Oxidation of C1 compounds by Pseudomonas sp. MS

Pseudomonas sp. MS is capable of growth on a number of compounds containing only C₁ groups. They include trimethylsulphonium salts, methylamine, dimethylamine and trimethylamine. Although formaldehyde and formate will not support growth they are rapidly oxidized by intact cells. Methanol neither supports growth nor is oxidized. A particulate fraction of the cell oxidizes methylamine to carbon dioxide in the absence of any external electron acceptor. Formaldehyde and formate are more slowly oxidized to carbon dioxide by the particulate fraction, although they do not appear to be free intermediates in the oxidation of methylamine. Soluble NAD-linked formaldehyde dehydrogenase and formate dehydrogenase are also present. The particulate methylamine oxidase is induced by growth on methylamine, dimethylamine and trimethylamine, whereas the soluble formaldehyde dehydrogenase and formate dehydrogenase are induced by trimethylsulphonium nitrate as well as the aforementioned amines.     

Microbial growth on oxalate by a route not involving glyoxylate carboligase

1. The metabolism of oxalate by the pink-pigmented organisms, Pseudomonas AM1, Pseudomonas AM2, Protaminobacter ruber and Pseudomonas extorquens has been compared with that of the non-pigmented Pseudomonas oxalaticus. 2. During growth on oxalate, all the organisms contain oxalyl-CoA decarboxylase, formate dehydrogenase and oxalyl-CoA reductase. This is consistent with oxidation of oxalate to carbon dioxide taking place via oxalyl-CoA, formyl-CoA and formate as intermediates, and also reduction of oxalate to glyoxylate taking place via oxalyl-CoA. 3. The pink-pigmented organisms, when grown on oxalate, contain l-serine–glyoxylate aminotransferase and hydroxypyruvate reductase but do not contain glyoxylate carboligase. The converse of this obtains in oxalate-grown Ps. oxalaticus. This indicates that, in contrast with Ps. oxalaticus, synthesis of C₃ compounds from oxalate by the pink-pigmented organisms occurs by a variant of the `serine pathway' used by Pseudomonas AM1 during growth on C₁ compounds. 4. Evidence in favour of this scheme is provided by the finding that a mutant of Pseudomonas AM1 that lacks hydroxypyruvate reductase is not able to grow on oxalate.     

Choice between autotrophy and heterotrophy in Pseudomonas oxalaticus. Utilization of oxalate by cells after adaptation from growth on formate to growth on oxalate

1. The labelling patterns of phosphoglycerate obtained from formate-grown or oxalate-grown Pseudomonas oxalaticus after exposure for 15sec. to [¹⁴C]formate or [¹⁴C]oxalate respectively were determined. 2. The phosphoglycerate obtained from the formate-grown cells contained 78% of the radioactivity in the carboxyl group. This is in accord with that predicted for operation of the ribulose diphosphate cycle of carbon dioxide fixation. 3. The labelling pattern of the phosphoglycerate obtained from the oxalate-grown cells approached uniformity, as predicted for the heterotrophic pathway of oxalate assimilation. The departure from complete uniformity may have been due to concurrent ¹⁴CO₂ fixation into C₄ dicarboxylic acids. 4. The labelling pattern of phosphoglycerate obtained from cells that had just started to grow on oxalate after adaptation from formate was determined after incubation of the cells for 15sec. with [¹⁴C]oxalate. This pattern approached uniformity. 5. The pathway of incorporation of ¹⁴CO₂ into cells that had just started to grow on oxalate after adaptation from formate, in the presence of either formate or oxalate as energy source, was studied by chromatographic and radio-autographic analysis. 6. It is concluded from the isotopic data that a mixed heterotrophic–autotrophic metabolism, with the former mode predominating, operates in the initial stages of growth on oxalate after adaptation from growth on formate.     

Oxalate, formate, formamide, and methanol metabolism in Thiobacillus novellus.

Thiobacillus novellus was able to grow with oxalate, formate, formamide, and methanol as sole sources of carbon and energy. Extensive growth on methanol required yeast extract or vitamins. Glyoxylate carboligase was detected in extracts of oxalate-grown cells. Ribulose bisphosphate carboxylase was found in extracts of cells grown on formate, formamide, and thiosulfate. These data indicate that oxalate is utilized heterotrophically in the glycerate pathway, and formate and formamide are utilized autotrophically in the ribulose bisphosphate pathway. Nicotinamide adenine dinucleotide-linked formate dehydrogenase was present in extracts of oxalate-, formate-, formamide-, and methanol-grown cells but was absent in thiosulfate- and acetate-grown cells.     

The respiratory system of the marine bacterium Beneckea natriegens. II. Terminal branching of respiration to oxygen and resistance to inhibition by cyanide

1. Cell-free extracts of the marine bacterium Beneckea natriegens, derived by sonication, were separated into particulate and supernatant fractions by centrifugation at 150 000 × g.2. NADH, succinate, d(?)- and l(+)-lactate oxidase and dehydrogenase activities were located in the particles, with 2- to 3-fold increases in specific activity over the cell free extract. The d(?)- and l(+)-lactate dehydrogenases were NAD⁺ and NADP⁺ independent. Ascorbate-N,N,N′,N′-tetramethylphenylenediamine (TMPD) oxidase was also present in the particulate fraction; it was 7–12 times more active than the physiological substrate oxidases.3. Ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide. Succinate, NADH, d(?)-lactate and l(+)-lactate oxidases were inhibited in a biphasic manner, with 10 μM cyanide causing only 10–50 % inhibition; further inhibition required more than 0.5 mM cyanide, and 10 mM cyanide caused over 90 % inhibition. Low sulphide (5 μM) and azide (2 mM) concentrations also totally inhibited ascorbate-TMPD oxidase, but only partially inhibited the other oxidases. High concentrations of sulphide but not azide caused a second phase inhibition of NADH, succinate, d(?)-lactate and l(+)-lactate oxidases.4. Low oxidase activities of the physiological substrates, obtained by using non-saturating substrate concentrations, were more inhibited by 10 μM cyanide and 2 mM azide than high oxidase rates, yet ascorbate-TMPD oxidase was completely inhibited by 10 μM cyanide over a wide range of rates of oxidation.5. These results indicate terminal branching of the respiratory system. Ascorbate-TMPD is oxidised by one pathway only, whilst NADH, succinate, d(?)-lactate and l(+)-lactate are oxidised via both pathways. Respiration of the latter substrates occurs preferentially by the pathway associated with ascorbate-TMPD oxidase and which is sensitive to low concentrations of cyanide, azide and sulphide.6. The apparent K_m for O₂ for each of the two pathways was detected using ascorbate-TMPD and NADH or succinate plus 10 μM cyanide respectively. The former pathway had an apparent K_m of 8–17 (average 10.6) μM and the latter 2.2–4.0 (average 3.0) μM O₂.     

Studies on the metabolism of a sulfur-oxidizing bacterium IX. Electron transfer and the terminal oxidase system in sulfite oxidation of Thiobacillus thiooxidans

The nature of the electron transfer and terminal oxidase(s)in the sulfite-oxidizing system of Thiobacillus thiooxidnaswas studied in detail with various artificial electron donorsand inhibitors. Thionine, when reduced by ascorbate, was mosteffectively oxidized by whole cells and the particulate fractionof the various artificial electron donors. p-PD and TMPD werescarcely oxidized by either intact cells or the particulatefraction. The optimum pH of the thionine-oxidizing activity by the particulatefraction was 7.0 and that of the sulfite-oxidizing activitywas 6.8. The K_m values for thionine and sulfite were 7.6x10^–5Mand 1.6xl0^–4M, respectively. Sulfite oxidase activity in the particulate fraction was markedlyinhibited by amytal, rotenone, quinacrine-HGl and 2,4-DNP. HOQNOinhibited sulfite oxidase activity completely, but had no effecton thionine oxidase activity. Cyanide- and azide-insensitive respirations were present inthe particulate fraction. Thionine oxidase activity was inhibitedphoto-irreversibly with carbon monoxide, while sulfite oxidaseactivity showed photo-reversible carbon monooxide inhibition.The presence of two carbon monoxide-binding pigments was confirmedin the particulate fraction by a spectrophotometric study. (Received May 16, 1975; )     

Electrically Contacted Bienzyme‐Functionalized Mesoporous Carbon Nanoparticle Electrodes: Applications for the Development of Dual Amperometric Biosensors and Multifuel‐Driven Biofuel Cells

The capping of electron relay units in mesoporous carbon nanoparticles (MPC NPs) by crosslinking of different enzymes on MPC NPs matrices leads to integrated electrically contacted bienzyme electrodes acting as dual biosensors or as functional bienzyme anodes and cathodes for biofuel cells. The capping of ferrocene methanol and methylene blue in MPC NPs by the crosslinking of glucose oxidase (GOx) and horseradish peroxidase (HRP) yields a functional sensing electrode for both glucose and H₂O₂, which also acts as a bienzyme cascaded system for the indirect detection of glucose. A MPC NP matrix, loaded with ferrocene methanol and capped by GOx/lactate oxidase (LOx), is implemented for the oxidation and detection of both glucose and lactate. Similarly, MPC NPs, loaded with 2,2′‐azino‐bis(3‐ethylbenzo­thiazoline‐6‐sulphonic acid), are capped with bilirubin oxidase (BOD) and catalase (Cat), to yield a bienzyme O₂ reduction cathode. A biofuel cell that uses the bienzyme GOx/LOx anode and the BOD/Cat cathode, glucose and/or lactate as fuels, and O₂ and/or H₂O₂ as oxidizers is assembled, revealing a power efficiency of ≈90 μW cm^?2 in the presence of the two fuels. The study demonstrates that multienzyme MPC NP electrodes may improve the performance of biofuel cells by oxidizing mixtures of fuels in biomass.     

Effects of dithiothreitol on dopa oxidase activity from potato tubers

A filtrate, prepared from potato tuber by grinding in an isotonic medium, has been separated into a particulate and a ‘soluble’ fraction by ultracentrifugation. Following dialysis and lyophilization, both fractions catalysed the oxidation of l-DOPA, with approximately 30% of the l-DOPA: oxygen-oxidoreductase (EC 1.14.18.1; DOPA oxidase) activity being associated with the particulate fraction. When dithiothreitol (DTT, 10^?2M was included in the grinding medium, much lower yields of DOPA oxidase were obtained and 80% appeared to be associated with the particulate fraction. DTT proved to be a powerful inhibitor of DOPA oxidase. With concentrations of DTT causing only partial inhibition, the kinetics of the inhibited rate of dopachrome formation from l-DOPA were complex. When oxygen consumption was measured inhibition was not transient. The degree of inhibition was inversely related to the DOPA oxidase activity, indicating interaction of a product of this activity with DTT. Direct determination of -SH groups in DTT using 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) showed that they were all oxidised during the initial phase of inhibition of dopachrome formation. It is concluded that the first phase of inhibition involves oxidation of DTT by an intermediate between l-DOPA and dopachrome. The second phase of inhibition also appeared to require -SH groups initially, since trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT) caused very little inhibition at all.     

Cytochrome c-linked reactions in Rhodopseudomonas palustris grown photosynthetically on thiosulfate

The nonsulfur purple bacterium Rps. palustris was adapted to grow photoautotrophically with thiosulfate as substrate. An isolated cell-free fraction catalyzed the enzymatic transfer of electrons from thiosulfate to endogenous and/or added mammalian cytochrome c. Antimycin A, NOQNO, rotenone, amytal and atebrin did not inhibit the thiosulfate-cytochrome c reductase. The products of thiosulfate oxidation were primarily tetrathionate, trithionate, and sulfate, suggesting oxidation via the polythionate pathway. Succinate, formate and NADH were also effective electron donors in this system showing Michaelis constants of 40, 30 and 0.025 mm, respectively for cytochrome c reduction. The NADH-cytochrome c reductase was not inhibited by flavoprotein inhibitors and by Antimycin A or NOQNO. The cell-free extracts also contained an active cytochrome c-O₂ oxidoreductase which was inhibited by cyanide, azide and EDTA, and these inhibitions were overcome by the addition of Cu²⁺. The oxidase activity was stimulated by the addition of uncoupling agents such as CCCP and DNP, as well as by Antimycin A and NOQNO. Reduced + CO minus reduced difference absorption spectra revealed the presence of cytochrome components of the a and o types which may function as the terminal oxidase(s).     

Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation

Industrial biocatalytic reduction processes require the efficient regeneration of reduced cofactors for the asymmetric reduction of prochiral compounds to chiral intermediates which are needed for the production of fine chemicals and drugs. Here, we present a new engineering strategy for improved NADH regeneration based on the Pichia pastoris methanol oxidation pathway. Studying the kinetic properties of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) and using the derived kinetic data for subsequent kinetic simulations of NADH formation rates led to the identification of FLD activity to constitute the main bottleneck for efficient NADH recycling via the methanol dissimilation pathway. The simulation results were confirmed constructing a recombinant P. pastoris strain overexpressing P. pastoris FLD and the highly active NADH-dependent butanediol dehydrogenase from S. cerevisiae. Employing the engineered strain, significantly improved butanediol production rates were achieved in whole-cell biotransformations.     

A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in Syntrophomonas wolfei

In syntrophic conversion of butyrate to methane and CO₂, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO₂ reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate.In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases.Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.     

The seed proteins of chick pea: Comparative studies ofCicer arietinum,C. reticulatum andC. echinospermum (Leguminosae)

Seed proteins fromCicer arietinum L.,C. reticulatum Ladiz. andC. echinospermum Davis were extracted and separated into water soluble (albumin) and water insoluble (globulin) fractions. These were analysed using three polyacrylamide gel systems: uniform pore slab gels, gradient gels and SDS disc gels. For all three species, albumins constitute just over one-third of total protein. Minor differences in the composition of this fraction were observed. Within the globulin fraction, seven disulphide-linked polypeptides were found. Four of these resemble the major polypeptide of legumin, consisting of constant small subunit (21,000 daltons) linked to variable large subunit (46,000, 41,000, 39,000 or 36,000 daltons), forming polypeptides of 67,000 (I), 62,000 (II), 60,000 (III) and 57,000 (IV) daltons respectively. Polypeptide I was prominent in both wild species, but absent fromC. arietinum. Polypeptides II and III were equally prominent inC. arietinum andC. reticulatum. Polypeptide IV was more prominent inC. echinospermum, which was deficient in polypeptide III. Polypeptides V (45,000 daltons) and VI (43,000 daltons), apparently composed of two equal subunits, were present in trace amounts in both wild species, but well represented inC. arietinum Polypeptide VII of 45,000 daltons (31,000 + 14,000) was present in all three species.     

Respiratory components and oxidase activities in Alcaligenes eutrophus

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H₂ oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H₂ oxidase by 50% and solubilizes the bulk of the ATPase.2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa₃, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm).3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2–3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H₂ oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20–100% stimulation of oxygen uptake).4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.     

Influence of nitrate on oxalate- and glyoxylate-dependent growth and acetogenesis by Moorella thermoacetica

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [(14)C]bicarbonate or [(14)C]oxalate showed that CO(2) reduction to acetate and biomass or oxalate oxidation to CO(2) was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO(2) into biomass than oxalate-grown cells. CO(2) conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO(2) as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.     

Use of selenite,selenide, and selenocysteine for the synthesis of formate dehydrogenase by a cysteine-requiring mutant ofEscherichia coli K-12

The forms of Se in the Se-dependent enzyme formate dehydrogenase is known to be selenocysteine, but the way this amino acid enters the polypeptide chain has not been established. Through the use of a cysteine-requiring mutant ofEscherichia coli K-12 that could also grow in the presence of glutathione, we were able to study the effect of selenite, selenide, andl-selenocysteine, each at a concentration of 0.1 μM, on the synthesis of formate dehydrogenase. The three forms of Se served equally well for inducing formate dehydrogenase activity, measured by dichlorophenol-indophenol reduction mediated by phenazine methosulfate. It is known that selenite can be reduced to selenide by the action of glutathione reductase, present inE. coli, and that selenocysteine is converted to elemental Se by the action of selenocysteine lyase, also present in the mutant. Elemental Se is then reduced nonenzymatically to hydrogen selenide. The conversion of both selenite and selenocysteine to selenide and the ability of each form of Se to induce the synthesis of equal levels of formate dehydrogenase suggest that the incorporation of Se into formate dehydrogenase is accomplished by a posttranslational mechanism.     

Carbonic anhydrase activity in halophilic anaerobes from soda lakes

The activity and cellular localization of carbonic anhydrase (CA) in two alkaliphilic anaerobes growing in soda lakes at pH 9–10 were studied. CA activity in the cell extracts of the acetogenic bacterium Natroniella acetigena was comparable to that of neutrophilic acetogens. Hydrogenotrophically grown cells of Desulfonatronum lacustre exhibited higher CA activity compared to the cells grown on medium with formate. High CA activity in the cytoplasmic fraction and the absence of high activity in the extracellular fraction were demonstrated. We propose that the cytoplasmic CA in alkaliphilic sulfate-reducers participates in conversion of bicarbonate to CO₂, which is reduced in the cell to acetate via the acetyl-CoA pathway.     

Oxidation of C1 Compounds by Particulate fractions from Methylococcus capsulatus: distribution and properties of methane-dependent reduced nicotinamide adenine dinucleotide oxidase (methane hydroxylase).

Cell-free particulate fractions of extracts from the obligate methylotroph Methylococcus capsulatus catalyze the reduced nicotinamide adenine dinucleotide (NADH) and O2-dependent oxidation of methane (methane hydroxylase). The only oxidation product detected was formate. These preparations also catalyze the oxidation of methanol and formaldehyde to formate in the presence or absence of phenazine methosulphate with oxygen as the terminal electron acceptor. Methane hydroxylase activity cannot be reproducibly obtained from disintegrated cell suspensions even though the whole cells actively respired when methane was presented as a substrate. Varying the disintegration method or extraction medium had no significant effect on the activities obtained. When active particles were obtained, hydroxylase activity was stable at 0 C for days. Methane hydroxylase assays were made by measuring the methane-dependent oxidation of NADH by O2. In separate experiments, methane consumption and the accumulation of formate were also demonstrated. Formate is not oxidized by these particulate fractions. The effects of particle concentration, temperature, pH, and phosphate concentration on enzymic activity are described. Ethane is utilized in the presence of NADH and O2. The stoichiometric relationships of the reaction(s) with methane as substrate were not established since (i) the presumed initial product, methanol, is also oxidized to formate, and (ii) the contribution that NADH oxidase activity makes to the observed consumption of reactants could not be assessed in the presence of methane. Studies with known inhibitors of electron transport systems indicate that the path of electron flow from NADH to oxygen is different for the NADH oxidase, methane hydroxylase, and methanol oxidase activities.     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.     

Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii

Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R₃” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN₃, and NH₂OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.