[Show abstract][Hide abstract] ABSTRACT: Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.
No preview · Article · Apr 2010 · Journal of Bioenergetics
[Show abstract][Hide abstract] ABSTRACT: X-ray structures of bovine heart cytochrome c oxidase have suggested that the enzyme, which reduces O(2) in a process coupled with a proton pumping process, contains a proton pumping pathway (H-pathway) composed of a hydrogen bond network and a water channel located in tandem across the enzyme. The hydrogen bond network includes the peptide bond between Tyr-440 and Ser-441, which could facilitate unidirectional proton transfer. Replacement of a possible proton-ejecting aspartate (Asp-51) at one end of the H-pathway with asparagine, using a stable bovine gene expression system, abolishes the proton pumping activity without influencing the O(2) reduction function. Blockage of either the water channel by a double mutation (Val386Leu and Met390Trp) or proton transfer through the peptide by a Ser441Pro mutation was found to abolish the proton pumping activity without impairment of the O(2) reduction activity. These results significantly strengthen the proposal that H-pathway is involved in proton pumping.
Preview · Article · Apr 2007 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.
[Show abstract][Hide abstract] ABSTRACT: The 1.9 A resolution X-ray structure of the O2 reduction site of bovine heart cytochrome c oxidase in the fully reduced state indicates trigonal planar coordination of CuB by three histidine residues. One of the three histidine residues has a covalent link to a tyrosine residue to ensure retention of the tyrosine at the O2 reduction site. These moieties facilitate a four electron reduction of O2, and prevent formation of active oxygen species. The combination of a redox-coupled conformational change of an aspartate residue (Asp51) located near the intermembrane surface of the enzyme molecule and the existence of a hydrogen bond network connecting Asp51 to the matrix surface suggest that the proton-pumping process is mediated at Asp51. Mutation analyses using a gene expression system of the Asp51-containing enzyme subunit yield results in support of the proposal that Asp51 plays a critical role in the proton pumping process.
Preview · Article · May 2006 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 --> Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-A resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.
Full-text · Article · Jan 2004 · Proceedings of the National Academy of Sciences