Tetramethyl-p-phenylenediamine Oxidase Research Articles

Modifications of the Aerobic Respiratory Chain of Paracoccus Denitrificans in Response to Superoxide Oxidative Stress.

Paracoccus denitrificans is a strictly respiring bacterium with a core respiratory chain similar to that of mammalian mitochondria. As such, it continuously produces and has to cope with superoxide and other reactive oxygen species. In this work, the effects of artificially imposed superoxide stress on electron transport were examined. Exposure of aerobically growing cells to paraquat resulted in decreased activities of NADH dehydrogenase, succinate dehydrogenase, and N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) oxidase. Concomitantly, the total NAD(H) pool size in cells was approximately halved, but the NADH/NAD+ ratio increased twofold, thus partly compensating for inactivation losses of the dehydrogenase. The inactivation of respiratory dehydrogenases, but not of TMPD oxidase, also took place upon treatment of the membrane fraction with xanthine/xanthine oxidase. The decrease in dehydrogenase activities could be fully rescued by anaerobic incubation of membranes in a mixture containing 2-mercaptoethanol, sulfide and ferrous iron, which suggests iron–sulfur clusters as targets for superoxide. By using cyanide titration, a stress-sensitive contribution to the total TMPD oxidase activity was identified and attributed to the cbb3-type terminal oxidase. This response (measured by both enzymatic activity and mRNA level) was abolished in a mutant defective for the FnrP transcription factor. Therefore, our results provide evidence of oxidative stress perception by FnrP.

A cb-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori.

A Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). When ascorbate was used as reductant, the Vmax and apparent Km values were 612 nmol electron min-1 (mg protein)-1 and 14 microM for yeast, and 419 nmol electron min-1 (mg protein)-1 and 19 microM for equine cytochrome c, respectively. For TMPD oxidation, the Vmax and Km values were 640 nmol electron min-1 (mg protein)-1 and 182 microM, respectively. These oxidase activities showed a high affinity for oxygen. Inhibition of both cytochrome-c and TMPD oxidase activities by 50% was caused by about 4 microM cyanide and about 0.5 mM azide. Redox difference spectra of the membrane solubilized with Triton X-100 showed b- or c-type cytochromes but not aa3-type cytochromes. c-type and a part of some b-type cytochromes were reduced with ascorbate plus TMPD. A CO difference spectrum revealed that protohaem, but not an aa3-type cytochrome, may be interacting with CO/oxygen. Only protohaem was detected in the haem fraction extracted from the membrane. Three polypeptides (60, 38 and 29 kDa) were found to be bearing haem c after SDS-PAGE of the membrane. From these results, it was suggested that the cbb3-type cytochrome-c oxidase, having a haem-copper binuclear centre like the cytochrome aa3-type oxidase, but differing in a few other properties, functions as a terminal oxidase in the respiratory chain of H. pylori.

Cytochromes c‐dependent aerobic respiration of Paracoccus denitrificans

AbstractTerminal parts of the respiratory chain of Paracoccus denitrificans containing cytochromes c have been investigated through the use of NNN′Nprime;‐tetramethyl‐p‐phenylenediamine (TMPD) as an electron donor. The cells have been shown to possess two major membrane bound TMPD oxidases characterized by their different sensitivities to cyanide. The more sensitive one, identified as cytochrome aa3, appears to be produced under oxygen‐rich growth conditions, whereas a less sensitive and as yet uncharacterized enzyme accompanied with only moderate amounts of cytochrome aa3 functions when oxygen becomes limiting. The soluble periplasmic cytochrome cd1 does not contribute significantly to the total oxygen consumption rate, although its maximal catalytic capacity may be high. The fact thap the activity of alternative TMPD oxidase is not proportional to cytochrome o content is discussed in relation to possible occurrence of two distinct cytochrome o‐type terminal oxidases.

Trypsin proteolysis of the cytochrome d complex of Escherichia coli selectively inhibits ubiquinol oxidase activity while not affecting N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity.

The cytochrome d complex is one of two membrane-bound terminal oxidases of the Escherichia coli aerobic respiratory chain. Previous studies have shown that this enzyme reconstituted into proteoliposomes rapidly oxidizes ubiquinol-8 as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane H+ electrochemical gradient. The enzyme also oxidizes the artificial reductant, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) with the generation of a H+ electrochemical gradient. In this work, it is established that trypsin digestion of the purified cytochrome d complex cleaves subunit I while subunit II is unaffected. Proteolysis of subunit I is correlated with loss of ubiquinol-8 and ubiquinol-1 oxidase activities. Trypsin digestion has no effect on TMPD oxidase activity. The cytochrome d complex is concluded to possess three distinct active sites for 1) ubiquinol oxidation, 2) TMPD oxidation, and 3) oxygen binding and reduction. Data also suggest that both sites of ubiquinol and TMPD oxidations are located on the periplasmic side of the E. coli membrane while the site of oxygen reduction is on the opposite side.

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.

Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane.

Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a1, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.

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.

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.

Survey of Oxidase-Positive and -Negative Bacteria Using a Quantitative Kovacs Oxidase Test

A manometric assay system employing ascorbate and N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD) was used to quantitate terminal oxidase activity in bacterial non-proliferating whole cells. A wide variety of physiologically diverse bacteria, all of which were grown heterotrophically, was tested by this assay. For this survey study, 79 bacterial strains, which represented 34 genera, were used. Turbidimetrically standardized resting (non-proliferating) cell suspensions were prepared from cells harvested at the late logarithmic growth phase; all cells were grown under identical nutritional conditions. The TMPD oxidase activity obtained quantitatively correlated exceptionally well with results of the standard Kovacs oxidase test. In fact, the increased sensitivity of this quantitative assay allowed for further reclassification within the two major divisions of Kovacs oxidase-positive and -negative groups. Groups I and II contained all of the oxidase-positive microorganisms and the bacteria listed in group I had the highest TMPD oxidase rates, the Qo2 values (microliters of O2 consumed per hour per milligram [dry weight] at 30 C) ranging from 393 to 2, 164. The organisms listed in group II still had moderately high TMPD oxidase activity, the Qo2 values ranging from 27 to 280. All oxidase-negative bacteria fell into groups III and IV. Bacteria in group III had low but still measurable TMPD oxidase rates, the Qo2 values ranging from 3 to 33, whereas the bacteria found in group IV were inert and unable to oxidize TMPD. A grouping analysis allowed for the resolution of that point which separates oxidase-positive from oxidase-negative bacteria. This point, for non-proliferating cells, was found to be an absolute TMPD oxidation Qo2 value of 33 (after correcting for the endogenous rate by subtraction) and a Qo2 (TMPD/endogenous) ratio of 5; the latter parameter indicated that the uncorrected TMPD oxidation Qo2 value had to be five times greater than the rate for endogenous respiration. All Kovacs oxidase-positive organisms were found to have TMPD oxidase Qo2 values greater than these two metabolic parameters, whereas all Kovacs oxidase-negative organisms had lower values.

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.

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.

Properties of electron transport particles from Halobacterium cutirubrum. The respiratory chain system

1. The electron transport particles (Type II) of Halobacterium cutirubrum were prepared by sedimentation at 79000 × g and by treatment with 70 mM MgCl2-100 mM Tris-HCl (pH 7.6). The negatively stained sample of the elexctron transport particles (Type II) showed vesicles of two sizes, 330 A and 165 A.2. Spectrophotometric investigations at 22° and −196° showed that the electron transport particles (Type II) contained a-, b- and c-type cytochromes with cytochrome o and cytochrome a3 possibly acting as oxidases. The cytochrome o content was about two and a half times that of cytochrome a3. Both cytochromes could be reduced by ascorbate. The c-type cytochrome could be reduced by ascorbate—tetramethyl-p-phenylenediamine (TMPD) but not by ascorbate alone.3. Polarographic studies showed that azide and cyanide blocked ascorbate—TMPD oxidase activity and that CO inhibited O2 uptake.4. The extracted o-type cytochrome could not be reduced by ascorbate or reoxidize reduced cytochrome c. Its dithionite-reduced minus oxidized spectrum showed maxima at 559, 525 and 428 mμ; its reduced pyridine hemochromogen spectrum showed maxima at 555, 524 and 418 mμ. Thus cytochrome o appears to be a b-type cytochrome.