L-malate oxidation by the electron transport fraction of Azotobacter vinelandii

The membrane-bound l-malate oxidoreductase of Azotobacter vinelandii strain O was found to be a flavoprotein-dependent enzyme associated with the electron transport system (R(3)) of this organism. The particulate R(3) fraction, which possessed the l-malate oxidoreductase, carried out the cyanide-sensitive oxidation of l-malate, d-lactate, reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, succinate, cytochrome c, tetramethyl-p-phenylenediamine, and p-phenylenediamine, with molecular O(2) as the terminal electron acceptor. d-Malate was not oxidized, but l-malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K(3), K(3)Fe(CN)(6), nitro blue tetrazolium, and dichloroindophenol all served as good terminal electron acceptors for the l-malate oxidoreductase. Cytochrome c was a poor electron acceptor. Extensive studies on the l-malate oxidase and PMS and K(3) reductases revealed that all were stimulated specifically by flavine adenine dinucleotide and nonspecifically by di- or trivalent cations, i.e., Ca(++), Ba(++), Mn(++), Mg(++), Fe(+++), Ni(++), and Al(+++). All these activities were markedly sensitive to ethylenediaminetetraacetate (EDTA). The V(max) values for the l-malate oxidase, PMS, and vitamin K(3) reductases were, respectively, 3.4, 15.1, and 45.5 mumoles of substrate oxidized per min per mg of protein at 37 C. Spectral studies revealed that the Azotobacter R(3) flavoprotein and cytochromes (a(2), a(1), b(1), c(4), and c(5)) were reduced by l-malate. l-Malate oxidase activity was sensitive to various inhibitors of the electron transport system, namely, p-chloromercuriphenylsulfonic acid, chlorpromazine, 2-n-heptyl-4-hydroxyquinoline-N-oxide, antimycin A, and KCN. Minor inhibitory effects were noted with the inhibitors 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, rotenone, and Amytal.     

Enzymatic Oxidation of Tetramethyl-p-Phenylenediamine and p-Phenylenediamine by the Electron Transport Particulate Fraction of Azotobacter vinelandii

The ability of the electron transport particulate fraction of Azotobacter vinelandii strain O to oxidize tetramethyl-p-phenylenediamine (TMPD) and p-phenylenediamine (PPD) was examined in detail. The highest specific activity for TMPD and PPD oxidation concentrated in the A. vinelandii O R(3) fraction. The A. vinelandii O R(3) fraction was used to develop a standard manometric assay which gave optimal oxidation rates for both of these dyes. The conditions of the assay and all essential related enzymatic kinetic parameters are presented. Other para derivatives of phenylenediamines also were oxidized readily, whereas ortho and meta derivatives were not. Hydroquinone, p-hydroxybenzoic acid, p-cresol, tyrosine, pyrogallol, pyrocatechol, and diphenylamine were not able to serve as electron donors for the A. vinelandii O R(3) system. The probable involvement of a particle-bound cytochrome oxidase is indicated by the marked sensitivity of both TMPD and PPD oxidation to cyanide, axide, phenylhydrazine, hydroxylamine, and, to a lesser degree, carbon monoxide.     

The Respiratory Chain of Plant Mitochondria: V. Reaction of Reduced Cytochromes a and a3 in Mung Bean Mitochondria with Oxygen in the Presence of Cyanide

The half-times of oxidation by oxygen pulses of reduced cytochromes a and a(3) in mung bean mitochondria made anaerobic with succinate have been measured by means of a rapid mixing flow apparatus coupled to a dual wave length spectrophotometer in the presence and absence of cyanide. The absorbance changes at 438 to 455 millimicrons and 603 to 620 millimicrons are suitable for recording the time course of cytochrome a oxidation; the half-time is 2.0 milliseconds at 24 Celsius. This half-time does not change over the range 0 to 300 mum KCN, but the fraction of cytochrome a oxidized falls to a limiting value of 0.3 at the higher cyanide concentrations. The absorbance changes at 445 to 455 millimicrons record the time course of both cytochrome a and cytochrome a(3) oxidation; the former contributes 60% of the absorbance change and the latter 40%. The half-time for a(3) oxidation is calculated as 0.9 milliseconds at 24 Celsius. This half-time increases slightly to 1.3 milliseconds at 300 mum KCN. Reduced cytochrome a(3), whether uncomplexed or complexed with cyanide, becomes fully oxidized. The dissociation constant for the reduced cytochrome a(3)-cyanide complex is estimated to be 30 mum, whereas that for the oxidized a(3)-cyanide complex which inhibits electron transport is estimated to be 2 mum. This suggests two different binding sites for cyanide on the reduced and oxidized forms of cytochrome a(3). The fact that a limiting fraction of reduced cytochrome a can be oxidized at high cyanide concentrations implies that there is no interference by cyanide with electron transport from a to a(3), if cyanide remains bound to the site it occupies on reduced a(3) after this carrier becomes oxidized on reaction with molecular oxygen. Rearrangement of cyanide from this noninhibitory site to the inhibitory site occurs rapidly enough to compete with cytochrome a oxidation. The half-time for the rearrangement is calculated to be 0.9 milliseconds.     

Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii.

The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azotobacter vinelandii concentrates in the membranous R3 fraction that is directly associated with Azotobacter electron transport function. Sonically disrupted Azotobacter cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R3 membranous fraction which sediments on ultracentrifugation at 144 000 X g for 2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activty occurred at pH 8.0; Mg2+ ion when added to the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATP stoichiometry was 1:1 on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0-60 min). The membrane-bound ATPase in the R3 fraction was stimulated 12-fold by treatment wiTH TRypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R3 electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R3 fraction had no effect on activity; overnight refrigeration at 4 degrees C, however, resulted in a 25% loss of activity as compared with a 14% loss when the R3 fraction was stored overnight at 25 degrees C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (-20 degrees C), and subsequent sonication failed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R3 electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation.     

Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii.

It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction in Azotobacter vinelandii strain O using turbidimetrically standarized resting cell suspensions. The Q(O2) value obtained for whole cell oxidation of ascorbate-TMPD appeared to reflect the full measure of the high respiratory oxidative capability usually exhibited by this genera of organisms. The Q(O2) value for the TMPD oxidase reaction ranged from 1,700 to 2,000 and this value was equivalent to that obtained for the oxidation of the growth substrate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cells was similar to that obtained for the "particulate" A. vinelandii electron transport particle, that fraction which TMPD oxidase activity is exclusively associated with. Under the conditions used, there appeared to be no permeability problems; TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a rate comparable to that of the growth substrate. This, however, was not true for the oxidation of another electron donor, 2,6-dichloroindophenol, whose whole cell Q(O2) values, under comparable conditions, were twofold lower. The TMPD oxidase activity in A. vinelandii whole cells was found to be affected by the physiological growth conditions, and resting cells obtained from cells grown on sucrose, either under nitrogen-fixing conditions or on nitrate as the combined nitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidase rates were also noted for chemically induced pleomorphic A. vinelandii cells, which suggests that modified growth conditions can (i) alter the nature of the intracellular terminal oxidase formed (or induced), or (ii) alter surface permeability, depending upon the growth conditions used. Preliminary studies on the quantitative TMPD oxidation reaction in mutant whole cells of both Azotobacter and a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxidase activity, showed this assay can be very useful for detecting respiratory deficiencies in the metabolism of whole cells.     

Effects of electron transport inhibitors and uncouplers on the oxidation of ferrous iron and compounds interacting with ferric iron in Acidithiobacillus ferrooxidans

Oxidation of Fe2+, ascorbic acid, propyl gallate, tiron, L-cysteine, and glutathione by Acidithiobacillus ferrooxidans was studied with respect to the effect of electron transport inhibitors and uncouplers on the rate of oxidation. All the oxidations were sensitive to inhibitors of cytochrome c oxidase, KCN, and NaN3. They were also partially inhibited by inhibitors of complex I and complex III of the electron transport system. Uncouplers at low concentrations stimulated the oxidation and inhibited it at higher concentrations. The oxidation rates of Fe2+ and L-cysteine inhibited by complex I and complex III inhibitors (amytal, rotenone, antimycin A, myxothiazol, and HQNO) were stimulated more extensively by uncouplers than the control rates. Atabrine, a flavin antagonist, was an exception, and atabrine-inhibited oxidation activities of all these compounds were further inhibited by uncouplers. A model for the electron transport pathways of A. ferrooxidans is proposed to account for these results. In the model these organic substrates reduce ferric iron on the surface of cells to ferrous iron, which is oxidized back to ferric iron through the Fe2+ oxidation pathway, leading to cytochrome oxidase to O2. Some of electrons enter the uphill (energy-requiring) electron transport pathway to reduce NAD+. Uncouplers at low concentrations stimulate Fe2+ oxidation by stimulating cytochrome oxidase by uncoupling. Higher concentrations lower deltap to the level insufficient to overcome the potentially uphill reaction at rusticyanin-cytochrome c4. Inhibition of uphill reactions at complex I and complex III leads to deltap accumulation and inhibition of cytochrome oxidase. Uncouplers remove the inhibition of deltap and stimulate the oxidation. Atabrine inhibition is not released by uncouplers, which implies a possibility of atabrine inhibition at a site other than complex I, but a site somehow involved in the Fe2+ oxidation pathway.     

Metabolism of Pipecolic Acid in a Pseudomonas Species IV. Electron Transport Particle of Pseudomonas putida

Baginsky, Marietta L. (University of California, San Francisco Medical Center, San Francisco), and Victor W. Rodwell. Metabolism of pipecolic acid in a Pseudomonas species. IV. Electron transport particle of Pseudomonas putida. J. Bacteriol. 92:424-432. 1966.-Enzymes of Pseudomonas putida P2 catalyzing oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate are located in a subcellular fraction sedimenting at 105,000 x g. Since this fraction resembles the mammalian electron transport particle in both chemical composition and enzymatic activities, it was termed Pseudomonas P2 electron transport particle (P2-ETP). P2-ETP contains flavin adenine dinucleotide, flavin mononucleotide, iron, copper, and both b- and c-type cytochromes. The reduced type b cytochrome has absorption maxima at 558 to 559, 530, and 427 mmu. Its oxidized pyridine hemochromogen has an absorption maximum at 406 mmu, with a shoulder at 564 mmu. On dithionite reduction, absorption bands with maxima at 556, 522, and 418 mmu are obtained. The reduced type c cytochrome has absorption maxima at 552, 520, and 422 mmu; its reduced pyridine hemochromogen has maxima at 551, 516 to 519, and 418 mmu. No type a cytochrome was detected. P2-ETP catalyzes oxidation of pipecolate and of reduced nicotinamide adenine dinucleotide (NADH(2)) by oxygen. It can also oxidize these compounds, as well as succinate and reduced nicotinamide adenine dinucleotide phosphate, with 2,6-dichlorophenol-indophenol as electron acceptor. Mammalian cytochrome c can be used as an alternate artificial electron acceptor for the oxidation of pipecolate and succinate, but not for oxidation of NADH(2).     

The methylamine oxidizing system of Pseudomonas AM1 reconstituted with purified components

The electron transport system coupled to the oxidation of methylamine in Pseudomonas AM1 was investigated by reconstituting it from the highly purified components. A mixture of methylamine dehydrogenase, cytochrome cH and cytochrome c oxidase (= cytochrome aa3) actively oxidized methylamine (161 mol of O2 consumed/mol of heme a of cytochrome c oxidase X min). In this system, addition of amicyanin did not affect the oxygen consumption rate. The oxygen consumption rate of the cell-free extract prepared from the cells cultivated in a copper-deficient medium was directly proportional to the amount of amicyanin added, and extrapolation to zero copper concentration gave a value of 28 mol of O2 consumed/mol of heme a of cytochrome c oxidase X min. These results suggest that methylamine oxidation in the bacterium can occur at least to some extent without participation of amicyanin.     

Hydrogen-oxidizing electron transport components in nitrogen-fixing Azotobacter vinelandii.

Membranes from N2-fixing Azotobacter vinelandii were isolated to identify electron transport components involved in H2 oxidation. We found direct evidence for the involvement of cytochromes b, c, and d in H2 oxidation by the use of H2-reduced minus O2-oxidized absorption difference spectra. Carbon monoxide spectra showed that H2 reduced cytochrome d but not cytochrome o. Inhibition of H2 oxidation by cyanide was monophasic with a high Ki (135 microM); this was attributed to cytochrome d. Cyanide inhibition of malate oxidation showed the presence of an additional, low Ki (0.1 microM cyanide) component in the membranes; this was attributed to cytochrome o. However, H2 oxidation was not sensitive to this cyanide concentration. Chlorpromazine (at 160 microM) markedly inhibited malate oxidation, but it did not greatly inhibit H2 oxidation. Irradiation of membranes with UV light inhibited H2 oxidation. Adding A. vinelandii Q8 to the UV-damaged membranes partially restored H2 oxidation activity, whereas addition of UV-treated Q8 did not increase the activity. 2-n-Heptyl-4-hydroxyquinoline-N-oxide inhibited both H2 and malate oxidation.     

The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex

The involvement of cytochromes in the electron-transport pathway to the periplasmic NO3- reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO3- reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO3(-)-oxidized spectra of cells had peaks in the alpha-band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c- and b-type cytochromes in the electron-transport pathway to NO3-. The total ferricyanide-oxidizable cytochrome that was also oxidized in the steady state by NO3- was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO3- reduction. Neither CN-, nor a previously characterized inhibitor of NO3- reduction, 2-n-heptyl-4-hydroxyquinoline N-oxide, prevented the oxidation of the cytochromes by NO3-. This suggested a site of action for these inhibitors on the reducing side of the b- and c-type cytochromes involved in electron transport to the NO3- reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c-type cytochrome with an alpha-band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO3- to a periplasmic fraction. The NO3(-)-oxidizable cytochrome co-purified with the periplasmic NO3- reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion-exchange chromatography. A NO3(-)-reductase-cytochrome-c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c-type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl-Sepharose hydrophobic chromatography column.     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. I. The interaction of cytochrome c2 with the reaction center

1. The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25% L-alpha-phosphatidylglycerol). 2. At low ionic strength, the kinetics of cytochrome-c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 microM, the process was strictly bimolecular (second-order rate constant, k = 1.7 x 10(9) M-1 s-1), while at higher concentrations a fast oxidation process (half-time lower than 20 microseconds) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 microM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction-center--cytochrome-c2 complex of about 10(5) M-1 was evaluated. From the fraction of photo-oxidized reaction centers promptly re-reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome-c2 site was exposed to the external compartment. 3. Both the second-order oxidation reaction and the formation of the reaction-center--cytochrome-c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCl, the value of the second-order rate constant was decreased to 7.0 x 10(7) M-1 s-1 and no fast oxidation of cytochrome c2 could be observed at 10 microM cytochrome c2. 4. The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double-flash experiments, varying the dark time between photoactivations over the range 30 microseconds to 5ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 x 10(3) s-1 and a lower limit for k2 of approximately 2 x 10(4) s-1 (corresponding to a second-order rate constant of approximately 3 x 10(9) M-1 s-1).     

Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.

During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex formation between flavodoxin and cytochrome c. Cross-linking studies

Complex formation between Azotobacter vinelandii flavodoxin and horse cytochrome c has been demonstrated through cross-linking studies with dimethyl suberimidate, dimethyl adipimidate, 1-ethyl-3-(3-di-methylaminopropyl)carbodiimide, and dimethyl-3,3'-dithiobispropionimidate. Essentially quantitative cross-linking of cytochrome c and flavodoxin was observed at low ionic strengths with the carbodiimide cross-linking reagent. An association constant of 4 X 10(4) M-1 was obtained between cytochrome c and flavodoxin at 88 mM ionic strength from analysis of the cross-linking studies. This value is similar to the association constant determined kinetically during the electron transfer reaction between cytochrome c and flavodoxin (Simondsen, R.P., Weber, P.C., Salemme, F.R., and Tollin, G. (1982) Biochemistry 21, 6366-6375), and suggests that the cross-linked complex may be similar to the precursor complex identified kinetically. A structural model for the flavodoxin-cytochrome c complex proposed by these workers is shown to be compatible with the present cross-linking results.     

The DeLey-Doudoroff Pathway of Galactose Metabolism in Azotobacter vinelandii

Azotobacter vinelandii cell extracts reduced NAD and oxidized d-galactose to galactonate that subsequently was converted to 2-keto-3-deoxy-galactonate. Further metabolism of 2-keto-3-deoxy-galactonate required the presence of ATP and resulted in the formation of pyruvate and glyceraldehyde 3-P. Radiorespirometry indicated a preferential release of CO(2) at the first carbon position of the d-galactose molecule. This suggested that Azotobacter vinelandii metabolizes d-galactose via the DeLey-Doudoroff pathway. The first enzyme of this pathway, d-galactose dehydrogenase, was partially characterized. It has a molecular weight of about 74,000 Da and an isoelectric point of 6.15. The pH optimum of the galactose dehydrogenase was about 9. The apparent K(m)s for NAD and d-galactose were 0.125 and 0.56 mM, respectively. Besides d-galactose, the active fraction of this galactose dehydrogenase also oxidized l-arabinose effectively. The electron acceptor for d-galactose or l-arabinose oxidation, NAD, could not be replaced by NADP. These substrate specificities were different from those reported in Pseudomonas saccharophila, Pseudomonas fluorescens, and Rhizobium meliloti.     

Crystal structure of Azotobacter cytochrome c5 at 2.5 A resolution

The crystal structure of cytochrome c5 from Azotobacter vinelandii has been solved and refined to an R value of 0.29 at 2.5 A resolution. The structure of the oxidized protein was solved using a monoclinic crystal form. The structure was solved by multiple isomorphous replacements, re-fit to a solvent-leveled multiple isomorphous replacement map, and refined by restrained least squares. The structure reveals monomers associated about the crystallographic 2-fold axis by hydrophobic contacts at the "exposed heme edge". The overall conformation for the monomer is similar to that of Pseudomonas aeruginosa cytochrome c551. However, relative to a common heme conformation, c5 and c551 differ by an average of 6.8 A over 82 alpha-carbon positions and the propionates of c5 are much more exposed to solvent. The shortest heme--heme contact at the "dimer" interface is 6.3 A (Fe to Fe 16.4 A). Alignment of c5 and c551 shows that the two cytochromes, in spite of sequence differences, have remarkably similar charge distributions. A disulfide stacks on a tyrosine between the N- and C-terminal helices.     

The interaction of metronidazole with electron carriers of the Azotobacter vinelandii respiratory particle fraction

Abstract Metronidazole, menadione bisulfite, and oxygen oxidized NADH- and dithionite-reduced flavin of the Azotobacter vinelandii respiratory particle fraction. The oxidation of NADH-reduced flavin by metronidazole was slow compared to the oxidations by menadione bisulfite and oxygen. Metronidazole caused spectral changes characteristic of cytochrome d oxidation, but the changes in NADH-reduced particles were slow.     

The methanol-oxidizing system of Methylobacterium extorquens AM1 reconstituted with purified constituents

The electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system. A mixture of 2.7 microM methanol dehydrogenase, 3.2 microM cytochrome cH, and 71 nM cytochrome c oxidase (= cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4 microM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min). Further addition of amicyanin to the above mixture did not affect the activity. Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture. On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M. extorquens AM1.     

Effects of dibromothymoquinone and bathophenanthroline on flash-induced cytochrome f oxidation in spinach chloroplasts

The effects of several electron transport inhibitors on themagnitude and kinetics of cytochrome f oxidation induced byflash illumination were studied in the - and -band regions.On the flash excitation only a fraction of cytochrome f presentin the chloroplasts was oxidized with a half time of 0.1 to0.3 msec and then reduced with a half time of 10 to 25 msec. Dibromothymoquinone (DBMIB) at concentrations which severelysuppressed the reduction of cytochrome f approximately doubledthe magnitude of cytochrome f oxidation caused by a flash, mainlyby inducing an additional slow oxidation of cytochrome f witha half time longer than 1 msec. Enhancement of the cytochromef oxidation was also observed in the presence of bathophenanthroline.Such enhanced oxidation in duced by the two inhibitors was largelydiminished with the addition of reduced 2,6-dichlorophenolindophenolwhich accelerated cytochrome f reduction. In contrast, the inhibitionof cytochrome f reduction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) was not associated with an increase in the magnitudeof cytochrome f oxidation. However, addition of DBMIB to theDCMU-poisoned chloroplasts enhanced cytochrome f oxidation,suggesting that this is related to a block of the electron transportbetween plastoquinone and cytochrome f. The results are explainedby assuming the occurrence of an electron carrier between plastoquinoneand cytochrome f. (Received May 10, 1978; )     

The Respiratory Chain of Plant Mitochondria: IV. Oxidation Rates of the Respiratory Carriers of Mung Bean Mitochondria in the Presence of Cyanide

The half-time for oxidation of cytochrome b(557) in mitochondria from etiolated mung bean (Phaseolus aureus) hypocotyls is 5.8 milliseconds at 24 Celsius in the absence or presence of 0.3 mm KCN, when the oxidation is carried out by injecting a small amount of oxygenated medium into a suspension of mitochondria made anaerobic in the presence of succinate plus malonate. Since oxygen is consumed by the alternate, cyanide-insensitive respiratory pathway of these mitochondria, cycles of oxidation and reduction can be obtained with the oxygen pulses when cyanide is present. Reduced cytochromes (a + a(3)) also become oxidized at nearly the uninhibited rate under these conditions, a(3) completely and a partially. The half-time for oxidation of c(547) is also unaffected by 0.3 mm KCN, but c(549) has a half-time equal to that of c(547) in the presence of KCN, compared to the shorter one observed in the absence of inhibitor. The maximum extent of oxidation of the cytochromes c is about 70% in the presence of 0.3 mm KCN; this oxidation is rapidly followed by an extensive reduction which is synchronous with the reduction of cytochrome a observed under the same conditions. In the presence of cyanide, it appears likely that the cytochromes c and b(557) are oxidized by cytochrome oxidase in oxygen pulse experiments, rather than by the alternate oxidase. The oxidation of cytochrome b(553) is partially inhibited by KCN, but complete oxidation is attained in the aerobic steady state with excess oxygen. If the oxygen pulse experiment is carried out in the presence of sufficient malonate so that entry of reducing equivalents into the respiratory chain occurs at a rate negligible compared to inter-carrier electron transport, the half-time for flavoprotein oxidation is unaffected by 0.3 mm KCN while that for ubiquinone oxidation is but 2-fold larger. The observed net oxidation rate of these two carriers in mung bean mitochondria is more sensitive to the entry rate of reducing equivalents, as set by succinate concentration and malonate to succinate ratio, then it is in skunk cabbage (Symplocarpus foetidus) mitochondria. These observations are interpreted in terms of a respiratory carrier Y, placed between flavoprotein plus ubiquinone and the cytochromes, which is the fork in the split respiratory pathway to the two terminal oxidases and which has lower electron transport capacity in mung bean mitochondria than in skunk cabbage mitochondria.     

Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD⁺, NADP⁺, FAD, or FMN for activity. O₂, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R₃ fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a₂, a₁, b₁, c₄ and c₅) are reduced by the addition of l-glutamate.