Flavocytochrome b2: reactivity of its flavin with molecular oxygen

Flavocytochrome b2, a flavohemoprotein, catalyzes the oxidation of lactate at the expense of the physiological acceptor cytochrome c in the yeast mitochondrial intermembrane space. The mechanism of electron transfer from the substrate to monoelectronic acceptors via FMN and heme b2 has been intensively studied over the years. Each prosthetic group is bound to a separate domain, N-terminal for the heme, C-terminal for the flavin. Each domain belongs to a distinct evolutionary family. In particular, the flavodehydrogenase domain is homologous to a number of well-characterized l-2-hydroxy acid-oxidizing enzymes. Among these, some are oxidases for which the oxidative half-reaction produces hydrogen peroxide at the expense of oxygen. For bacterial mandelate dehydrogenase and flavocytochrome b2, in contrast, the oxidative half-reaction requires monoelectronic acceptors. Several crystal structures indicate an identical fold and a highly conserved active site among family members. All these enzymes form anionic semiquinones and bind sulfite, properties generally associated with oxidases, whereas electron transferases are expected to form neutral semiquinones and to yield superoxide anion. Thus, flavocytochrome b2 is a highly unusual dehydrogenase-electron transferase, and one may wonder how its flavin reacts with oxygen. In this work, we show that the separately engineered flavodehydrogenase domain produces superoxide anion in its slow reaction with oxygen. This reaction apparently also takes place in the holoenzyme when oxygen is the sole electron acceptor, because the heme domain autoxidation is also slow; this is not unexpected, in view of the heme domain mobility relative to the tetrameric flavodehydrogenase core (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863). Nevertheless, this reaction is so slow that it cannot compete with the normal electron flow in the presence of monoelectronic acceptors, such as ferricyanide and cytochrome c. An inspection of the available structures of family members does not provide a rationale for the difference between the oxidases and the electron transferases.     

Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding

The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.     

A New Method of Visualization of the Enzymatic Activity of Flavocytochrome b 2 in Electrophoretograms

A new method of visualization of the activity of flavocytochrome b ₂ (FCC b ₂; L-lactate : ferricytochrome c oxidoreductase, EC 1.1.2.3) in electrophoretograms was developed based on the interaction between ferrocyanide (generated during the enzymatic reaction) and Fe³⁺, resulting in the formation of intensely colored precipitates of Berlin blue. The main advantages of this method were its high sensitivity (less than 0.005 U FCC b ₂ was detected within a suitable time period) and the stability of the dye formed. The method developed can be used for determining FCC b ₂ activity in cell-free extracts (e.g., in the selection of FCC b ₂ producers) and monitoring chromatographic purification of proteins, as well as in other cases associated with FCC b ₂ assessment.     

Pressure modulation of cytochrome-to-cytochrome electron-transfer. Models and enzyme reactions.

The kinetics of electron-transfer involved in reactions of reduction of 2,6-dichlorophenol indophenol and Fe(CN)3-(6) by L-ascorbic acid and reduction of ferric cytochrome c by both L-ascorbic acid and reduced hydroxylamine oxidoreductase were studied as a function of three parameters: ionic strength, pressure (1-2000 bar) and temperature (4-20 degrees C) using the high-pressure stopped-flow method. From measurements, the thermodynamic parameters of activation volume (delta V++), and, when possible, activation enthalpy and entropy (delta H++ and delta S++) have been calculated. We found, for these four systems, that the pressure has revealed solvation effects involved in electron-transfer. For the reduction of ferric cytochrome c by reduced hydroxylamine oxidoreductase (a cytochrome-to-cytochrome electron-transfer), we have not obtained evidence for a conformational change.     

Cytochrome b2, an electron carrier between flavocytochrome b2 and cytochrome c. Rapid kinetic characterization of the electron-transfer parameters with ionic-strength-dependence.

The oxidation-reduction properties of free cytochrome b2 isolated by controlled proteolysis from flavocytochrome b2, i.e. the flavodehydrogenase-bound cytochrome b2, were investigated by using stopped-flow spectrophotometry. The rapid kinetics of the reduction of cytochrome b2 by flavocytochrome b2 in the presence of L-lactate are reported. The self-exchange rate constant between reduced cytochrome b2 bound to the flavodehydrogenase and free cytochrome b2 was determined to be 10(5) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. The specific electron-transfer reaction between reduced cytochrome b2 and cytochrome c was also studied, giving an apparent second-order rate constant of 10(7) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. This electron-exchange rate is slightly modulated by ionic strength, following the Debye-Hückel relationship with a charge factor Z1Z2 = -1.9. Comparison of these data with those for the reduction of cytochrome c by flavodehydrogenase-bound cytochrome b2 [Capeillère-Blandin (1982) Eur. J. Biochem. 128, 533-542] leads to the conclusion that the intramolecular electron exchange between haem b2 and haem c within the reaction complex occurs at a rate very similar to that determined experimentally in presence of the flavodehydrogenase domain. The low reaction rate observed with free cytochrome b2 is ascribed to the low stability of the reaction complex formed between free cytochrome b2 and cytochrome c.     

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Oxidation of vitamin E during iron-catalyzed lipid peroxidation: evidence for electron-transfer reactions of the tocopheroxyl radical.

Incubation of phosphatidylcholine liposomes containing the biological antioxidant alpha-tocopherol (alpha-TH) with xanthine, xanthine oxidase, and FeCl2 caused alpha-TH oxidation to alpha-tocopherol quinone (alpha-TQ) and 8a-hydroperoxytocopherone (2). In addition, 4a,5-epoxy-8a-hydroperoxytocopherone (3), 7,8-epoxy-8a-hydroperoxytocopherone (4), and their respective hydrolysis products 2,3-epoxy-alpha-tocopherol quinone (6) and 5,6-epoxy-alpha-tocopherol quinone (7) also were formed. alpha-TQ was the major product at less than 20% alpha-TH oxidation, whereas epoxides were the predominant products when alpha-TH was more extensively oxidized. 8a-(Alkyldioxy)tocopherones 1, which are formed when peroxyl radicals oxidize alpha-TH in other systems and which are precursors to alpha-TQ, were not found. 8a-Hydroxytocopherone (5), rather than 8a-(alkyldioxy)tocopherones 1, appeared to be the precursor to alpha-TQ. Approximately 30% of the alpha-TH consumed was regenerated by treatment of samples with ascorbic acid or nordehydroguaiaretic acid (NDGA) at pH 3, but not at pH 7. The stability of the ascorbic acid- and NDGA-reducible species and pH dependence for regeneration matched those of 8a-hydroxytocopherone (5) and contrasted with the properties of the tocopheroxyl radical (alpha-T.). Incubation of liposomes containing alpha-TH with the diphenylpicrylhydrazyl (DPPH) radical, which oxidizes alpha-TH to alpha-T. in high yield, formed an ascorbic acid-reducible species with properties identical to those of compound 5. The results indicate that phospholipid peroxyl radicals oxidize alpha-T. to epoxides, 8a-hydroperoxytocopherone (2), and the tocopherone cation (alpha-T+), which hydrolyzes to 5, the immediate precursor to alpha-TQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the prosthetic groups of dimethylamine dehydrogenase from Hyphomicrobium X.

Trimethylamine dehydrogenase (TMADH) and dimethylamine dehydrogenase (DMADH) were purified from Hyphomicrobium X. The absorbance spectra of the two enzymes were similar with λmax = 443 nm for TMADH and 440 nm for DMADH. DMADH had an apparent molecular weight of 138,000 daltons and was composed of two subunits of similar molecular weights. DMADH contained 3.91 atoms S and 4.55 atoms Fe per mole of the enzyme. Both DMADH and TMADH contained a covalently bound yellow coenzyme. The coenzyme-peptides obtained from DMADH and TMADH of Hyphomicrobium X by tryptic-chymotryptic digestion were partially purified and found to differ electrophoretically and chromatographically from the coenzyme-peptide obtained similarly from TMADH of bacterium W₃A₁. After digestion with aminopeptidase M the aminoacyl-coenzymes from the three enzymes had identical spectral, electrophoretic and chromatographic properties. DMADH is only the second enzyme yet found to contain 6-S-cysteinyl-FMN as coenzyme. Dissimilarities between the coenzyme-peptides of DMADH and TMADH from either Hyphomicrobium X or bacterium W₃A₁ are consequently located in the peptide component.     

The multiplicity and stoichiometry of the prosthetic groups in QH2: cytochrome c oxidoreductase as studied by EPR.

1. The EPR signal in the g = 2 region of the reduced QH2: cytochrome c oxidoreductase as present in submitochondrial particles and the isolated enzyme is an overlap of two signals in a 1 : 1 weighted ratio. Both signals are due to [2Fe-2S]+1 centers. 2. From the signal intensity it is computed that the concentration of each Fe-S center is half that of cytochrome c1. 3. The line shape of one of the Fe-S centers, defined as center 1, is reversibly dependent on the redox state of the b-c1 complex. The change of the line shape cannot be correlated with changes of the redox state of any of the cytochromes in QH2: cytochrome c oxidoreductase. 4. Lie the optical spectrum, the EPR spectrum of the cytochromes is composed of the absorption of at least three different b cytochromes and cytochrome c1. 5. The molar ratio of the prosthetic groups was found to be c1 : b-562 : b-566 : b-558 : center 1 : center 2 = 2 : 2 : 1 : 1 : 1 : 1. The consequences of this stoichiometry are discussed in relation to the basic enzymic unit of QH2 : cytochrome c oxidoreductase.     

Kinetic studies of the reactions of pentacyanonitrosylferrate(2−) with ligands containing acidic methylene groups

The kinetic studies of the addition reactions of pentacyanonitrosylferrate(2−) (NP) with acidic methylene ligands of the form CH₂LL′ (L, L′ = -CN, -CONH₂; -CN, -CN; CH₃CO-, CH₃CO-) have been carried out in basic solutions. The increase in the second order rate constants for the formation of Fe(CN)₅N(O)CLL′⁴⁻ complexes with the increase of OH⁻ concentration indicated that ^?CHLL′ is the reactive species of the reactions. In addition to the pK_a, the lability of the methylene protons of the ligands in aqueous solution also governs the reactivity of the reactions. The nitrosation products were rather unstable with respect to the dissociation into and oxime compounds. The kinetic measurements showed that the rates of dissociation were first order and were rather insensitive to the hydroxide concentration for the ligands under study.     

Effect of mutations in the cytochrome b ef loop on the electron-transfer reactions of the Rieske iron-sulfur protein in the cytochrome bc1 complex

Long-range movement of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c1 redox centers plays a key role in electron transfer within the cyt bc1 complex. A series of 21 mutants in the cyt b ef loop of Rhodobacter sphaeroides cyt bc1 were prepared to examine the role of this loop in controlling the capture and release of the ISP from cyt b. Electron transfer in the cyt bc1 complex was studied using a ruthenium dimer to rapidly photo-oxidize cyt c1 within 1 mus and initiate the reaction. The rate constant for electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c1 was k1 = 60 000 s-1. Famoxadone binding to the Qo site decreases k1 to 5400 s-1, indicating that a conformational change on the surface of cyt b decreases the rate of release of the ISP from cyt b. The mutation I292A on the surface of the ISP-binding crater decreased k1 to 4400 s-1, while the addition of famoxadone further decreased it to 3000 s-1. The mutation L286A at the tip of the ef loop decreased k1 to 33 000 s-1, but famoxadone binding caused no further decrease, suggesting that this mutation blocked the conformational change induced by famoxadone. Studies of all of the mutants provide further evidence that the ef loop plays an important role in regulating the domain movement of the ISP to facilitate productive electron transfer and prevent short-circuit reactions.     

The carbohydrate prosthetic groups of rat fibrinogen plasmic fragment E.

Rat fibrinogen plasmic fragment E was found to contain one oligosaccharide chain per gamma-chain attached by a glycosylamine linkage. The oligosaccharide was composed of 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues. The probable sequence from the nonreducing end was sialic acid leads to galactose beta leads to mannose alpha leads to mannose alpha leads to glucosamine leads to glucosamine. No difference in the rate of clearance from the rat circulation could be detected between native and desialated fragment E. A non-denaturing method for the purification of fragment E is described.     

Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents.

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2'-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.     

Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5).