MiP2005 - Session 5 - Young Investigator Presentation

Mitochondrial Physiology Network 10.9: 62 (2005) - download pdf


ph dependence of the various phases of the proton pump of cytochrome c oxidase.

G Capitanio1, Pietro Luca Martino1, N Capitanio2, E De Nitto1, S Papa1,3

1Dept. Medical Biochemistry, Biology and Physics, Univ. Bari, Bari; 2Dept. Biomedical Science, University of Foggia, Foggia; 3Inst. Bioenergetics and Biomembranes, CNR, Bari, Italy. - luca_mp@libero.it

    Cytochrome c oxidase (COX) catalyses the reduction of dioxygen to water by ferrocytochrome c. This reaction  is coupled to the translocation of up to 1H+/e- , 4H+/O2  across the coupling membrane from the inner to the outer aqueous phase [1,2]. The catalytic cycle of COX can be divided into two phases: a reductive phase and an oxidative phase. A study is presented on the pH-dependence of proton transfer of COX reconstituted in vesicles,  associated with the oxidation phase, the reduction phase and the oxidation-rereduction rapid transition.

    The reconstitution of cytochrome c oxidase, purified from beef heart mitochondria [3], in phospholipid vesicles was performed by the cholate dialysis method [4] at different internal pH. Simultaneous recording of absorbance and pH changes were carried out with a diode-array spectrophotometer (settled in the multiwavelength mode) and a combined electrode respectively [5].

    The experimental data show (1) an increase of  the H+/COX ratio from 0 at acidic external pH to 2 at alkaline external pH in the reductive phase; (2) a decrease of  the H+/COX ratio from 2.7 at acidic external pH to 1.7 at alkaline external pH in the aerobic oxidative phase. Experiments carried out when changing the internal or the external pH, indicate that the H+/COX ratio depends essentially on the external pH. In the rapid oxidation-rereduction transition the results reveal a bell-shaped external pH-dependence of the H+/COX ratio with a maximum of 4 at pH 7.25 (see also [1]).

    The results are discussed in terms of presently debated models, in which roles of cooperative H+/e- coupling are envisaged at the metal centers and H2O formation at the a3-CuB  binuclear center [2,6,7].

1.    Verkhovsky MI et al. (1999) Nature 400: 480-483.

2.    Bloch D et al. (2004) PNAS 101: 529-533.

3.    Errede B et al. (1978) Methods Enzymol. 53: 40-47.

4.    Casey RP et al. (1979) Biochem. J. 182: 149-156.

5.    Papa S et al. (1986) Methods Enzymol. 126: 331-343.

6.    Wikstrom M et al. (2003) Biochim. Biophys. Acta 1604: 61-65.

7.    Papa S (2005) Biochemistry (Moscow) 70: 178-186.

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Mitochondrial Physiology