High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: New insights into the active site and the proton transfer pathways

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 proteinprotein 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 twodimensional 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.cytochrome aa3 | electron transfer | energy transduction | membrane protein | respiration

Section: Discussionsupporting

confidence: 55%

Functional interactions between membrane-bound transporters and membranes

Öjemyr

Lee

Gennis

et al. 2010

“…The alterations include movement of the heme a 3 porphyrin ring, its farnesyl tail, and helix VIII, as well as loss of a key water linking the D path to the active site and formation of a new water chain into the active site (3). These changes upon reduction were an unexpected finding because other structural studies of bacterial oxidases (12,31,32) have reported no change, and in the bovine CcO the changes are fewer and mainly involve different regions than those seen in RsCcO (33), with the exception of Ser425 (bov Ser382). The significance of our recent findings (3) is potentially profound, as they suggest a unique mechanism of gating of the two proton pathways.…”

Section: S142mentioning

confidence: 78%

“…Removal of the carboxyl at position 132 strongly inhibits enzyme activity (2% of wild-type RsCcO) and proton pumping (8). A chain of waters between D132 and E286 can be seen in the D pathway of high-resolution structures of aa 3 -type CcO (10)(11)(12). Computational studies and molecular dynamics simulations suggest that these waters may serve in both proton transfer and proton storage (13)(14)(15).…”

Section: Resultsmentioning

confidence: 99%

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

Liu

Qin

Ferguson‐Miller

2011

Crystal structures in both oxidized and reduced forms are reported for two bacterial cytochrome c oxidase mutants that define the D and K proton paths, showing conformational change in response to reduction and the loss of strategic waters that can account for inhibition of proton transfer. In the oxidized state both mutants of the Rhodobacter sphaeroides enzyme, D132A and K362M, show overall structures similar to wild type, indicating no long-range effects of mutation. In the reduced state, the mutants show an altered conformation similar to that seen in reduced wild type, confirming this reproducible, reversible response to reduction. In the strongly inhibited D132A mutant, positions of residues and waters in the D pathway are unaffected except in the entry region close to the mutation, where a chloride ion replaces the missing carboxyl and a 2-Å shift in N207 results in loss of its associated water. In K362M, the methionine occupies the same position as the original lysine, but K362-and T359-associated waters in the wild-type structure are missing, likely accounting for the severe inhibition. Spectra of oxidized frozen crystals taken during X-ray radiation show metal center reduction, but indicate development of a strained configuration that only relaxes to a native form upon annealing. Resistance of the frozen crystal to structural change clarifies why the oxidized conformation is observable and supports the conclusion that the reduced conformation has functional significance. A mechanism is described that explains the conformational change and the incomplete response of the D-path mutant.conformational gating | membrane protein structure | proton path mutants | X-ray reduction | water chain C ytochrome c oxidase (CcO) is a major contributor to and regulator of energy conservation in eukaryotic and many prokaryotic organisms. It uses four electrons from cytochrome c and four protons from the inner side of the membrane to reduce O 2 to water (1). In each catalytic cycle, four protons are also translocated across the membrane to generate a membrane potential, but the mechanistic details of coupling between proton pumping and the electron transfer events are still not fully understood (1, 2). Our recently solved crystal structures of the fully reduced form of Rhodobacter sphaeroides (Rs) CcO reveal conformational changes that were not previously observed (3), introducing mechanistic possibilities for coupling and gating of proton transfer, the significance of which we further elucidate in this report.Bacterial forms of CcO are similar in many respects to the mammalian mitochondrial CcO and their simpler subunit composition and ease of mutation have made them useful model systems for studying the mechanism of energy conservation, assuming it to be conserved. It has become increasingly clear, however, that within the large superfamily of heme-copper oxidases some bacterial forms may have solved the proton pumping problem in different ways (4, 5), and even those most closely related to eukaryotic oxidases (the aa ...

“…An equivalent H pathway is present but not continuous in bacterial CcOs, but mutations in this region show no effect on coupling (11,12). However, no structural changes have been seen in the bacterial D channel (7,13), and to date only changes in the K and H pathways have been discerned in structures of the Rb. sphaeroides CcO (7).…”

mentioning

confidence: 99%

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Maréchal

Rich

2011

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm −1 range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(−), and 3,607(+) cm −1 . A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D 2 O and H 2 18 O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.energy coupling | mitochondria | respiratory chain | complex IV M itochondrial cytochrome c oxidase (CcO) catalyses electron transfer from cytochrome c to molecular oxygen, conserving the released energy as coupled transmembrane proton transfers. It is a member of a homologous "superfamily" of oxidases that includes diverse bacterial forms; all types in the CcO subgroup contain a common "catalytic core" of subunits I, II, and III with equivalent metal centers of Cu A , heme a. and a binuclear center (BNC) that is formed from heme a 3 and Cu B (1, 2). Structures of bovine mitochondrial (3, 4) and various bacterial (5-7) CcOs have been solved at atomic resolution and show common details in the binuclear heme a 3 ∕Cu B catalytic site, other cofactors, key amino acids, and several unusual structural features. These structural data, coupled with a substantial body of biophysical and spectroscopic studies, have provided detailed information on internal electron transfer and the chemical catalytic cycle of oxygen reduction.Crucially, however, these structural data have not provided a single picture for all forms of CcO of the pathway(s) for coupled proton transfer and, therefore, the interplay between electron transfer/oxygen chemistry and the intraprotein proton transfer reactions that result in proton-motive action. Crystal structures of bacteri...