Relationships between membrane-bound cytochrome o from Vitreoscilla and that of Escherichia coli

The cytochrome o terminal oxidases from the bacteria Vitreoscilla and Escherichia coli are structurally and functionally related. They have similar optical spectra, both exhibit ubiquinol-1 oxidase activity and are inhibited similarly. Both enzymes contain four subunits by SDS-polyacrylamide gel electrophoresis analysis and contain protoheme IX and Cu2+ prosthetic groups. Antibodies raised against the oxidase purified from E. coli crossreact with the Vitreoscilla oxidase.     

Metabolic role of flavoprotein in terminal oxidation by cytochrome o from Vitreoscilla

NADH-cytochrome o reductase is associated with purified preparationsof cytochrome o, and these preparations can be separated into"reductase-enriched" and "reductase-poor" fractions by columnchromatography. Direct evidence for the presence of flavin inthese preparations was obtained from fluorescence spectra, andthe intensity of the fluorescence maxima was greater in reductase-enrichedpreparations of cytochrome o than in reductase-poor ones. Exogenouslyadded flavin stimulated the rate of NADH oxidation by molecularoxygen that is catalyzed by preparations of cytochrome o, morestimulation being observed with "reductase-poor" than with "reductase-enriched"preparations. Since reduction of cytochrome o in an aerobicsolution was also stimulated by added flavin, the primary effectof the latter is on the NADH-cytochrome o reductase side ofthe cytochrome. Possible explanations for the observed stimulationof the reduction of cytochrome o in aerobic solutions in thepresence of exogenous flavin are 1) reconstitution of flavin-deficientreductase, 2) flavin acting as a mobile electron carrier betweenthe reductase and the cytochrome, 3) cytochrome o being reducedby superoxide anion generated as an intermediate in the reactionof reduced flavin with oxygen. More direct evidence for theparticipation of a flavor-protein in the reduction of cytochromeo was the observed photochemical reduction of cytochrome o inan anaerobic CO atmosphere without added flavin using EDTA asan electron donor. (Received July 18, 1977; )     

Purification and partial characterization of the membrane-bound cytochrome o(561,564) from Vitreoscilla

Cytochrome o(561,564) terminal oxidase was solubilized from the membrane fraction of the bacterium Vitreoscilla sp., strain C1, and purified by differential pH dialysis, gel filtration chromatography, and ion-exchange chromatography. Subunit molecular weights, determined on sodium dodecyl sulfate-polyacrylamide gels by the Ferguson plot method, were 49,500 and 23,500. There were two protohemes IX, two coppers, and 45 mol of phosphorus per mole of protomer (73,000). The molecular weight of the cytochrome o complex estimated by chromatography on Sephacryl-400 in deoxycholate was 265,000, which is consistent with the enzyme complex under these conditions being a dimer (146,000) with the remaining molecular weight contribution arising from bound phospholipid, deoxycholate, and possibly other, smaller subunits. Difference spectra of the dithionite-reduced enzyme have split alpha absorption maxima at 561 and 564 nm at room temperature and 558 and 561 nm at 77 K. The CO difference spectrum at room temperature has absorption maxima at 570, 534, and 416 nm. Dissociation constants for CO and cyanide binding to the reduced and oxidized forms of the oxidase are 5.2 microM and 3.5 mM, respectively. The hemes in the cytochrome are one electron accepting centers, both with midpoint potentials around +165 mV at pH 7.0. The enzyme is highly autoxidizable, and its menadiol oxidizing activity is stimulated by phospholipids.     

Biphasic recombination of photodissociated CO compound of cytochrome o(s) from Vitreoscilla

The soluble cytochrome o from Vitreoscilla contains two identical subunits and two hemes. The reduced form binds 2 mol of CO in a cooperative manner with a Hill coefficient near 2 (Tyree, B., and Webster, D. A. (1978) J. Biol. Chem. 253, 6988-6991). This carbonyl compound was photolysed with a dye laser and recombination followed at 437 or 420 nm where maximal absorbance changes were registered. Recombination kinetics were biphasic, and the fast phase was approximately 10 times the rate of the slow phase. Apparent rate constants of both phases showed a nonlinear dependence on CO concentration, respectively, in conformity with a reaction scheme which assumes the transient formation of an intermediate species in both slow and fast reactions. A study of temperature dependence of the reactions gave EA = 2.7 kcal/mol for the slow reaction and EA = 3.2 kcal/mol for the fast reaction below 23 degrees C; above this temperature the slope of the Arrhenius plot for the fast reaction became positive. Maximal rates for both phases were around pH 6.5 and fell to approximately 40% of maximal at pH 12. The binding reaction was affected by even a low concentration of sodium dodecyl sulfate (0.0025%), which changed both the kinetic constant of each phase and the relative contribution of each phase to the reaction. A model which assumes the existence of fast and slow reaction conformers in equilibrium is proposed.     

Oxygenated cytochrome o (Vitreoscilla) formed by treating oxidized cytochrome with superoxide anion

Oxygenated cytochrome o can be formed experimentally in twoways, i) by reaction of reduced cytochrome o with molecularoxygen, or ii) by reaction of oxidized cytochrome o with superoxideanion generated by the action of the xanthine oxidase system.It is thermodynamically feasible for oxidized cytochrome o plusO₂–, and reduced cytochrome o plus O₂ to appear as intermediatesin reactions i) and ii), respectively. Superoxide dismutase completely inhibits the xanthine oxidase-catalyzedconversion of oxidized cytochrome o into the oxygenated formbut it has relatively little effect on the oxygenated cytochromeo formation in the reaction system consisting of NADH, NADH-cytochromeo reductase, and cytochrome o. Thus, if superoxide anion doesplay a significant role in the latter system it must be efficientlycoupled to react with cytochrome o and inaccessible to superoxidedismutase. Direct electron transfer from the reductase to thecytochrome without the involvement of superoxide anion is analternative mechanism. (Received December 16, 1976; )     

The formation of hydrogen peroxide during the oxidation of reduced nicotinamide adenine dinucleotide by cytochrome o from Vitreoscilla.

The formation of hydrogen peroxide during the oxidation of NADH by purified preparations of cytochrome o has been demonstrated by employing three independent methods: polarographic, colorimetric, and fluorometric. The first two methods were used to assay for the accumulation of hydrogen peroxide and showed that hydrogen peroxide did accumulate as a product, but only about 30% of the oxygen consumed or 15 to 20% of the NADH oxidized was recoverable as hydrogen peroxide. This lack of 1:1 stoichiometry was not due to residual catalase activity in these preparations which could be eliminated by freeze-thawing. Thus, hydrogen peroxide may not be the sole or primary product of the NADH-cytochrome o oxidase reaction. The fluorometric assay could be coupled directly to the NADH-cytochrome o oxidase reaction in one medium, and this method showed that hydrogen peroxide was generated continuously from the beginning of the reaction in a 1:1 stoichiometry, hydrogen peroxide generated to NADH oxidized. This result suggests that hydrogen peroxide is an intermediate that can be trapped efficiently under the conditions of the fluorometric assay, whereas under the conditions of the first two assays most of the hydrogen peroxide generated undergoes further reaction. Exogenously added FAD or FMN increased the percentage of hydrogen peroxide that accumulated in the NADHcytochrome o oxidase reaction. Flavin is believed to act on the reductase side of cytochrome o so the increased percentage of hydrogen peroxide is not likely to result from the direct reaction of reduced flavin with oxygen.     

Physiological role of oxygenated cytochrome o: observations on whole-cell suspensions of Vitreoscilla.

The form of cytochrome o that predominates in Vitreoscilla cells having various levels of respiratory activity was studied by using untreated, frozen-thawed, and starved cells, which had respiratory rates decreasing in the order given. Direct spectral observation revealed that the oxygenated form of cytochrome o predominated during the aerobic steady-state oxidation of endogenous substrate or exogenous glutamate in untreated and frozen-thawed cells and was replaced by the reduced form when the cell suspensions became anaerobic. The respiratory rates, estimated inversely from the time of duration of the steady state, were correlated to the rates of oxygen consumption for the various cells. Oxidized cytochrome o predominated in aerobic starved cells. These results indicate the involvement of three forms of cytochrome o--oxidized, reduced, and oxygenated--in the catalytic and cyclic change of this cytochrome. The oxygenated form also appeared after the addition of hydrogen peroxide to the cells, but only the oxidized form appeared when ethyl hydrogen peroxide was added. The appearance of the oxygenated form with the addition of hydrogen peroxide was probably due to the reaction of the reduced cytochrome with the oxygen that had evolved by the action of catalase present in the cells.     

Photodissociation of oxygenated cytochrome o(s) (Vitreoscilla) and kinetic studies of reassociation

Oxygenated cytochrome o(s) from Vitreoscilla was photodissociated by a laser flash but the quantum yield was low. The rebinding of oxygen to the ferrous cytochrome proceeded monophasically, and the second order rate constant was 7.8 X 10(7) M-1 s-1, the off rate constant 5.6 X 10(3) s-1, and the calculated dissociation constant for the oxygenated compound 7.2 X 10(-5) M at pH 7.3 and 25 degrees C. Rapid scanning spectroscopy revealed the formation of chytochrome o-O2 directly from ferrous chytochrome o and oxygen without any evidence for an intermediary formation of Compound D, another type of oxygenated chytochrome o. Photodissociation in solution containing CO/O2 mixtures resulted in rapid binding of oxygen followed by slow replacement by CO. This property as well as the photodissociability of chytochrome o-O2 suggests that the heme iron of the compound is in the ferrous state. In addition, the primary oxygen compound was fairly stable and did not decay further in the absence of CO, in marked contrast with that of mammalian cytochrome oxidase primary oxygen compound which rapidly decayed. This result suggests a possible role of this cytochrome as an oxygen carrier or storage.     

Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla

Cytochrome o, a protoheme IX pigment, has been proposed as the terminal oxidase of the filamentous bacterium, Vitreoscilla. Aerobic and anaerobic photolysis of CO-liganded whole cells demonstrated the presence of a second CO-reactive pigment, cytochrome o'. At temperatures lower than -100 degrees C, anaerobic photolysis dissociated only about 25% of the total CO-liganded components to reveal the unliganded cytochrome o'. At these temperatures, the photolysis of cytochrome o could not be demonstrated. At warmer temperatures, recombination of CO with the reduced cytochrome o' occurred with an apparent energy of activation of 5.8 kcal/mol. Aerobic photolysis of whole cells demonstrated two oxygen-bound intermediates. At temperatures lower than -95 degrees C, a spectrally distinct compound with absorption maxima at 428, 534, and 564 nm appeared (form I'); the apparent second order rate constant (k+1) for the formation of this intermediate was found to be 9.1 M-1 s-1, the reverse rate (k-1) was 9.9 X 10(-5) s-1, and the equilibrium constant (Kd) was 1.1 X 10(-5) M. This oxygen intermediate of cytochrome o' is spectrally and kinetically similar to the oxygen intermediate of cytochrome o seen in Escherichia coli. At temperatures warmer than -90 degrees C, photolysis of aerobic samples resulted in the immediate formation of a second oxygen-bound intermediate (form I) with absorption maxima at 422, 534, and 564 nm. This second intermediate results from the binding of oxygen to the cytochrome o (oxygenated cytochrome o). These data support the proposal that whole cells of Vitreoscilla contain two alternative pathways of electron transport, one terminating with cytochrome o and the other with cytochrome o'.     

The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii.

Oxidation-reduction titrations of Azotobacter vinelandii cytochrome o + c4 and cytochrome o were performed with simultaneous potential and absorbance measurements under anaerobic conditions. Cytochrome c4 has a midpoint potential (Em, 7.4) of 260mV and purified cytochrome o has an Em, 7.4 of -18mV. Little change in the midpoint potential of cytochrome o was observed when titrated in the pH range 6.2--9.8.     

Biochemical and biophysical properties of cytochrome o of Azotobacter vinelandii

Cytochrome o, solubilized from the membrane of Azotobacter vinelandii, has been purified to homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The detergent-containing cytochrome o is composed of one polypeptide chain with a molecular weight of 28 000-29 000, associated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exists as a dimer by gel filtration analysis. The amino analysis which reveals the majority of residues are of hydrophobic nature. The cytochrome o oxidase contains protoheme as its prosthetic group and about 20-40% of phospholipids. The phospholipids are identified as phosphatidylethanolamine and phosphatidylglycerol by radioautographic analysis using 2-dimensional thin-layer chromatography. No copper or nonheme iron can be detected in the purified oxidase preparation by atomic absorption and chemical analyses. Oxidation-reduction titration shows this membrane-bound cytochrome o to be a low-potential component, and Em was determined to be -18 mV in the purified form and -30 mV in the membrane-bound form. Both forms bind CO with a reduced absorption peak at 559 and 557-558 nm in the native and solubilized forms, respectively. A high-spin (g = 6.0) form is assigned to the oxidized cytochrome o by electron paramagnetic resonance analysis, and KCN abolishes this high-spin signal. CO titration of purified cytochrome o in the anaerobic conditions shows the enzyme binds one CO per four protohemes and a dissociation constant is estimated to be 3.2 microM for CO. Cyanide reacts with purified cytochrome o in both oxidized and CO-bound forms, identified by specific spectral compounds absorbed at the Soret region. Cytochrome c, often co-purified with cytochrome c from the membrane, cannot serve as a reductant for cytochrome o in vitro, due to the apparent potential difference of about 300 mV. Upon separation, both cytochrome o and cytochrome c4 show a great tendency of aggregation. Furthermore, the oxidase activity (measured by tetramethyl-p-phenylenediamine oxidation rate) decreases as the cytochrome c concentration is decreased by ammonium sulfate fractionation. All these suggest the structural and functional complex nature of cytochrome c4 and cytochrome o in the membrane of A. vinelandii.     

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth. (c) 1996 John Wiley & Sons, Inc.     

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth.