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K Krab

Publications and source records attributed to K Krab.

11 recordsLinked to original sources
PubMed1987

Principles of coupling between electron transfer and proton translocation with special reference to proton-translocation mechanisms in cytochrome oxidase.

The recent general acceptance of the proton-pumping function of cytochrome oxidase has stimulated discussion and experiment on possible underlying molecular mechanisms. Adequate experimental design requires clear understanding of the theoretical principles governing such a linked function. The increasing structural knowledge of cytochrome oxidase also contributes to a present-day requirement of more precise chemical and physical description of redox-linked proton translocation, which is the fundamental process underlying conservation of energy from aerobic metabolism in all eukaryotes and many bacteria. This essay is based on our original theoretical treatment of this problem, which is expanded here to include discussion of more recent analyses by others, classification of different types of coupling principles, as well as some concrete proposed molecular mechanisms. The latter will be analysed qualitatively, and in some cases quantitatively where this is possible, using a common theoretical framework to help comparison between models. Experimental findings relevant to this problem will be critically reviewed, and some suggestions will be made to stimulate further experiments dedicated to clarify the problem.

Biological Transport↗
PubMed1986

Determination of the stoichiometry of redox-linked proton translocation from the kinetics of pulse experiments. A simulation study.

We have previously published a simple kinetic model to analyse possible pitfalls in kinetic measurements of H+/O ratios in mitochondria [(1984) FEBS Lett. 178, 187-192]. While this model demonstrated how relative electrode response times may affect the results, it did not adequately describe the kinetics of proton back-diffusion across the membrane. Here this model is further developed and improved, and shown to give a good quantitative description of both oxygen-pulse type experiments as well as of experiments where the reaction is started by photolysis of the cytochrome c oxidase-CO complex. Simulations based on this model reveal that the extrapolation procedure used by Lehninger et al. [e.g. (1984) J. Biol. Chem. 259, 4802-4811] to estimate the H+/O ratio will tend to yield overestimated values. This is mainly due to the back-diffusion of protons into the mitochondria, which is not correctly accounted for by this extrapolation.

Carbon Monoxide↗
PubMed1986

The semiquinone cycle. A hypothesis of electron transfer and proton translocation in cytochrome bc-type complexes.

The Q cycle and the b cycle are the main current models of action of the cytochrome bc-type complexes of mitochondria, bacteria, and chloroplasts. Both are based on the concept, proposed in 1972, of two sequential one-electron oxidations of (ubi)quinol along two discrete pathways which operate at different redox potentials, and with bound semiubiquinone as an intermediate. The models differ in two respects, viz. in the pathway of electron transfer and the principle of linkage of electron transfer to proton translocation. In this article we outline a new model, called the semiquinone or, simply, SQ cycle, which is based on the electron transfer principles of the b cycle but which incorporates the Q cycle concept of direct coupling between electron transfer and proton translocation through action of ubiquinone.

Benzoquinones↗
PubMed1985

The use of carotenoids and oxonol VI as probes for membrane potential in proteoliposomes.

Carotenoids present in lipids extracted from the cyanobacterium Synechococcus 6716 indicate trans-membrane potential in proteoliposomes reconstituted from these lipids and the ATPase complex isolated from the same organism. A carotenoid absorbance band shift to a longer wavelength is obtained with valinomycin-induced potassium ion diffusion potentials, irrespective of the polarity of the potassium gradient. In contrast to this, the (externally added) probe oxonol VI only shows an absorbance band shift when the external potassium ion concentration is higher than the internal one. In liposomes without ATPase complex, no carotenoid absorbance band shifts were observed.

Adenosine Triphosphatases↗
PubMed1979

On the stoichiometry and thermodynamics of proton-pumping cytochrome c oxidase in mitochondria.

Different approaches have been used to evaluate the stoichiometry of proton translocation linked to cytochrome c oxidase in rat liver mitochondria. A mathematical model was designed that successfully describes the kinetics of redox-linked proton translocation provided that the rate of electron transfer is not too high. With ascorbate as reductant, an essentially pH-independent (in the pH range 6--8.5) proton ejection stoichiometry (H+/e-) is obtained from either initial rates of H+ ejection (0.86 +/- 0.12), or the model (0.87 +/- 0.14). Similar results are obtained with either ferrocyanide, N.N.N',N'-tetramethyl-p-phenylenediamine or externally added cytochrome c mediating between ascorbate and cytochrome c in rotenone- and antimycin-inhibited mitochondria. Oxygen pulse experiments with ferrocytochrome c as substrate show fully uncoupler-sensitive redox-linked proton ejection with a stoichiometry of 0.78 +/- 0.14. With murexide to measure Ca2+ uptake during oxidation of ferrocyanide, we found a stoichiometry of two positive charges taken up/electron transferred, confirming earlier findings. These results provide strong evidence that cytochrome c oxidase functions as a redox-linked proton pump with a stoichiometry of one H+ ejected and two charges translocated/electron transferred. The thermodynamic consequences of the proton pump are discussed and a maximal P/O ratio of 1 1/3 for 'site 3' is predicted in agreement with state 4 redox potentials and phosphate potential.

Animals↗
PubMed1979

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake.

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N',N'-tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c. 2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa3 by ferrocyanide. 3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake. 4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.

Cyanides↗
PubMed1978

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles.

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated. Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H+ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K+. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons. The proton ejection is the result of true translocation of H+ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system. Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a H+/e- quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions. It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H+.

Aerobiosis↗
PubMed1977

Uncoupling of mitochondrial respiration by ADP.

Even when oxidative phosphorylation is blocked completely by addition of high concentrations of oligomycin plus aurovertin, the addition of ADP to a suspension of mitochondria containing a high concentration of ATP inside the mitochondria induces a stimulation of respiration and oxidation of nicotinamide nucleotide. It is concluded that transport of ADP into mitochondria with a high endogenous ATP/ADP ratio requires energy.

Adenosine Diphosphate↗