Principles of Molecular Bioenergetics and The Proton Pump of Cytochrome Oxidase

ROBERT B. GENNIS

University of Illinois, Department of Biochemistry, 600 South Mathews Street, Urbana, IL 61801

1 Introduction: General Principles of Bioenergetic Systems

All of the bioenergetic enzymes described in this book can couple an exergonic or free-energy yielding reaction to the electrogenic movement of charged species across the membrane, generating a protonmotive force. In the case of bacteriorhodopsin, the driving reaction is the absorption of a photon, for the bc1 complex, the oxidation of ubiquinol by cytochrome c is the driving force, and for the respiratory oxidases, the reduction of O2 to H2O provides the impetus. In this book, the principles utilized by a number of these systems are detailed with an emphasis on recent structural studies. It is convenient to classify two classes of mechanisms used to generate a trans-membrane voltage:

(1) Mechanisms utilizing an oxidoreduction loop.

(2) True ion (proton) pumps.

1.1 Oxidoreduction Loops

The principle of coupling different chemical reactions is central to biology and is accomplished in a number of ways. Many of the systems that generate a protonmotive force can be understood in terms of Mitchell's chemiosmotic oxidoreduction loop. This is illustrated by the example shown in Figure 1, which shows a redox loop formed from the anaerobic respiratory system comprised of formate dehydrogenase and nitrate reductase enzymes from E. coli. Recently, the structures of each of these two enzymes were determined. The topology of the catalytic active sites assures that the net reaction results in the generation of a protonmotive force. Formate dehydrogenase oxidizes formate on the periplasmic side of the membrane (the positive or P-side) and electrons are delivered through a series of metal centers to a menaquinone reductase site located near the cytoplasmic surface (the negative or N-side of the membrane). The formate dehydrogenase, thus, separates the oxidative and reductive half-reactions on opposite sides of the membrane. Protons are released in the periplasm upon formate oxidation and protons are taken up from the cytoplasm upon the reduction of menaquinone. The actual charge crossing the chemiosmotic barrier is the electron.

Reduced menaquinol is a neutral, hydrophobic compound and can diffuse freely within and across the membrane bilayer. The nitrate reductase enzyme has a menaquinol oxidation site located near the periplasm, whereas the site where nitrate is reduced to nitrite is located on the opposite side of the membrane. Electrons are transferred across the membrane between these active sites to couple the two half-reactions catalyzed by the enzyme (see Figure 1). The full reaction of nitrate reductase, therefore, is coupled to the release of protons in the periplasm, the uptake of protons from the cytoplasm and the transfer of charges, in the form of electrons, across the membrane.

The net reaction of both of these enzymes together results in the transfer of four protons from the cytoplasm to the periplasm for each formate oxidized and nitrate reduced. Points to note are

(1) The actual charges crossing the membrane are electrons and not protons.

(2) The net transfer of protons is due to the vectorial placement of the enzyme active sites so that the oxidation and reduction half-reactions occur on oppo site sides of the membrane.

(3) The protons are directly involved in the substrate chemistry.

(4) The two enzymes are coupled by a neutral, hydrophobic hydrogen carrier, in this case, menaquinol.

(5) The generation of the protonmotive force cannot be decoupled from the chemical reaction without changing the 'wiring'. The topology of the active sites, located on opposite sides of the membrane, and the uptake/release of protons from/to the N/P side of the membrane enforce this coupling so that the chemistry cannot proceed without generating a transmembrane voltage.

These are general features of Mitchell's initial proposal for how the protonmotive force is generated, and the structural and functional studies on these and other systems have supported this proposal.

The photosynthetic reaction center and the bc1 (and b6f) complex can be understood as variations of this same general principle, illustrated schematically in Figure 2. In the case of the reaction center, the absorption of a photon results in electron transfer across the membrane leading to charge separation. The reductive and oxidative reactions that follow occur on opposite sides of the membrane. The direction of electron flow is from the P-side to the N-side of the membrane, as is also the case for nitrate reductase and formate dehydrogenase. The reduction of ubiquinone in the bacterial reaction center utilizes protons from the N-side of the membrane (bacterial cytoplasm for the prokaryotic enzyme) and the protons are delivered to the buried QB active site through a proton-conducting channel. In essence, the proton is transferred electrogenically towards the P-side of the membrane to 'meet' the electron at the quinone reduction site. Both proton and electron transfers contribute to the voltage generation by the reaction. The charge movement across the membrane is, thus,...