The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 A resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a(3) iron and Cu(B) of the active site is fitted best by a peroxo-group or a chloride ion. Two waters or OH(-) groups do not fit, one water (or OH(-)) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu(B) ligand His326 is present in the imidazolate form.

We present a molecular dynamics study of cytochrome c oxidase from Paracoccus denitrificans in the fully oxidized state, embedded in a fully hydrated dimyristoylphosphatidylcholine lipid bilayer membrane. Parallel simulations with different levels of protein hydration, 1.125 ns each in length, were carried out under conditions of constant temperature and pressure using three-dimensional periodic boundary conditions and full electrostatics to investigate the distribution and dynamics of water molecules and their corresponding hydrogen-bonded networks inside cytochrome c oxidase. The majority of the water molecules had residence times shorter than 100 ps, but a few water molecules are fixed inside the protein for up to 1.125 ns. The hydrogen-bonded network in cytochrome c oxidase is not uniformly distributed, and the degree of water arrangement is variable. The average number of solvent sites in the proton-conducting K- and D-pathways was determined. In contrast to single water files in narrow geometries we observe significant diffusion of individual water molecules along these pathways. The highly fluctuating hydrogen-bonded networks, combined with the significant diffusion of individual water molecules, provide a basis for the transfer of protons in cytochrome c oxidase, therefore leading to a better understanding of the mechanism of proton pumping.

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. Protonations and deprotonations of individual amino acid residues and of clusters formed by these residues play an important role in activating these enzymes and in the mechanism of transport. We have used multiconformation continuum electrostatics method to investigate the protonation states of residues in the sodium proton exchanger NhaA from Escherichia coli, the structure of which has been determined recently by x-ray crystallography. Our calculations identify four clusters of electrostatically tightly interacting residues as well as long-range interactions between residues required for activation. The importance of many of these residues has been demonstrated by the characterization of site-directed mutants. A number of residues with extreme pK a values, including several of the ''pH sensor,'' can only undergo protonation͞depro-tonation reactions subsequent to conformational changes. The results of the calculations provide valuable information on the activation of the antiporter and the role of individual amino acid residues, and provide a solid framework for further experiments.hydrogen-bonded networks ͉ multiconformers ͉ water binding sites

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