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.     

Comparative Cytochrome Oxidase and Superoxide Dismutase Analyses on Strains of Azotobacter vinelandii and Other Related Free-Living Nitrogen-Fixing Bacteria

Quantitative N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase and superoxide dismutase (SOD) analyses were performed on representative organisms of the family Azotobacteraceae. Azotobacter vinelandii, Azotobacter chroococcum, Azotobacter paspali, and Derxia gummosa exhibited high quantitative TMPD oxidase activities, and their extracts possessed very active and electrophoretically homogeneous (single gel band) Fe-type SODs. Azomonas macrocytogenes extracts had similar single Fe-type SODs, and their cells exhibited no TMPD-dependent cytochrome oxidase activity. Nitrogen-fixing cells of Beijerinckia indica, Beijerinckia derxii, and Beijerinckia mobilis exhibited minimal TMPD oxidation capabilities (rates equivalent to the TMPD autooxidation reaction), and these extracts also possessed very active SODs but only of the Mn metallotype.     

Use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria.

It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5. The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels.     

Use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria.

It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5. The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels.     

Alternative Function of the Electron Transport System in Azotobacter vinelandii: Removal of Excess Reductant by the Cytochrome d Pathway

The N(inf2)-fixing bacterium Azotobacter vinelandii was grown in an O(inf2)-regulated chemostat with glucose or galactose as substrate. Increasing the O(inf2) partial pressure resulted in identical synthesis of the noncoupled cytochrome d terminal oxidase, which is consistent with the hypothesis that A. vinelandii uses high rates of respiration to protect the nitrogenase from oxygen. However, cell growth on glucose showed a lower yield of biomass, higher glycolytic rate, higher respiratory rate, and lower cytochrome o content than cell growth on galactose. Elemental analysis indicated no appreciable change in the C-to-N ratio of cell cultures, suggesting that the major composition of the cell was not influenced by the carbon source. A poor coordination of glucose and nitrogen metabolisms in A. vinelandii was suggested. The rapid hydrolysis of glucose resulted in carbonaceous accumulation in cells. Thus, Azotobacter species must induce a futile electron transport to protect cells from the high rates of glucose uptake and glycolysis.     

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.     

Construction and characterization of a mutant of alkaliphilic Bacillus firmus OF4 with a disrupted cta operon and purification of a novel cytochrome bd.

The caa3-type terminal oxidase of Bacillus firmus OF4 has been proposed to play an important role in the growth and bioenergetics of this alkaliphile (A. A. Guffanti and T. A. Krulwich, J. Biol. Chem. 267:9580-9588, 1992). A mutant strain was generated in which the cta operon encoding the oxidase was disrupted by insertion of a spectinomycin resistance cassette. The mutant was unable to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Absorption spectra of membranes confirmed the loss of the enzyme and indicated the presence of a cytochrome bd-type terminal oxidase. The mutant could grow on glucose but was unable to grow on malate or other nonfermentative carbon sources, despite the presence of the cytochrome bd. The cytochrome bd was purified from the mutant. The enzyme consisted of two subunits and, with menadiol as substrate, consumed oxygen with a specific activity of 12 micromol of O2 x min(-1) x mg(-1). In contrast to both cytochromes bd of Escherichia coli, the enzyme did not utilize TMPD as an electron source. A number of additional features, including subunit size and spectral properties, distinguish this cytochrome bd from its counterparts in E. coli and Azotobacter vinelandii.     

Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii.

The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given.     

Hyperproduction of Poly-beta-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD

The transformation of Azotobacter vinelandii UW with A. vinelandii 113 DNA resulted in the formation of rifampin-resistant colonies, 13% of which also inherited a previously unrecognized mutation in the respiratory NADH oxidase. These transformants produced colonies with a white-sectored phenotype after prolonged incubation. Cells from these sectors were separated and purified by streaking and were named UWD. The dense white phenotype was due to the production of a large amount of poly-beta-hydroxybutyrate during the exponential growth of strain UWD. The polymer accounted for 65 or 75% of the cell dry weight after 24 h of incubation of cultures containing glucose and either ammonium acetate or N(2), respectively, as the nitrogen source. Under the same conditions, strain UW cells contained 22 to 25% poly-beta-hydroxybutyrate, but O(2)-limited growth was required for these optimal production values. Polymer production was not dependent on O(2) limitation in strain UWD, but the efficiency of conversion of glucose to poly-beta-hydroxybutyrate was enhanced in O(2)-limited cultures. Conversion efficiencies were >0.25 and 0.33 mg of poly-beta-hydroxybutyrate per mg of glucose consumed under vigorous- and low-aeration conditions, respectively, compared with an efficiency of 0.05 achieved by strain UW. Strain UWD, therefore, appeared to from poly-beta-hydroxybutyrate under novel conditions, which may be useful in designing new methods for the industrial production of biodegradable plastics.     

Cytochrome c oxidation by the electron transport fraction of Azotobacter vinelandii

The spectrophotometric oxidation of horse heart ferrocytochrome c was examined by use of the particulate electron transport fraction (R(3)) of Azotobacter vinelandii strain O. Unlike cytochrome c, purified preparations of native Azotobacter cytochromes c(4) + c(5) were oxidized only slowly by the electron transport fraction. The oxidation of mammalian cytochrome c proceeded at an appreciable rate and displayed "apparent" first-order kinetics at a pH optimum of 9.0 with tris(hydroxymethyl)aminomethane-chloride buffer. The calculated V(max) value was 0.22 mumole of cytochrome c oxidized per min per mg of protein (25 C) and a K(m) value for cytochrome c of 2.3 x 10(-5)m was obtained. Ferricytochrome c was a "strict" competitive inhibitor for this oxidation. Cytochrome c oxidation by the Azotobacter electron transport system was markedly sensitive to cyanide, azide, and hydroxylamine, although carbon monoxide inhibition could not be demonstrated. It was sensitive also to high concentrations of phosphate, ethylenediaminetetraacetate, and some metal cations. "Aging" or prolonged storage of the Azotobacter R(3) fraction, at 4 C for 10 days, resulted in a threefold increase in specific activity. The cytochrome c peroxidase type of reaction did not occur with the R(3) electron transport fraction.     

Mannuronan C-5 epimerases and cellular differentiation of Azotobacter vinelandii

Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a co-polymer of β- d -mannuronic acid (M) and α- l -guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the epimerase-encoded R-modules. This gene is located within the epimerase gene cluster of Azotobacter vinelandii .     

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.     

Cytochrome bd from Azotobacter vinelandii: evidence for high-affinity oxygen binding

Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements of O2 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give Kd(O2) = 0.5 microM, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 microM); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with O2 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate of O2 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.     

Internal Membrane Control in Azotobacter vinelandii

Azotobacter vinelandii was grown on N(2), NH(4) (+), or NO(3) (-), and an internal membrane network was observed by electron microscopy of thin sections of cells. Cells obtained in early exponential growth contained less internal membrane than did cells from cultures in late exponential growth. It seems likely that O(2) has a role in regulating the amount of internal membrane structure.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. II. Ionically adsorbed cells

Continuous culture studies of Azotobacter vinelandii cells immobilized by ionic adsorption to Cellex E anion exchange resin were conducted under oxygen-limited conditions for comparison to free-cell cultures. Immobilization had little effect upon the specific respiration and sucrose consumption rates as compared to free cells. However, maxima in specific nitrogen fixation rate and nitrogenase activity as a function of dissolved oxygen occurred at a C(O(2) ) value of approximately 0.005 mM as opposed to 0.02 mM for free cells. Further, in contrast to free-cell culture, most of the fixed nitrogen appeared in the medium rather than within intact cells. There were strong indications that reproduction of bound cells often resulted in cell lysis accounting for the fixed nitrogen content in solution.     

Phospholipids of Azotobacter vinelandii

Analyses of resting cells of Azotobacter vinelandii revealed that numerous phospholipids were present that did not concentrate in the membranous R(3) fraction which carried out electron transport function.     

The cytochrome cbo from the obligate methylotroph Methylobacillus flagellatus KT is a cytochrome-c oxidase

The oxidase cho of Methylobacillus flagellatus KT was purified to homogeneity by nondenaturing gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cbo with a pH optimum of 8.3. When TMPD served as electron donor for the oxidase cho, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and of only ascorbate. The kinetic constants, determined at pH 7.0, were as follows: oxidation by the enzyme of reduced TMPD at pH 7.0 was characterized by KM = 0.86 mM and Vmax = 1.1 mumol O2/(min mg protein), and oxidation of reduced cytochrome c from horse heart was characterized by KM = 0.09 mM and Vmax = 0.9 mumol O2/(min mg protein) Cyanide inhibited ascorbate/TMPD oxidase activity (Ki = 4.5-5.0 microM). The soluble cytochrome cH (12 kDa) partially purified from M. flagellatus KT was found to serve as the natural electron donor for the oxidase cbo.     

Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: A cytochrome o mutant is aero-tolerant during nitrogen fixation

Abstract The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo -type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o . However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd ) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o . Cytochrome o -deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd , which is required for respiratory protection of oxygen-labile nitrogenase.     

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N',N'-tetramethyl-p-phenyl-enediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT--cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (approximately 70% of the total with resting and approximately 85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.     

Respiratory properties of cysts and vegetative cells of Azotobacter vinelandii

Abstract The respiratory activity of cysts of Azotobacter vinelandii has been compared with that of vegetative cells. Whole cysts had a much reduced respiratory activity which was less sensitive to KCN. Substrate oxidation rates by membrane preparations from cysts were reduced approximately 10-fold and sensitivity to KCN was decreased by a similar factor. Difference spectra of cyst membranes revealed changes in cytochrome content. Cytochrome oxidase d was apparently absent, cytochrome a ₁ levels were approximately halved whilst those of cytochrome oxidase o were almost doubled. Cytochromes of the b and c -type were present in similar amounts to those in vegetative cells.