Active Transport of Ions across Biomembranes : A Kinetic Study of Cytochrome c Oxidase Reconstituted into Phospholipid Vesicles

Abstract: Ion transport across membranes is of uttermost importance for us. It is the foundation for signaling of various kinds e.g. in the nerve-system. Furthermore, energy from photosynthesis and metabolism is conserved in electrochemical gradients across membranes, maintained by ion pumps. In this thesis I discuss mechanisms of how protons and other ions are translocated across biomembranes against their concentration gradients. I have studied one specific proton pump, cytochrome c oxidase (CytcO) and in the summary I also compare CytcO with two other pumps for which a wealth of structural and functional information has recently been obtained. The data in the articles presented in this thesis support a model were proton pumping can be achieved without simultaneous oxidation of heme a or electron transfer (paper I); where a proton is transferred to the catalytic site before the pump site is protonated (paper IV); and where proton release is preceded by a conformational change (paper II). These observations could be explained by a model involving a conformational change of the pump element, recently proposed from our laboratory1. Furthermore the results from the papers in this thesis show that proton uptake precedes proton release in D2O (paper II). The kinetics of electron transfers linked to proton pumping is solely determined by the pH on the N-side of the membrane (paper III). Finally Zn2+ added on the P-side of the membrane inhibits a specific reaction step (paper IV). In the three pumps described here conformational changes, modulating ion affinities, and the opening and closing of gates, seem to be involved in driving the ions across the membrane. 1. Brzezinski, P. & Larsson, G. (2003) Biochim. Biophys. Acta 1605, 1-13.