Reactions of electron-transfer flavoprotein and electron-transfer flavoprotein: ubiquinone oxidoreductase.

Electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-Q oxidoreductase) catalyses the re-oxidation of reduced electron-transfer flavoprotein (ETF) with ubiquinone-1 (Q-1) as the electron acceptor. A kinetic assay for the enzyme was devised in which glutaryl-CoA in the presence of glutaryl-CoA dehydrogenase was used to reduce ETFox. and the reduction of Q-1 was monitored at 275 nm. The partial reactions involved in the overall assay system were examined. Glutaryl-CoA dehydrogenase catalyses the rapid reduction of ETFox. to the anionic semiquinone (ETF.-), but reduces ETF.- to the fully reduced form (ETFhq) at a rate that is about 6-fold lower. ETF.-, but not ETFhq, is directly re-oxidized by Q-1 at a rate that, depending on the steady-state concentration of ETF.-, may contribute significantly to the overall reaction. ETF-Q oxidoreductase catalyses rapid disproportionation of ETF.- with an equilibrium constant of about 1.0 at pH 7.8. In the presence of Q-1 it also catalyses the re-oxidation of ETFhq at a rate that is faster than that of the overall reaction. Rapid-scan experiments indicated the formation of ETF.-, but its fractional concentration in the early stages of the re-oxidation of ETFhq is low. The data indicate that the re-oxidation of ETFhq proceeds at a rate that is adequate to account for the overall rate of electron transfer from glutaryl-CoA to Q-1. An unusual property of ETF-Q oxidoreductase seems to be that it not only catalyses the re-oxidation of the reduced forms of ETF but also facilitates the complete reduction of ETFox. to ETFhq by disproportionation of the radical.     

Turning a monocovalent flavoprotein into a bicovalent flavoprotein by structure-inspired mutagenesis

A recently discovered class of bicovalent flavoproteins is an interesting group of enzymes because of their unusual cofactor binding mode, their open active sites and the bulky substrates they can accept. Through a sequence comparison study we have identified a conserved sequence region in bicovalent flavoproteins that is different from monocovalent flavoproteins. Based on this and the available structural information we have designed mutants of the prototype monocovalent flavoprotein, 6-hydroxy-d-nicotine oxidase (6HDNO), in order to introduce a second cofactor-protein linkage. Two amino acid replacements, namely histidine 130 to a cysteine and leucine 138 to a histidine, were sufficient to create a bicovalent 6HDNO. The introduced cysteine forms a covalent bond with FAD as found in natural bicovalent flavoproteins, while the second mutation was found to be essential to facilitate the formation of the cysteinyl linkage. This points to an important role of the introduced histidine in stabilizing a negative charge of the isoalloxazine ring during covalent flavinylation. The His130Cys/Leu138His 6HDNO is still active and shows a higher midpoint redox potential when compared to wild-type 6HDNO. This agrees well with the previous studies that have shown that bicovalent flavoenzymes have extremely high redox potentials     

Estrogen 1,2-epoxides or estrogen quinones/semiquinones

Metabolic activation of estradiol leading to the formation of catechol estrogens is a prerequisite for its genotoxic activity. Both estrogen-o-quinones/semiquinones and estrogen 1,2-epoxides have been proposed to be responsible for this activity. Incubations of [3H]estradiol and [3H]1 alpha,2 alpha-epoxy-4-estrene-3-one-17 beta-ol (ketotautomer of estradiol 1,2-epoxide) with rat liver microsomal and cytosol preparations were carried out in the presence of SKF 525A, ascorbic acid, glutathione and cysteine. Ascorbic acid decreased binding to proteins and aqueous-soluble fraction with both [3H] estradiol and [3H]epoxyestrenolone in incubations with microsomes but no effect with cytosol fraction. Incubations of microsomes with thiols gave water-soluble metabolites which were characterized as 1(4)-thioether derivatives of 2-hydroxyestradiol and incubations of [3H]epoxyestrenolone with cytosol and thiols gave estradiol-2-thioether. Incubations with ascorbic acid and thiols resulted in decreased formation of water-soluble metabolites in microsomal incubations but not in cytosol incubations. These studies indicate that the major pathway for irreversible binding of estrogens to macromolecules involves estrogen-o-quinones/semiquinones and not estrogen 1, 2-epoxide.     

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.     

Fluorescence spectroscopic detection of mitochondrial flavoprotein redox oscillations and transient reduction of the NADPH oxidase-associated flavoprotein in leukocytes

Steady-state and time-resolved fluorescence spectroscopy and fluorescence microscopy of leukocyte flavoproteins have been performed. Both living human peripheral blood monocytes and neutrophils have been utilized as experimental models, as the former relies much more heavily on mitochondrial metabolism for energy production than the latter. We confirm previous studies indicating that cellular flavoproteins absorb at 460 nm and emit at 530 nm, very similar to that of the FAD moiety. Furthermore, the emission properties of intracellular flavoproteins were altered by the metabolic inhibitors rotenone, antimycin A, azide, cyanide, DNP (2,4-dinitrophenol), and FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone]. Kinetic studies revealed flavoprotein emission oscillations in both monocytes and neutrophils. The flavoprotein intensity oscillations correlated with the physiological status of the cell and the nature of membrane receptor ligation. Microscopy revealed the presence of flavoprotein fluorescence in association with the plasma membrane, intracellular granules and distributed throughout the cytoplasm, presumably within mitochondria. Metabolic inhibitors such as cyanide suggest that the plasma membrane and granular components are cyanide-insensitive and therefore are likely associated with the flavoprotein component of the NADPH oxidase, which is located in these two compartments. This interpretation was found to be consistent with structural localization of the NADPH oxidase using an antibody molecule specific for this protein. Using peripheral blood neutrophils, which display less active mitochondria, and time-resolved emission spectroscopy, we show that the NADPH oxidase-associated flavoprotein undergoes a periodic transient reduction of about 54±2 ms in living cells. This finding is consistent with prior studies indicating that propagating substrate (NADPH) waves periodically promote electron transport across the NADPH oxidase.     

Reactivity of semiquinones with ascorbate and the ascorbate radical as studied by pulse radiolysis

Free-radical interactions between hydroquinones (QH2) and ascorbate (AscH-) have a profound impact in many biological situations. Despite the obvious biological significance, not much is known about the kinetics of reactions of QH2 and AscH- with their corresponding free radicals, i.e., semiquinones, Q1.-, and the ascorbate radical, Asc.-. Furthermore, a general approach to reliably measure rate constants for the above reactions is fraught with complications. In this work, the kinetic behavior of Q.- and Asc.-, after pulse radiolytic oxidation of mixtures of a series of alkyl- and methoxysubstituted hydroquinones and ascorbate by azide radicals in aqueous buffer, pH 7.40, was monitored in submillisecond range by time-resolved UV spectroscopy. Rate constants for reactions of Q.- with AscH-(reaction [1]) and Asc.- (reaction [2]) were directly determined by using new kinetic procedures which distinguished between reactions [1] and [2]. The results show that the rate constants for reaction [2] vary only within a narrow range from 1.2 x 10(8) to 2.5 x 10(8) M(-1) s(-1) and do not display any pronounced correlation with Q.- structures. In contrast, the value of k1 for nonsubstituted Q.- was found to be (1.8 +/- 0.2) x 10(5) M(-1) s(-1) and decreases with the number of alkyl and methoxy substituents as well as with the decrease of the one-electron reduction potential E(Q.-/QH2).     

Activation of a flavoprotein by proteolysis

Chymotryptic digestion of brain pyridoxine-5-P oxidase brings about a 4-fold enhancement of the catalytic power (Vmax/KM) using pyridoxine-5-P as substrate in the assay mixtures. The chymotrypsin-treated enzyme is less susceptible to inhibition by pyridoxal-5-P than the native enzyme. Fragments arising from limited proteolysis were separated by affinity chromatography using P-pyridoxal-Sepharose as supporting matrix. Catalytically active fractions, eluted by pyridoxine-5-P (5mM), displayed three bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of the three protein bands are considerably lower than 28 kDa, the molecular mass of monomeric pyridoxine-5-P oxidase. Spectroscopic studies, absorption, fluorescence, and circular dichroism revealed that the microenvironment surrounding the cofactor flavin mononucleotide is not perturbed by limited proteolysis.     

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.     

Subunit structure of electron transfer flavoprotein

The electron transfer flavoprotein from pig liver mitochondria is a 57,000-dalton electron transferase which links several primary flavoprotein dehydrogenases with the mitochondrial electron transport system. The protein was previously reported to be a dimer of apparently identical subunits. There are conflicting estimates in the literature regarding the FAD content of the protein. The results presented here clearly show that the protein contains nonidentical subunits based on polyacrylamide gel electrophoresis in the presence of 8 M urea and sodium dodecyl sulfate. The molecular weights of the subunits are 31,000 and 27,000. Analysis of peptides generated by cleavage of the subunits with cyanogen bromide show that the subunits have different primary structures. This result and amino acid analyses of the protein and the purified subunits show that the heterogeneity cannot be due to proteolysis. Using an experimentally determined molar extinction coefficient for the protein-bound flavin, a minimum Mr = 55,000 was calculated, indicating that the protein contains 1 mol of FAD/mol of protein.     

Intermediates in flavoprotein catalyzed hydroxylations

The reaction of molecular oxygen with the complex of reduced p-hydroxybenzoate hydroxylase and 2,4-dihydroxybenzoate has been followed by rapid reaction techniques. During the reaction, which produces stoichiometric amounts of oxidized enzyme and the hydroxylated product, 2,3,4-trihydroxybenzoate, three spectroscopically distinguishable intermediates have been detected and characterized.     

Hydrogen bonding of flavoprotein. I. Effect of hydrogen bonding on electronic spectra of flavoprotein.

The effect of hydrogen bonding on the transition energy and the oscillator strength of the isoalloxazine nucleus of flavins was studied by the molecular orbital method. Among the possible hydrogen bondings examined, characteristic spectral shifts were found for the hydrogen bondings at N(1) and N(5) of the nucleus. The hydrogen bonding at N(1) resulted in the shift of the first absorption band towards blue and that of the second one towards red. On the other hand, the hydrogen bonding at N(5) resulted in the shifts of both the first and the second band towards red. The spectral characteristics reported on Clostridium MP and Desulfovibrio vulgaris flavodoxin coincided with the calculated results. The application of the calculated results to D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) led to the conclusion that hydrogen bonding occurs at O(12), N(3)H, O(14) and N(5) of the isoalloxazine nucleus. The occurrence of hydrogen bondings at O(12), N(3)H, and O(14) is favorable for N(5) of the isoalloxazine nucleus to accept electron from an electron donor.     

The growing VAO flavoprotein family

The VAO flavoprotein family is a rapidly growing family of oxidoreductases that favor the covalent binding of the FAD cofactor. In this review we report on the catalytic properties of some newly discovered VAO family members and their mode of flavin binding. Covalent binding of the flavin is a self-catalytic post-translational modification primarily taking place in oxidases. Covalent flavinylation increases the redox potential of the cofactor and thus its oxidation power. Recent findings have revealed that some members of the VAO family anchor the flavin via a dual covalent linkage (6-S-cysteinyl-8α-N1-histidyl FAD). Some VAO-type aldonolactone oxidoreductases favor the non-covalent binding of the flavin cofactor. These enzymes act as dehydrogenases, using cytochrome c as electron acceptor.     

Identification of ADP in the iron-sulfur flavoprotein trimethylamine dehydrogenase

Analysis of the 2.4-A resolution electron density map of trimethylamine dehydrogenase has revealed the unexpected presence of one molecule of ADP/subunit. This binding has been confirmed chemically. The binding site is located at the analogous position of the ADP moiety of FAD in glutathione reductase, the FAD and NADPH binding domains of which resemble two of the domains of trimethylamine dehydrogenase. Comparison of the environments of the ADP moieties in the two proteins indicates that 32 residues in 6 peptides are in equivalent positions with a root mean square deviation for C alpha positions of 1.11 A. Twelve of these amino acids are identical, based on the electron density-derived "x-ray" sequence of trimethylamine dehydrogenase. Detailed analysis of the environment of the ADP moiety indicates that most of the conserved residues are not in direct contact with the cofactor. Some of them probably represent the "fingerprint" of the beta alpha beta binding fold found in dinucleotide binding proteins, but the remaining conserved residues may indicate a closer evolutionary relationship between these two proteins.