8-Hydroxy-5-deazaflavin-dependent electron transfer in the extreme halophile Halobacterium cutirubrum

Abstract Cell extracts of the extreme halophile Halobacterium cutirubrum were found to contain 8-hydroxy-5-deazaflavin as well as 8-OH-5-deazaflavin: NADPH oxidoreductase activity. The oxidoreductase was partially purified and showed maximum activity at pH 5.4, which is unusually low for halobacteria, and 5.3 M NaCl, close to the intracellular salt concentration. The results indicate the presence of an 8-OH-5-deazaflavin-dependent electron transfer system in a nonmethanogenic organism.     

Purification and properties of a nad: 3α-hydroxy-5α-pregnan-20-one-oxidoreductase from rat liver microsomes

From rat liver microsorties a NAD: 3α-hydroxy-5α-pregnan-20-one oxidoreductase was isolated and purified up to a specific activity of 73 nmol/min.mg by affinity chromatography and DEAE-cellulose chromatography. Various Km-values have been determined. The enzyme exhibits highest affinity for 5α-pregnane-3,20-dione and NADH. The 3-oxo group of 5α-dihydrocortisone (17, 21-dihydroxy-5α-pregnane-3,11,20-trione) was not reduced by the purified enzyme preparation and NADH and no dehydrogenation with NAD was observed of 3α, 11β, 17, 21-tetrahydroxy-5α-pregnan-20-one. The optimal pH for the hydrogenation of the 3-oxo group was at pH 5.3 and for the dehydrogenation at pH 8.9. Disc gel electrophoresis in presence of 0.1% sodium dodecylsulfate yielded a homogeneous preparation.     

Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta

Glycerol-3-phosphate oxidoreductase (sn-glycerol 3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6-trinitrobenzenehexamethylenediamine-Sepharose, DEAE-Sephadex A-50 and 5'-AMP-Sepharose 4B approximately 15800-fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 mumol NADH min-1 mg protein-1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 +/- 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 +/- 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 +/- 0.09. The pH optimum of the placental enzyme was in the range 7.4-8.1 for dihydroxyacetone phosphate reduction and 8.7-9.2 for sn-glycerol 3-phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn-glycerol 3-phosphate and NAD+ were 26 microM, 5 microM, 143 microM and 36 microM respectively. The activity ratio of cytoplasmic glycerol-3-phosphate oxidoreductase to mitochondrial glycerol-3-phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol-3-phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn-glycerol 3-phosphate. The possible physiological role of glycerol-3-phosphate oxidoreductase in placental metabolism is discussed.     

DNA photoreactivating enzyme from the cyanobacterium Anacystis nidulans

Photoreactivating enzyme, which specifically monomerizes pyrimidine dimers in UV-irradiated DNA, was purified 21,000-fold from the cyanobacterium Anacystis nidulans to apparent homogeneity with 41% overall yield. The enzyme consists of a single protein chain with 53,000 molecular weight. Maximal activity was found at pH 6.2 and 0.1 M NaCl. Purified photoreactivating enzyme exhibits a marked absorption spectrum with a main band in the blue region (maximum 437 nm), a protein band (maximum 266 nm), and a low intensity band above 500 nm. The molar extinction coefficient of native enzyme was estimated 53,000 at 437 nm. The action spectrum for photoreactivation shows maximal activity at 440 nm and correlates closely with the 437-nm absorption band. The enzyme contains two different intrinsic chromophores in equimolar amounts, which were identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) and (reduced) FAD. The low intensity absorption band of native photoreactivating enzyme exhibits a shoulder at 498 and maxima at 588 and 634 nm. This band is attributed to a neutral FAD semiquinone radical which accounts for the major part of the FAD present in dark equilibrated enzyme. Preillumination at 585 nm bleaches the semiquinone spectrum due to formation of fully reduced FAD, but exposure to air in the dark restores the spectrum completely. On preillumination at 437 nm the disappearance of FAD semiquinone is more rapid, indicating that the photoreduction is sensitized by the 8-hydroxy-5-deazaflavin chromophore. The 8-hydroxy-5-deazaflavin and possibly also the reduced FAD chromophore appear to act as a primary photon acceptor in the photoreactivation process.     

Roles of FAD and 8-hydroxy-5-deazaflavin chromophores in photoreactivation by Anacystis nidulans DNA photolyase.

DNA photolyase from the cyanobacterium Anacystis nidulans contains two chromophores, flavin adenine dinucleotide (FADH2) and 8-hydroxy-5-deazaflavin (8-HDF) (Eker, A. P. M., Kooiman, P., Hessels, J. K. C., and Yasui, A. (1990) J. Biol. Chem. 265, 8009-8015). While evidence exists that the flavin chromophore (in FADH2 form) can catalyze photorepair directly and that the 8-HDF chromophore is the major photosensitizer in photoreactivation it was not known whether 8-HDF splits pyrimidine dimer directly or indirectly through energy transfer to FADH2 at the catalytic center. We constructed a plasmid which over-produces the A. nidulans photolyase in Escherichia coli and purified the enzyme from this organism. Apoenzyme was prepared and enzyme containing stoichiometric amounts of either or both chromophores was reconstituted. The substrate binding and catalytic activities of the apoenzyme (apoE), E-FADH2, E-8-HDF, E-FAD(ox)-8-HDF, and E-FADH2-8-HDF were investigated. We found that FAD is required for substrate binding and catalysis and that 8-HDF is not essential for binding DNA, and participates in catalysis only through energy transfer to FADH2. The quantum yields of energy transfer from 8-HDF to FADH2 and of electron transfer from FADH2 to thymine dimer are near unity.     

Localization of the F420-reducing hydrogenase in Methanococcus voltae cells by immuno-gold technique

The F₄₂₀-reducing hydrogenase of Methanococcus voltae, which takes part in the terminal reduction step of methanogenesis, was localized in situ in ultrathin sections. This result was obtained by the immuno-gold technique using a high titer antiserum raised against the purified enzyme. Its specifity for the hydrogenase was shown by Western blot analysis. The hydrogenase of M. voltae was found to be membrane-associated.Abbreviations ELISA Enzyme linked immuno sorbent assay - F₄₂₀ 8-hydroxy-5-deazaflavin     

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.     

The pathway of electron transfer in NADH:Q oxidoreductase

The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.     

Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.     

Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine under anaerobic conditions by reductively activated nitro 5-deazaflavin derivatives.

Electrolytically reduced 6- and 8-nitro-5-deazaflavin derivatives have been found to interact to react specifically with guanine base by means of cyclic voltammetry. Electrolytic reductions of 6- and 8-nitro-5-deazaflavin derivatives in the presence of the 2'-deoxyguanosine under anaerobic conditions resulted in prominent formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine.     

A selenium-containing hydrogenase from Methanococcus vannielii. Identification of the selenium moiety as a selenocysteine residue

A 75Se-labeled hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii cells grown in the presence of [75Se]selenite. The molecular weight of the enzyme was estimated as 340,000 by gel filtration. The enzyme tends to aggregate and occurs also as a larger protein species (Mr = 1.3 x 10(6)). The same phenomenon was observed on native gel electrophoretic analysis. Hydrogenase activity exhibited by these two protein bands was proportional to protein and 75Se content. Both molecular species reduce the natural cofactor, 8-hydroxy-5-deazaflavin, and tetrazolium dyes with molecular hydrogen. Sodium dodecyl sulfate-gel electrophoresis of 75Se-labeled enzyme showed that 75Se is present exclusively in an Mr = 42,000 subunit. A value of 3.8 g atoms of selenium/mol of enzyme (Mr = 340,000) was determined by atomic absorption analysis. The chemical form of selenium in the enzyme was shown to be selenocysteine. This was identified as the [75Se]carboxymethyl and [75Se]carboxyethyl derivatives in acid hydrolysates of alkylated 75Se-labeled protein. The hydrogenase is extremely oxygen-sensitive but can be reactivated by incubation with molecular hydrogen and dithiothreitol.     

Photochemical system for regenerating NADPH from NADP with use of immobilized chloroplasts

Spinach chloroplasts were immobilized in 2% agar gel. Crude ferredoxin and NADP–ferredoxin oxidoreductase isolated from spinach were used as electron carriers. The activity of the NADP reduction by immobilized chloroplasts increased with increasing ferredoxin concentration and the maximum activity was obtained at 8μM ferredoxin. The saturation of NADP reduction was observed at a light intensity of over 1000 lx. The optimum pH and temperature of NADP reduction were 8 and 25°C, respectively. The reduced NADP in a reaction medium increased linearly with increasing reaction time under illumination. NADP was continuously reduced for 2 hr with a hollow-fiber reactor containing immobilized chloroplasts. NADPH and NADP were separated with a hollow-fiber dialyzer from ferredoxin and NADP–ferredoxin oxidoreductase, which were reused. The conversion ratio of NADP to NADPH was from 40 to 80%.     

Carbon monoxide:methylene blue oxidoreductase from Pseudomonas carboxydovorans.

The enzyme carbon monoxide:methylene blue oxidoreductase from CO autotrophically grown cells of Pseudomonas carboxydovorans strain OM5, was purified to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. The enzyme was stable for at least 6 days, had a molecular weight of 230,000, gave a single protein and activity band on polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis revealed a single band of molecular weight 107,000. Carbon monoxide:methylene blue oxidoreductase did not catalyze reduction of pyridine or flavin nucleotides but catalyzed the oxidation of CO to CO2 in the presence of methylene blue, thionine, toluylene blue, dichlorophenolindophenol, or pyocyanine under strictly anaerobic conditions. The visible spectrum revealed maxima at 405 and 470 nm. The millimolar extinction coefficients were 43.9 (405 nm) and 395.5 (275 nm), respectively. Absorption at 470 nm decreased in the presence of dithionite, and the spectrum was not affected by the substrate CO. Maximum reaction rates were found at pH 7.0 and 63 degrees C; temperature dependence followed the Arrhenius equation, with an activation energy (delta H degree) of 36.8 kJ/mol (8.8 kcal/mol). The apparent Km was 53 microM for CO. The purified enzyme was incapable of oxidizing methane, methanol, or formaldehyde in the presence of methylene blue as electron acceptor.     

Purification and properties of N5, N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of N5,N10-methylenetrahydromethanopterin (CH2 = H4MPT) to N5-methyltetrahydromethanopterin (CH3-H4MPT) is an intermediate step in methanogenesis from CO2 and H2. The reaction is catalyzed by CH2 = H4MPT reductase. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) was found to be specific for reduced coenzyme F420 as electron donor; neither NADH or NADPH nor reduced viologen dyes could substitute for the reduced 5-deazaflavin. The reductase was purified over 100-fold to apparent homogeneity. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band at the 36-kDa position. The apparent molecular mass of the native enzyme was determined by gel filtration to be in the order of 150 kDa. The purified enzyme was colourless. It did not contain flavin or iron. The ultraviolet visible spectrum was almost identical to that of albumin, suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentration at different constant concentrations of the second substrate yielded straight lines intersecting at one point on the abscissa to the left of the vertical axis. This intersecting pattern is characteristic of a ternary complex catalytic mechanism. The Km for CH2 = H4MPT and for the reduced coenzyme F420 were determined to be 0.3 mM and 3 microM, respectively. Vmax was 6000 mumol.min-1.mg protein-1 (kcat = 3600 s-1). The CH2 = H4MPT reductase was stable in the presence of air; at 4 C less than 10% activity was lost within 24 h.     

Promotion of radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by nitro 5-deazaflavin derivatives.

6-Nitro- and 8-nitro-5-deazaflavin derivatives have been found to enhance prominently the radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at the expense of formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine nucleosides (FapydGuo) both in deaerated and in N(2)O saturated aqueous 2'-deoxyguanosine solutions. The radiosensitizing capacity of a 9-nitro-5-deazflavin derivative was observed only in the N(2)O saturated aqueous solutions.     

Effect of temperature on the spectral properties of coenzyme F420 and related compounds.

The uv-visible spectra of 7,8-didemethyl-8-hydroxy-5-deazaflavin-5'-phosphoryllactyl glutamate (coenzyme F420), a naturally occurring 5-deazaflavin derivative, in three different buffers changed with a rise in temperature; the effect on the extinction coefficient at 420 nm (epsilon 420) was as follows: In phosphate-buffered solutions at pH less than 7.5, the epsilon 420 increased (at pH 5.0 for a temperature shift from 15 to 60 degrees C, delta epsilon 420 was +87%), but between pH 7.5 and 8, epsilon 420 changed very little. At pH greater than 8.0 in phosphate- or borate-buffered solutions, epsilon 420 decreased slightly. In morpholineethanesulfonic acid (Mes)-buffered F420 solutions at pH 5 and 5.5, epsilon 420 changed very little, whereas at pH 6-8, the epsilon 420 decreased. Absorbance of F420 at 401 nm in phosphate buffer at pH 5 to 9 was not significantly affected by temperature. Changes in epsilon 420 due to temperature change corresponded to changes in the pKa of 8-OH of the deazaflavin molecule; studies with adenylated F420 showed that the 8-OH of F420 was responsible for these changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soluble NADH-cytochrome b5 reductase from rabbit liver cytosol: partial purification and characterization.

A soluble form of NADH-cytochrome b5 reductase (NADH: ferricytochrome b5 oxidoreductase, EC 1.6.2.2) was found in the cytosolic fraction of rabbit liver. The partially purified enzyme was strictly specific for NADH. It catalyzed the reduction of several substrates such as the methemoglobin-ferrocyanide complex (Hegesh, E. and Avron, M. (1967) Biochim. Biophys. Acta 146, 91-101) (apparent Km: 8 micrometer), potassium ferricyanide (apparent Km: 10 micrometer) and ferricytochrome b5 (apparent Km: 15 micrometer). Upon acrylamide gel isoelectro-focusing followed by specific staining, the enzyme was resolved into four bands (isoelectric pH: 7.05, 6.70, 6.50 and 6.30). The optimum pH of activity with ferricytochrome b5 as a substrate was 6.5. The estimated molecular weight was 25 000--30 000. The enzyme was unsensitive to cyanide. It was strongly inhibited by p-hydroxymercuribenzoate. The cytosolic liver cytochrome b5 reductase was immunologically related to the soluble cytochrome b5 reductase from human and rabbit red-cells, and to the microsomal cytochrome b5 reductase from rabbit liver.     

Purification of the solubilized NADPH:O2 oxidoreductase of human neutrophils. Isolation of its catalytically inactive cytochrome b and flavoprotein redox centers

The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.     

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.