Klaas Krab

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+.

Respiration-Linked H+ Translocation in Mitochondria: Stoichiometry and Mechanism

Publisher Summary The conservation of redox energy in respiration and photosynthetic electron transport takes place by a process in which the net translocation of hydrogen ions across an energy-transducing biomembrane plays an essential part. This chapter describes the mechanism of respiration-linked H + translocation in mitochondria: stoichiometry. It discusses the molecular mechanisms of proton translocation. The chapter reviews the standpoints on the stoichiometry of mitochondrial proton translocation for the different segments of the respiratory chain. In a redox loop, the proton is translocated by electroneutral “symport” with the electron, whereas the charge is translocated in a separate molecular event by electron translocation in the opposite direction. The proton-electron symport in a redox loop is the resultant of direct linkage between the two particles as in classical reduced hydrogen carriers, such as ubiquinol. The number of known “classical” hydrogen carriers is limited in the respiratory chain

Relationship between chemiosmotic flows and thermodynamic forces in oxidative phosphorylation

A set of equations has been derived, describing quantitatively the relationships between flows and thermodynamic forces in the chemiosmotic model of oxidative phosphorylation. Experimental tests of these equations give information on the stoichiometric coupling constants between the different flows.

Cytochrome c oxidase is a proton pump: a rejoinder to recent criticism.

Based on experimental evidence from this laboratory, we have suggested that mitochondrial cytochrome c oxidase (EC 1.9.3.1.) functions as a redoxlinked proton pump 11-51, and not merely as an electron translocator as postulated by Mitchell [6], The evidence for our proposal has been found in experiments with intact mitochondria, sonicated submitochondrial particles, and with phospholipid vesicles inlaid with the isolated and purified enzyme. In all these systems the results have been consistent, suggesting that the cytochrome c oxidase reaction may be characterised by the equation:

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.

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.

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria

In a recent communication Lehninger and co‐workers (Costa L.E., Reynaferje B., and Lehninger A.L. (1984) J. Biol. Chem. 259, 4802‐4811) reported values approaching 8 for the H+/O ratio of vectorial proton ejection from rat liver mitochondria respiring with succinate. Here we present a rigorous analysis of these measurements which reveals that they may significantly overestimate the true H+/O stoicheiometry.

Redox-Linked Proton Pumps in Mitochondria

During the past 15 years it has become evident that the conservation of oxidation-reduction energy, linked to respiratory and photosynthetic processes, is based upon seperation of positive and negative electrical charges across biological membranes. This widely accepted view originated principally with the proposal by Mitchell (1,2) of the chemiosmotic theory of oxidative and photosynthetic phosphorylation.