Functional interactions between membrane-bound transporters and membranes

Abstract

One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated protein-protein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the two-dimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery.

Fig. 1.

Structure of cytochrome c oxidase from R. sphaeroides. The overall structure (four subunits) is shown in the background (PDB ID code 1M56). The redox-active cofactors, Cu_A, heme a, heme a₃, and Cu_B, are shown together with a number of amino acid residues that participate in proton transfer through the K-proton-transfer pathway. Residue Ser^I299 is not part of the proton pathway, but it is hydrogen-bonded to Lys362 (via a water molecule), which is a key element of the pathway. The thick () and thin arrows () indicate rapid proton transfer in the membrane-reconstituted CytcO and slower proton transfer in detergent-solubilized CytcO, respectively. The arrow within CytcO indicates proton transfer via Lys^I362 within the K proton pathway.

Fig. 2.

PCET for wild-type and mutant forms of CytcO in DDM and reconstituted into SUVs. Absorbance changes at 598 nm after flash photolysis of CO from the two electron reduced CytcO are shown. (A) Wild-type CytcO, (B) Ser^I299Glu mutant CytcO, (C) Ser^I299Gly CytcO, (D) Glu^II101Ala CytcO, and (E) Glu^II101Asp CytcO. Experimental conditions: 0.1 M Ches pH 9.5. For the detergent-solubilized enzyme 0.05% DDM was used, T ≅ 22 C. The traces have been normalized such that the total absorbance changes are equal (ca. 1 μM reacting CytcO).