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Q H Gibson

Publications and source records attributed to Q H Gibson.

At least 73 records · Page 4Linked to original sources
PubMed1983

Turnover of cytochrome c oxidase from Paracoccus denitrificans.

The heme aa3 type cytochrome oxidase from Paracoccus denitrificans incorporated into vesicles with phospholipid reacts during turnover much as the oxidase from mitochondria does. The spectrophotometric changes observed at various wavelengths are closely similar, and the rate is about one-half of that for beef heart oxidase under the same conditions. The rate of appearance of oxidized cytochrome c on initiation of the reaction is also similar and depends on the previous treatment of the oxidase as described by Antonini, E., Brunori, M., Colosimo, A., Greenwood, C. and Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-3132. In terms of their model the resting Paracoccus enzyme is converted to the pulsed form during turnover. The effect is observed with both cytochrome c and hexamine ruthenium as reductants. With the latter a 60-fold increase in rate is observed.

Anaerobiosis↗
PubMed1983

Oxygen activation by cytochrome oxidase: a new spectral intermediate observed by flow-flash.

The reaction of cytochrome-c-oxidase with O2 has been reinvestigated at 2 degrees C by a flow laser pulsed method. The experiments indicate that a new spectral intermediate, populated with a rate constant of approximately 5000/s, is observed in a wavelength range in which heme a is the only chromophore (445 and 430 nm). The results are interpreted with reference to previous data obtained in the near infrared region, and it is suggested that breakage of the (enzyme bound) dioxygen bond is associated to a spectral perturbation of the cytochrome a3-CuB binuclear center.

Animals↗
PubMed1982

Spectra of intermediates in oxidation and reduction of cytochrome c oxidase.

Two kinetic components with distinct difference spectra occur during reduction of cytochrome c oxidase by ruthenium hexamine. They are attributed to reduction of heme a (fast phase) and heme a3 (slow phase) (Scott, R. A., and Gray, H. B. (1980) J. Am. Chem. Soc. 102, 3219-3774). Two spectra seen during oxidation of cytochrome c oxidase by molecular oxygen have also been attributed to oxidation of hemes a3 and a (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787). We now report that spectra for the reductive and oxidative reactions obtained with the same preparations and the same apparatus under similar conditions are significantly different. The reactions appear to populate different reaction intermediates. Reconstitution into phospholipid vesicles does not affect these two spectra significantly. During turnover, the chief intermediates are those of the reductive pathway (Scott and Gray type intermediates). Reduction of heme a3 occurs approximately 70 times faster after turnover than the reduction of the resting enzyme. This is probably a dramatic "pulsing" effect (Wilson, M. T., Peterson, J., Antonini, E., Brunori, M., Colosimo, A., and Wyman, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 7115-7118).

Electron Transport Complex IV↗
PubMed1982

Cooperative ligand binding to hemoglobin. Effects of temperature and pH on a hemoglobin with spectrophotometrically distinct chains (Tunnus thynnus).

Binding of CO to the hemoglobin of Tunnus thynnus has been studied by kinetic and static methods in the range pH 6.75-8.0 and at temperatures between 1.5-40 degrees C. The results may be described in terms of the two-state model of cooperativity (Monod. J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) extended to include chain differences. The rate constants for CO binding to alpha and beta chains in the R state, at 20 degrees C, were 8.4 and 1.9 microM-1 s-1 with associated apparent activation energies of 3.5 and 10.5 kcal mol-1. Dissociation from the R state was monophasic with a rate constant, at 20 degrees C, of 0.016 s-1 and Ea of 26.4 kcal mol-1. The difference spectrum for binding of CO to the alpha chain in the T state is displaced 2 nm to the red as compared to the spectra associated with CO binding to the beta chain in the T state and to both chains in the R state. The proportion of T state hemoglobin present at different fractional saturations can thus be measured and compared with the results predicted from the model. When the temperature is raised, the value of L decreases greatly with an enthalpy of +80 kcal mol-1. The effects of the change in L override the decrease in intrinsic CO affinity of T. thynnus hemoglobin in the R and T states and result in a reverse temperature dependence of CO equilibria compared to mammalian hemoglobins.

Animals↗
PubMed1981

Reaction of oxygen with cytochrome c oxidase from Paracoccus denitrificans.

The reaction of reduced cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans (American Type Culture Collection 13543) with dioxygen has been followed by laser flash photolysis of the CO derivative. In detergent-stabilized solutions the reaction showed at least two distinct kinetic components, the faster of which was oxygen concentration dependent and had a rate of approximately 60 X 10(6) M-1 s-1. The slower reaction was independent of oxygen concentration and had a rate of 9 X 10(2) s-1. These rates are about 1.5 times greater than comparable rates for ox heart oxidase reported by C. Greenwood and Q. H. Gibson (J. Biol. Chem. (1967) 242, 1782-1787). The kinetic components have markedly different optical spectra which agree precisely in form with those for ox heart enzyme (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787) but are shifted by 2 nm toward the red. In phospholipid vesicles, the spectral contribution of the faster component was augmented. The dissociation constant for CO at 20 degrees C is 1.6 microM, 6 times greater than for the ox heart enzyme. The bacterial enzyme binds one CO per 2 heme a. The enzyme has an absorption band at 830 nm in the oxidized form similar to that of the ox heart enzyme.

Carbon Monoxide↗
PubMed1981

Studies on carbon monoxide binding by shark haemoglobin.

The kinetics of the reactions of Pacific-porbeagle haemoglobin with CO were studied by flash-photolysis and stopped-flow methods, and the equilibrium binding curves for CO were measured in spectrophotometric titrations. Measurements were made in the pH range 6-8 and in the temperature range 0-40 degrees C. The results are discussed in terms of the allosteric model proposed by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88-118]. Within this framework the results indicate that in the R-state the haem groups fall into two classes of different reactivity with different spectral characteristics, but that in the T-state the groups may be essentially equivalent. The physiological importance of the temperature-insensitivity of the equilibrium ligand-binding curves for porbeagle haemoglobin is discussed.

Animals↗
PubMed1981

Asynchronous ligand binding and proton release in a root effect hemoglobin.

CO binding to the Root effect hemoglobin of menhaden, Brevoortia tyrannus, has been studied by flash photolysis and equilibrium measurements in [bis(2-hydroxyethyl)amino]Tris(hydroxymethyl)methane and Tris buffers, containing 0.2 M NaCl, between pH 6.0 and 8.0. The equilibrium and kinetic data were analyzed according to the two-state model, extended to include chain differences. The calculated value of the allosteric constant, L, varied from 3 X 10(6) at pH 6.0 to 20 at pH 8.0, lower at each pH value than that computed for phosphate buffer. In addition, the intrinsic rate constants of both T and R states were found to vary with pH. The kinetics of CO binding and of proton release, followed by absorbance changes in the pH indicator dye phenol red, were observed in 0.2 M NaCl, at pH values ranging from 6.3 to 7.8. Proton release lags behind CO binding across this pH range, the larger lags occurring at lower pH; this suggests that some proton release is associated with quaternary conformational change. The CO binding progress curves in unbuffered solution were simulated by the two-state model; in these calculations the value of L was systematically changed during the course of the reaction. The time courses of reaction intermediates, obtained from these computations, were then used to represent the kinetics of proton release. A simple model, assuming that proton release accompanies quaternary conformational transition but a modified model, incorporating pH dependence of the intrinsic T and R state affinities, describes proton release across the pH range studied.

Animals↗
PubMed1981

Allosteric properties of carbamylated hemoglobins.

The spectra, ligand-binding properties, and conformational change of carbamylated human hemoglobin A, specifically modified at its alpha-NH2 groups, have been compared with those of hemoglobin A. In the Soret region, the spectra of all deoxyhemoglobins are identical. The Soret bands of carbonmonoxy alpha 2c beta 2 and alpha 2 beta 2c are shifted in opposite directions relative to hemoglobin A, alpha 2c beta 2 having its Soret band at the longer wavelength. Carbamylation of either chain slows oxygen dissociation from the alpha chain in the R-state, although the effect is larger for alpha 2c beta 2 and alpha 2c beta 2c than alpha 2 beta 2c. Carbamylation of either chain also slowed oxygen binding to the alpha chain while the rate for the beta chain is not changed significantly, indicating that the chains influence each other in the R-state. The rates of oxygen binding after full and partial laser photolysis of oxygen-saturated alpha 2c beta 2 appear identical. The rate of oxygen rebinding for alpha 2 beta 2c, after partial (10%) photolysis is the same as that for alpha 2c beta 2, but following full photolysis, biphasic rebinding was observed due to the appearance of T-state molecules. Carbon monoxide binding studies under photostationary conditions suggested an L value for alpha 2c beta 2c smaller than that for alpha 2 beta 2c and hemoglobin A. The R to T transition of the deoxy form of alpha 2c beta 2 is abut 10 times slower than hemoglobin A. Carbamylation of the beta chain does not slow the transition, indicating that carbamylation of the chain affects the allosteric equilibrium in deoxyhemoglobin. To a reasonable approximation with minimum assumptions, all of the observations made in this study may be rationalized using the two-state model of Monod, J., Wyman, J., and Changeux, J. P. ((1965) J. Mol. Biol. 12, 88-118).

Allosteric Site↗
PubMed1981

Multiple T state conformations in a fish hemoglobin. Carbon monoxide binding to hemoglobin of Thunnus thynnus.

The blood of the Atlantic bluefin tuna (Thunnus thynnus) contains four hemoglobin components separable by chromatography on diethylaminoethylcellulose. These components are stable and functionally identical in their reactions with carbon monoxide. At low pH they remain in the T state even when liganded, and show two kinetic components in binding and in dissociation, with rates of 1) 1.2 microM-1 s-1 and 0.095 s-1 and 2) 0.013 microM-1 s-1 and 0.195 s-1, respectively, at pH 6, 20 degrees C, 0.1 m KPi. These components have difference spectra separated by more than 2 nm and are present in equal amounts. After CO has bound, there is a conformation change to an altered T state, in terms of the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118), with a half-time of 65 s. At equilibrium, one-third of the slow kinetic component is changed into the new conformer, which binds CO at a rate of 0.14 microM-1 s-1.

Animals↗
PubMed1981

Tetramer-dimer dissociation of carboxyhemoglobin in the absence of dithionite.

The generally accepted value for the tetramer-dimer dissociation constant KL4,2 of carboxyhemoglobin in pH 7.0 phosphate buffer lies in the range 1--2 micrometers. Previous determinations of the quantity have generally involved addition of dithionite to samples to exclude oxygen. We report flash photolysis experiments on carboxyhemoglobin in the absence of dithionite which suggest that KL4,2 is 0.2 +/- 0.05 micrometer. Addition of dithionite to our samples resulted in an order of magnitude increase in KL4,2 in good agreement with previously published results. The mechanism of this increase in dissociation has not been determined with certainty. However, impurities, possibly metal ions, are required in addition to dithionite to produce this effect. Dithionite did not increase KL4,2 for phosphate buffer solutions treated with Chelex 100 analytical grade chelating resin. Addition of bovine serum albumin to untreated buffer solutions before addition of dithionite was found to prevent increased dissociation. The sulfhydryl-reducing agents dithiothreitol and beta-mercaptoethanol were found to protect against the effect of dithionite and to reverse its effect on KL4,2 if they were added after the dithionite. The interaction of the unknown impurities with dithionite to produce increased values of KL4,2 could be mimicked by addition of CU2+ ions in concentrations of less than 1 micrometer to buffer treated with Chelex 100 resin.

Carboxyhemoglobin↗
PubMed1980

The role of diffusion in limiting the rate of ligand binding to hemoglobin.

The Root effect hemoglobin from Thunnus thynnus, the Atlantic bluefin tuna, remains in the T state at acid pH even when liganded. There are two components in the binding of CO to the T state with rates of 0.01 and 1.0 X 10(6) M-1 S-1 at 20 degrees C, 0.1 M KPi, pH 6.0, and two components in binding to the R state with rates of 1.7 and 8.5 X 10(6) M-1 S-1 as measured by partial flash photolysis at 20 degrees C, 0.05 M Tris-HCl, pH 8.0. A single rate of NO binding of 1.8 X 10(7) M-1 S-1 is found at both pH 6 and pH 8. The rate of binding of NO to dimethyldeuterohemedisulfonate is much higher still at 5 X 10(8) M-1 S-1. The observed rate of NO binding to the protein is not affected by chain differences or by the R to T transition. It may measure the rate of diffusion of ligand to the vicinity of the heme site. The T state components have different NO dissociation velocity constants of 0.004 and 0.07 S-1. The apparent quantum yields for the T state components are approximately 0.035 and 0.0008; the high affinity component has the lower yield. A two-step scheme is used to explain the results. The presence of chain differences and allosteric states is reflected in the intrinsic binding rates of both NO and CO to the heme group.

Animals↗
PubMed1979

Dependence of the quantum efficiency for photolysis of carboxyhemoglobin on the degree of ligation.

A combined stopped flow-laser photolysis apparatus was used to measure the quantum efficiency for removal of carbon monoxide bound to human hemoglobin as a function of fractional CO saturation. This flow-flash technique allows the properties of partially liganded hemoglobin molecules, which are sparsely populated under equilibrium conditions, to be conveniently studied. Experiments performed at pH 7 and 20 degrees C both in the presence and absence of phosphates gave a similar dependence of quantum efficiency on fractional saturation. The observed quantum efficiency was 0.90 +/- 0.06 at 10% saturation and decreased to 0.47 +/- 0.02 as full saturation was approached. An allosteric model in which Hb(CO)1 has a quantum efficiency of 0.99 while other liganded species have quantum efficiencies of 0.47 was used to produce a good simulation of the results.

Carbon Monoxide↗
PubMed1978

Carbon monoxide binding to a fish hemoglobin under photostationary conditions.

Determinations of carbon monoxide binding curves for hemoglobin from Brevoortia tyrannus under equilibrium and photostationary conditions show that in the light, the curve is shifted to the right and altered in shape. The Bohr effect is much less in the light. The kinetics of the transition between equilibrium and photostationary states has been examined. All of the results are satisfactorily described using the two-state model of Monod, J. Wyman, J., and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118 with the assumption that light produces an additive increase in the rate of dissociation of ligand from the R and T states.

Animals↗