Lars Chr. Petersen

The effect of inhibitors on the oxygen kinetics of cytochrome c oxidase

1. The oxygen kinetics of purified beef heart cytochrome c oxidase were investigated. 2. The effect of addition of various fixed concentrations of the inhibitors CO, HN3, HCOOH, HCN and H2S on the double reciprocal plot of respiration rate against oxygen concentration was studied. 3. CO is strictly competitive, azide and formate are uncompetitive, and cyanide and sulfide are non-competitive inhibitors towards oxygen. 4. Binding constants for the various inhibitors from secondary plots of the oxygen kinetics at pH 7.4 are: CO: Ki = 0.32 micronM, azide: Ki = 33 micronM; formate: Ki = 15 mM; cyanide: Ki = 0.2 micronM and sulfide: Ki = 0.2 micronM. 5. The possible significance of these results in the elucidation of the reaction mechanism is discussed.

Haem—haem interactions in cytochrome aa3 during the anaerobic-aerobic transition

Abstract A biphasic response is seen at both 445 and 605 nm as the ascorbate—cytochrome c —cytochrome aa 3 system is taken slowly from the anaerobic to the aerobic state. At low oxygen tensions the 445 nm band is more reduced while at high oxygen tensions the 605 nm band is more reduced. It is suggested that the redox potential for cytochrome a (contributing 70% at 605–630 nm and 50% at 445–455 nm) is a function of the redox state of cytochrome a 3 . This model can account for both the aerobic/anaerobic data and for observations of interactions in the anaerobic system alone (Leigh, Jr, J.S., Wilson, D.F., Owen, C.S. and King, T.E. (1974) Arch. Biochem. Biophys. 160, 476–486).

Steady-state kinetics of laccase from Rhus vernicifera

Abstract The steady-state kinetics of laccase (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase, EC 1.14.18.1) from the lacquer tree Rhus vernicifera is investigated using the respirograph method to produce Lineweaver-Burk plots of oxygen consumption rate against oxygen concentration. A ping-pong mechanism is established. The kinetic constants obtained according to the model is in close agreement with the corresponding values obtained from earlier studies on the transient reactions between the reduced enzyme and oxygen (Andreasson, L.-E.; Branden, R.; and Reinhammar, B. (1976) Biochim. Biophys. Acta 438, 370–379) and between the oxidized enzyme and reducing substrates (Andreason, L.-E.; and Reinhammar, B. (1976) Biochim. Biophys. Acta 445, 579–597).

The effect of oxygen concentration on the steady-state kinetics of the solubilized cytochrome c oxidase

1. The steady-state kinetics of ascorbate oxidation as a function of oxygen concentration was measured with a solubilized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparation. 2. Linear double reciprocal plots were obtained at various fixed concentrations of ascrobate, cytochrome c and cytochrome aa3. 3. The results are interpreted in terms of an oxidase model similar to that put forward by Minnaert in 1961 (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23-34). 4. The Km for oxygen at infinite cytochrome c concentration is 0.95 muM and the intramolecular rate constant for the transfer of electrons from cytochrome c to cytochome aa3 is 400 s(-1). According to the model, this implies that the second order rate constant for the reaction between oxygen and the oxidase is 9.5 X 10(7)M(-1)-s(-1).

The reaction between oxidized cytochrome c and reduced cytochrome c oxidase

The elucidation of the reaction mechanism of isolated cytochrome c oxidase has been approached by methods involving equilibrium studies, steadystate and transient-state kinetics (see [l] and [2] for reviews). The reaction of the,oxidase with its natural substrate, ferrocytochrome c, has for obvious reasons received a good deal of attention. The transient-state kinetics of this reaction, investigated in the presence and absence of oxygen, consist of several phases, the first of which is a second-order reaction, with a rate constant of 106-10’ M-’ s-’ [3-71. Direct measurement of the stoichiometry of the reaction, using the product, ferricytochrome c, as a probe, indicates that only one of the possible four electron acceptors of the oxidase, cytochrome a, is reduced in the burst phase [5,7]. The establishment of a quasi-equilibrium permits the calculation of an equilibrium constant for the redox rea.ction. This corresponds to an oxidation-reduction potential for cytochrome a of 285 mV, which is significantly different from the value found in potentiometric titrations. Together with other apparently conflicting observations concerning the properties of the prosthetic groups in cytochrome c oxidase (see [2]) this finding emphasizes the complexity of the enzyme. In an attempt to investigate further the oxidation-reduction sites of the enzyme, we report in this paper studies on the reaction between ferricytochrome c

The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes

The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.