Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 Å resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned. Well defined detergent head groups (maltose) are found associated with aromatic residues in a manner similar to phospholipid head groups, likely contributing to the success of alkyl glycoside detergents in supporting membrane protein activity and crystallizability. Other significant features of this structure include the following: finding of a previously unreported crystal contact mediated by cadmium and an engineered histidine tag; documentation of the unique His-Tyr covalent linkage close to the active site; remarkable conservation of a chain of waters in one proton pathway (D-path); and discovery of an inhibitory cadmium-binding site at the entrance to another proton path (K-path). These observations provide important insight into CcO structure and mechanism, as well as the significance of bound lipid in membrane proteins.cadmium binding ͉ membrane protein structure ͉ proton-conducting water chains C ytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in eukaryotes and many bacteria. It provides the final electron sink by accepting electrons from cytochrome c and reducing oxygen to water (1). The energy generated from this reaction is used to translocate protons across the membrane against the membrane potential and pH gradient (2). The proton gradient so formed is then used to make ATP, a universal energy source. CcO is an intrinsic membrane protein with varying numbers of subunits from 3 in some bacteria to 13 in mammalian mitochondria. However, only subunits I and II, which contain the redox active heme and metal centers and are highly conserved among different species, are required for electron transfer and proton pumping activities. Other subunits, particularly the highly conserved subunit III, likely play key roles in stabilizing and regulating the enzyme activity and energy metabolism in general.Over the past decade, several x-ray crystal structures of aa 3 -type CcOs from bovine heart mitochondria and bacteria have been determined (3-8). However, the mechanism of vectorial translocation of protons by CcO remains to be solved (9). It is now recognized that water molecules play a critical role in facilitating and controlling proton transfer (10). Thus, highresolution crystal structures of different redox states and key mutants with defined waters are necessary to elucidate the energy conservation process. However, achieving a reproducible high-resolution structure of membrane proteins remains a ...

A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase, but none in bacterial oxidases. Comparing the x-ray crystal structures of the reduced (at 2.15 Å resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a 3 involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high resolution structures of oxidized Rhodobacter sphaeroides cytochrome c oxidase, a water molecule is observed in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path. In the reduced and reduced plus cyanide structures, this water molecule disappears, implying disruption of proton conduction from the D path under conditions when the K path is open, thus providing a mechanism for alternating access to the active site.Cytochrome c oxidase (CcO) 1 , the terminal enzyme of the electron transfer chain, provides the final electron sink by accepting electrons from reduced cytochrome c and passing them sequentially through Cu A , heme a and finally to its active site, composed of heme a 3 and Cu B . Oxygen binds at this binuclear center, accepts the electrons and is reduced to water. The energy released from oxygen reduction is utilized by CcO to translocate protons from the N (negative)-side of the membrane to the P(positive)-side, to generate an transmembrane proton electrochemical gradient, which is then used for ATP synthesis (for recent reviews, see ref.( 1-3 )). Extensive kinetic and spectroscopic studies have identified intermediate states during the chemical catalysis, yet the details of the oxygen reduction process are not entirely understood. Even more elusive and controversial is the proton pumping mechanism. † This work is supported by National Institutes of Health Grant GM26916 (S. Supporting Information Available: Schematic drawing of the microspectrophotometer used for spectral analysis of single crystal ( Figure S1), comparison of the distances between heme a 3 farnesyl-OH and Y288 I -OH, as well as the distances between Cu B and heme a 3 Fe in different forms of RsCcO (Table SI). This materials is available free of charge via the Internet at http://pubs.acs.org. Two types of protons, termed pump and substrate protons, are taken up from the N-side of the membrane. During each catalytic cycle, four pump protons are...

Like many voltage-sensitive ion pumps, cytochrome c oxidase is inhibited by zinc. Binding of zinc to the outside surface of Rhodobacter sphaeroides cytochrome c oxidase inhibits the enzyme with a K I of < 5 M when the enzyme is reconstituted into phospholipid vesicles in the presence of a membrane potential. In the absence of a membrane potential and a pH gradient, millimolar concentrations of zinc are required to inhibit. This differential inhibition causes a dramatic increase in the respiratory control ratio from 6 to 40 for wild-type oxidase. 2؉ . Proton pumping is slower and less efficient with zinc. The results suggest that zinc inhibits proton movement through a proton exit path, which can allow proton back-leak at high membrane potentials. The physiological and mechanistic significance of proton movement in the exit pathway and its blockage by zinc is discussed in terms of regulation of the efficiency of energy transduction.

A conserved, crystallographically-defined bile acid binding site was originally identified in the membrane domain of mammalian and bacterial cytochrome c oxidase (CcO). Current studies show other amphipathic molecules including detergents, fatty acids, steroids, and porphyrins bind to this site and affect the already 50% inhibited activity of the E101A mutant of Rhodobacter sphaeroides CcO, as well as altering the activity of wildtype and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine and CHOBIMALT detergents further inhibit RsCcO E101A, with lesser inhibition observed in wildtype. The detergent inhibition is overcome in the presence of μM concentrations of steroids and porphyrin analogs including deoxycholate, cholesteryl hemisuccinate, bilirubin, and protoporphyrin IX. In addition to alleviating detergent inhibition, amphipathic carboxylates including arachidonic, docosahexanoic, and phytanic acids stimulate the activity of E101A to wildtype levels by providing the missing carboxyl group. Computational modeling of dodecyl maltoside, bilirubin, and protoporphyrin IX into the conserved steroid site shows energetically favorable binding modes for these ligands and suggests that a groove at the interface of subunit I and II, including the entrance to the K-path and helix VIII of subunit I, mediates the observed competitive ligand interactions involving two overlapping sites. Spectral analysis indicates that ligand binding to this region affects CcO activity by altering the K path dependent electron transfer equilibrium between heme a and heme a3. The high affinity and specificity of a number of compounds for this region, and its conservation and impact on CcO activity, support its physiological significance.

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.