Effect of quinones and phenols on the triple‐enzyme bioluminescent system with protease

The study addressed the effects of redox‐active compounds on trypsin activity. Series of organic oxidizers (quinones) and reducers (phenols) were chosen as model redox‐active compounds. Trypsin activity was quanti?ed by bioluminescent technique. Interactions of these compounds with trypsin were studied by ?uorescent and light absorption methods. Luminescence intensity decay constants in the reduced nicotinamidadeninedinucleotide (NADH): ?avinmononucleotide (FMN)‐oxidoreductase (R)–luciferase (L)–trypsin (T) (R + L + T) triple‐enzyme system were calculated and compared in the presence of different concentrations of quinones and phenols. The triple‐enzyme system was shown to be sensitive to quinones and not sensitive to phenols. It has been found that the effects produced by quinones on the coupled enzyme system (R + L) and on the trypsin molecule (T) are not related. The conclusions were extrapolated to the properties of other proteases and antiproteases. Copyright © 2003 John Wiley & Sons, Ltd.     

Effect of quinones on enzymatic bioluminescence of NADH-dependent systems

The effects of a number of quinones on the bioluminescence characteristics of a three-component enzymatic system containing alcohol dehydrogenase, bacterial luciferase, and NADH-FMN oxidoreductase were studied to find the most sensitive kinetic parameters of the system intended to be used in biological testing. Both direct and back reactions catalyzed by alcohol dehydrogenase were studied in the presence and in the absence of quinones. The kinetic parameters of the bioluminescent system were found to depend on the redox potentials and concentrations of quinones. The quinone-induced effects were shown to be associated with changes in the NAD+/NADH ratio in the chain of NADH-dependent enzymes. The three-enzyme system based on alcohol dehydrogenase is suggested as a bioluminescence test for ecological monitoring of waste water.     

Effects of quinones on NADH-dependent enzymatic bioluminescent systems

The effects of a number of quinones on the bioluminescence characteristics of a three-component enzymatic system containing alcohol dehydrogenase, bacterial luciferase, and NADH-FMN oxidoreductase were studied to find the most sensitive kinetic parameters of the system intended to be used in biological testing. Both direct and back reactions catalyzed by alcohol dehydrogenase were studied in the presence and in the absence of quinones. The kinetic parameters of the bioluminescent system were found to depend on the redox potentials and concentrations of quinones. The quinone-induced effects were shown to be associated with changes in the NAD⁺/NADH ratio in the chain of NADH-dependent enzymes. The three-enzyme system based on alcohol dehydrogenase is suggested as a bioluminescence test for ecological monitoring of waste water.     

Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster

Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster. A difference between SQR and QFR is that the redox potential of the [3Fe-4S] cluster in SQR is 140 mV higher than that found in QFR. This may reflect the character of the different quinones with which the two enzymes preferentially react. To investigate how the environment around the [3Fe-4S] cluster affects its redox properties and catalysis with quinones, a conserved amino acid proximal to the cluster was mutated in both enzymes. It was found that substitution of SdhB His-207 by threonine (as found in QFR) resulted in a 70-mV lowering of the redox potential of the cluster as measured by EPR. The converse substitution in QFR raised the redox potential of the cluster. X-ray structural analysis suggests that placing a charged residue near the [3Fe-4S] cluster is a primary reason for the alteration in redox potential with the hydrogen bonding environment having a lesser effect. Steady state enzyme kinetic characterization of the mutant enzymes shows that the redox properties of the [3Fe-4S] cluster have only a minor effect on catalysis.     

The organisation of proton motive and non-proton motive redox loops in prokaryotic respiratory systems

Respiration is fundamental to the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. In principle, the free energy of a redox reaction catalysed by a membrane-bound electron transport chain is transduced via the generation of an electrochemical ion (usually proton) gradient across a coupling membrane that drives ATP synthesis. The proton motive force (pmf) can be built up by different mechanisms like proton pumping, quinone/quinol cycling or by a redox loop. The latter couples electron transport to a net proton transfer across the membrane without proton pumping. Instead, charge separation is achieved by quinone-reactive enzymes or enzyme complexes whose active sites for substrates and quinones are situated on different sides of the coupling membrane. The necessary transmembrane electron transport is usually accomplished by the presence of two haem groups that face opposite sides of the membrane. There are many different enzyme complexes that are part of redox loops and their catalysed redox reactions can be either electrogenic, electroneutral (non-proton motive) or even pmf-consuming. This article gives conceptual classification of different operational organisations of redox loops and uses this as a platform from which to explore the biodiversity of quinone/quinol-cycling redox systems.     

Redox and addition chemistry of quinoid compounds and its biological implications

The overall biological activity of quinones is a function of the physico-chemical properties of these compounds, which manifest themselves in a critical bimolecular reaction with bioconstituents. Attempts have been made to characterize this bimolecular reaction as a function of the redox properties of quinones in relation to hydrophobic or hydrophilic environments. The inborn physico-chemical properties of quinones are discussed on the basis of their reduction potential and dissociation constants, as well as the effect of environmental factors on these properties. Emphasis is given on the effect of methyl-, methoxy-, hydroxy-, and glutathionyl substituents on the reduction potential of quinones and the subsequent electron transfer processes. The redox chemistry of quinoid compounds is surveyed in terms of a) reactions involving only electron transfer, as those accomplished during the enzymic reduction of quinones and the non-enzymic interaction with redox couples generating semiquinones, and b) nucleophilic addition reactions. The addition of nucleophiles, entailing either oxidation or reduction of the quinone, are exemplified in reactions with oxygen- or sulfur nucleophiles, respectively. The former yields quinone epoxides, whereas the latter yields thioether-hydroquinone adducts as primary molecular products. The subsequent chemistry of these products is examined in terms of enzymic reduction, autoxidation, cross-oxidation, disproportionation, and free radical interactions. The detailed chemical mechanisms by which quinoid compounds exert cytotoxic, mutagenic and carcinogenic effects are considered individually in relation to redox cycling, alterations of thiol balance and Ca++ homeostasis, and covalent binding.     

Structures of mammalian cytosolic quinone reductases

The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Energy coupling in type II topoisomerases: why do they hydrolyze ATP?

Type II topoisomerases are essential enzymes in all cells. They help to solve the topological problems of DNA by passing one double helix through a transient break in another, in a reaction coupled to the hydrolysis of ATP. Members of one class of the enzymes, DNA gyrases, are configured to carry out an intramolecular reaction, removing positive supercoiling and introducing negative supercoiling into circular DNA using free energy derived from ATP hydrolysis. The nonsupercoiling class, including bacterial topoisomerase IV and eukaryotic topoisomerase II enzymes, can carry out both intra- and intermolecular reactions, and their primary role is the unlinking (decatenation) of daughter replicons before partition. In these enzymes, ATP hydrolysis is coupled to a reduction in DNA complexity (catenation, supercoiling, and knotting) below the level expected at equilibrium. This review discusses our current understanding of the mechanisms behind the coupling of the energy of ATP hydrolysis to topological changes catalyzed by both of these classes of enzyme.     

Accelerated Search Kinetics Mediated by Redox Reactions of DNA Repair Enzymes

A charge transport (CT) mechanism has been proposed in several articles to explain the localization of base excision repair (BER) enzymes to lesions on DNA. The CT mechanism relies on redox reactions of iron-sulfur cofactors that modify the enzyme's binding affinity. These redox reactions are mediated by the DNA strand and involve the exchange of electrons between BER enzymes along DNA. We propose a mathematical model that incorporates enzyme binding/unbinding, electron transport, and enzyme diffusion along DNA. Analysis of our model within a range of parameter values suggests that the redox reactions can increase desorption of BER enzymes not already bound to lesions, allowing the enzymes to be recycled—thus accelerating the overall search process. This acceleration mechanism is most effective when enzyme copy numbers and enzyme diffusivity along the DNA are small. Under such conditions, we find that CT BER enzymes find their targets more quickly than simple passive enzymes that simply attach to the DNA without desorbing.     

Interactions of quinones with thioredoxin reductase: a challenge to the antioxidant role of the mammalian selenoprotein

Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single- and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the k(ca)(t)/K(m) value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH(4)) and two-electron reduced (EH(2)) states in quinone reduction. The redox potential of the EH(2)/EH(4) couple of TrxR calculated according to the Haldane relationship with NADPH/NADP(+) was -0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate K(i) = 6.3 +/- 1 microm). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones.     

Bioluminescence characteristics of Lake Shira water

Bioluminescence bioassays based on luminous bacteria (Photobacterium phosphopreum) and coupled enzyme system NADH-FMN-oxidoreductase-luciferase were adapted for monitoring the saline-water conditions of Lake Shira (Khakasia, Siberia). The differences in bioluminescence responses have been found to be related to the salt composition and the oxidation-reduction properties of water. Bioluminescent kinetics parameters, which are mostly sensitive to pollution under conditions of saline water, have been observed. The enzymatic system in the presence of 1,4-benzoquinone are shown to be more sensitive to redox characteristics of the salt water than this in the absence of 1,4-benzoquinone. 1,4-benzoquinone should be applied for the preparation of a model solution for the monitoring of redox properties of the salt water. Using this technique, the results of bioluminescence analysis are used to construct a heterogeneity map that characterizes the spatial and temporal water quality of lake Shira. A partial map was based on the bioluminescence characteristics of water samples taken along the shoreline, sampling stations in the different places and in different depths of the lake. It has been demonstrated that the bioluminescence assay measurements must be done within two hours after the sampling time.     

Redox-shuttling between chloroplast and cytosol: integration of intra-chloroplast and extra-chloroplast metabolism

Reducing equivalents produced in the chloroplast are essential for many key cellular metabolic enzyme reactions. Two redox shuttle systems transfer reductant out of the chloroplast; these systems consist of metabolite transporters, coupled with stromal and cytosolic dehydrogenase isozymes. The transporters function in the redox shuttle and also operate as key enzymes in carbon/nitrogen metabolism. To maintain adequate levels of reductant and proper metabolic balance, the shuttle systems are finely controlled. Also, in the leaves of C(4) plants, cell-specific division of carbon and nitrogen assimilation includes cell-specific localization of the redox shuttle systems. The redox shuttle systems are tightly linked to cellular metabolic pathways and are essential for maintaining metabolic balance between energy and reducing equivalents.     

Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.     

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalyzed by a human placental NADPH-linked carbonyl reductase.

Polycyclic aromatic hydrocarbon quinones, hydroquinones, and glutathionyl adducts of quinones undergo oxidation-reduction (redox) cycling in the presence of NADPH and the NADPH-linked human placental carbonyl reductase. K-region and non-K-region o-quinones and their glutathione adducts are the best substrates of this enzyme; they are reduced to hydroquinones. Under aerobic conditions, the hydroquinones are autoxidized with the formation of potentially hazardous semiquinones and the superoxide anion. Because of these reactions it is unlikely that polycyclic aromatic hydrocarbon quinones or their glutathione adducts are inert products of detoxication in tissues that contain the carbonyl reductase or another enzyme with similar substrate specificity. If superoxide dismutase is added to reaction mixtures containing the carbonyl reductase and quinones, it inhibits redox cycling. Presumably this results from destruction of the superoxide anion which acts as a chain propagator in these reactions.     

Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids.

Substituted primary hydroxamic acids were found to inhibit the catalytic activity of a number of redox enzymes. The inhibition was not related to the nature of the metal-active site of the enzyme nor to the nature of the oxygen-containing substrate. Two easily available enzymes, mushroom tyrosinase (monophenol,dihydroyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which were potently inhibited by hydroxamic acids, were chosen for more detailed study. A kinetic analysis of the inhibitory effects on the partially purified tyrosinase of mushroom (Agaricus bispora) revealed that inhibition was reversible and competiitive with respect to reducing substrate concentration, but was not competitive with respect to molecular oxygen concentration. A spectrophotometric and EPR study of the binding of salicylhydroxamic acid to horseradish peroxidase revealed that his hydroxamic acid was bound to the enzyme in the same manner as a typical substrate, hydroquinone. Spectroscopic and thermodynamic measurements of the binding reactions suggested that this binding site is close, to but, not directly onto, the heme group of the enzyme. From these results it is concluded that the mode of inhibition of hydroxamic acid need not be, as generally supposed, by metal chelation, and mechanisms involving either hydrogen bonding at the reducing substrate binding site or the formation of a charge transfer complex between hydroxamic acid and an electron-accepting group in the enzyme are considered to be more feasible. The relevance of these findings to deductions on the nature of other hydroxamic acid-inhibitable systems is discussed.     

Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae

The reductive products of several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic. The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their deleterious metabolites. A "classical" nitroreductase has been purified from Enterobacter cloacae 587-fold using a protocol which yields approximately 1 mg of purified nitroreductase from 10 liters of cell culture. An analysis of the physical properties of the nitroreductase indicates that the enzyme is active as a monomer with a calculated molecular mass of 27 kDa. FMN has been identified as a required flavin cofactor and is present at a stoichiometry of 0.88 mol of FMN bound/mol of active enzyme. The enzyme was found capable of reducing nitrofurazone under aerobic conditions indicating that the mechanism involves an obligatory two-electron transfer. Thus, this enzyme can be classified as an oxygen-insensitive nitroreductase. The purified nitroreductase can utilize either NADH or NADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes as well as quinones. Studies in which the rates of nitroreduction for a series of para substituted nitrobenzene derivatives were determined suggest that a linear free energy relationship exists between the rate and the redox midpoint potential of the substrate.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.