDate not supplied

[Oxidative-reductive processes in diabetes mellitus].

Altogether 70 patients with diabetes mellitus of medium severity were examined to determine sulfhydryl (SH) groups, total glutathione (tG) and its individual forms, reduced (rG) and oxidized (oG), cytochrome oxidase activity (CCO), blood and urine V-O, and underoxidation coefficient of the urine with respect to the degree of hyperglycemia and glucosuria. Effect of sugar-reducing therapy on these parameters was studied in 26 subjects. A statistically significant reduction in SH groups balance, in oG content at the expense of rG, increase in CCO activity, in the blood and urine V-O, and in underoxidation coefficient of the urine were detected. The observed changes depend on the sugar content in the blood and urine, and do not disappear even when normoglycemia and aglycosuria are achieved after the use of sugar-reducing drugs.

Adult↗
PubMed1986

Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates.

We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.

Acyl Coenzyme A↗
PubMed1975

The kinetics of flavine oxidation--reduction. I. Dismutation in nonaqueous solvent.

The dismutation reactions of flavines in dimethylformanide have been investigated using the stopped-flow technique under anaerobic conditions. The ionization constants of fully reduced and oxidized tetraacetylriboflavine were measured spectrophotometrically in buffered dimethylformanide. The dismutation equilibrium of the flavine as a function of pH in dimethylformanide was roughly comparable to that reported in water and allowed the estimation of the pKa value of the flavosemiquinone. The dismutation kinetics of tetraacetylriboflavine in unbuffered dimethylformanide were investigated using the fully oxidized and reduced flavines in their neutral form at constant produce of concentrations and varying the reduction degree. The kinetics at very low reduction ratios (less than3%) were triphasic. The kinetic analysis of the initial and simultaneous formation of the anionic and neutral radicals revealed a second-order reaction. The electron transfer between the oxidized and reduced flavines was not directly coupled with prton exchange. The multiphasic time course of the reaction proceeded primarily from differences in the intrinsic rates of the direct and mixed backward dismutation reactions of the two radical species, and finally from a change in the equilibrium conditions resulting from the accumulation of anionic flavohydroquinone. An acidic-basic negative catalytic effect from the neutral flavohydroquinone appeared progressively as the reduction degree was increased. It was complete at reduction ratios higher than 30%, i.e. under conditions where the radical anion could not be observed at any reaction time. Acids with a pKa value lower than the second one of the flavosemiquinone exhibited a similar catalytic effect. These acidic-basic catalytic effects are associated with changes in the ionic state of labile intermediate dimers formed in the forward as well as in the backward direactions of the dismutation reaction. Such a transient complex revealed by the kinetic analysis could be observed directly by absorption spectroscopy in alkaline-buffered dimethylformanide. Its spectral characteristics, as well as the kinetic effects induced by substitution of the benzenoid part of the flavine, can hardly be taken into account by a quinhydrone-like structure for the intermediate dimers at any pH value. The experimental results favored a more specific interaction, possibly of covalent character, involving the benzenoid part of the isoalloxazine ring.

Chemical Phenomena↗
PubMed2001

Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization.

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor. In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1). Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.

Aldehydes↗
PubMed2001

Oxidation-reduction and activation properties of chloroplast fructose 1,6-bisphosphatase with mutated regulatory site.

The concentration of Mg(2+) required for optimal activity of chloroplast fructose 1,6-bisphosphatase (FBPase) decreases when a disulfide, located on a flexible loop containing three conserved cysteines, is reduced by the ferredoxin/thioredoxin system. Mutation of either one of two regulatory cysteines in this loop (Cys155 and Cys174 in spinach FBPase) produces an enzyme with a S(0.5) for Mg(2+) (0.6 mM) identical to that observed for the reduced WT enzyme and significantly lower than the S(0.5) of 12.2 mM of oxidized WT enzyme. E(m) for the regulatory disulfide in WT spinach FBPase is -305 mV at pH 7.0, with an E(m) vs pH dependence of -59 mV/pH unit, from pH 5.5 to 8.5. Aerobic storage of the C174S mutant produces a nonphysiological Cys155/Cys179 disulfide, rendering the enzyme partially dependent on activation by thioredoxin. Circular dichroism spectra and thiol titrations provide supporting evidence for the formation of nonphysiological disulfide bonds. Mutation of Cys179, the third conserved cysteine, produces FBPase that behaves very much like WT enzyme but which is more rapidly activated by thioredoxin f, perhaps because the E(m) of the regulatory disulfide in the mutant has been increased to -290 mV (isopotential with thioredoxin f). Structural changes in the regulatory loop lower S(0.5) for Mg(2+) to 3.2 mM for the oxidized C179S mutant. These results indicate that opening the regulatory disulfide bridge, either through reduction or mutation, produces structural changes that greatly decrease S(0.5) for Mg(2+) and that only two of the conserved cysteines play a physiological role in regulation of FBPase.

Catalytic Domain↗