No abstract

One of the key problems of molecular bioenergetics is the understanding of the function of redox-driven proton pumps on a molecular level. One such class of proton pumps are the heme-copper oxidases. These enzymes are integral membrane proteins in which proton translocation across the membrane is driven by electron transfer from a low-potential donor, such as, e.g. cytochrome c, to a high-potential acceptor, O(2). Proton pumping is associated with distinct exergonic reaction steps that involve gradual reduction of oxygen to water. During the process of O(2) reduction, unprotonated high pK(a) proton acceptors are created at the catalytic site. Initially, these proton acceptors become protonated as a result of intramolecular proton transfer from a residue(s) located in the membrane-spanning part of the enzyme, but removed from the catalytic site. This residue is then reprotonated from the bulk solution. In cytochrome c oxidase from Rhodobacter sphaeroides, the proton is initially transferred from a glutamate, E(I-286), which has an apparent pK(a) of 9.4. According to a recently published structure of the enzyme, the deprotonation of E(I-286) is likely to result in minor structural changes that propagate to protonatable groups on the proton output (positive) side of the protein. We propose that in this way, the free energy available from the O(2) reduction is conserved during the proton transfer. On the basis of the observation of these structural changes, a possible proton-pumping model is presented in this paper. Initially, the structural changes associated with deprotonation of E(I-286) result in the transfer of a proton to an acceptor for pumped protons from the input (negative) side of the membrane. After reprotonation of E(I-286) this acceptor releases a proton to the output side of the membrane.

Light-driven vectorial proton translocation is basic to the mechanism of energy transduction by photosynthetic systems. Bacteriorhodopsin (bR)1 is the simplest known light-driven proton pump and has long served as a model system for understanding how protons may be transported "up hill" against a transmembrane proton motive potential. bR contains seven transmembrane ␣-helices that surround a proton translocation channel lined with strategically placed charged residues (3). Depending upon their protonation states, which change in a well orchestrated cascade as a proton is transported across the cell membrane, these charged residues can serve as either proton donors or proton acceptors. Light activation of the chromophore, an all-trans-retinal molecule covalently attached to Lys-216 in helix G via a protonated Schiff base (the primary proton donor) results in the 13-cis-retinal configuration with two-thirds quantum efficiency. Steric conflicts and mechanical stress resulting from photoisomerization initiate a sequence of conformational changes that can be characterized spectroscopically and that perturb the local environment of several key residues, strongly affecting their pK a values and creating transient pathways for proton transfer.The specific spectral intermediates of the bR photocycle have been well characterized, and a common reaction scheme is: bR 570 3 K 590 7 L 550 7 M 412 7 N 560 7 O 640 3 bR 570 (sub-

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.