Cytochrome ba3, a terminal oxidase was isolated from the haloalkaliphilic archaeon Natronobacterium pharaonis. NH 2 -terminal sequence information of two subunits with apparent molecular masses of 40 and 36 kDa was used to generate a DNA probe by polymerase chain reaction. Cloning and sequencing of two overlapping genomic fragments revealed four genes forming a transcriptional unit. The policystronic messenger RNA of this cbaDBAC gene locus was identified by RNA analysis. The genes cbaC and cbaD code for small hydrophobic peptides with 81 and 54 amino acids. The genes cbaB and cbaA code for cytochrome oxidase subunit II (calculated molecular mass ϭ 18.6 kDa) and I (calculated molecular mass ϭ 63.8 kDa) respectively. Five potential Cu A ligands for subunit II and six His residues for subunit I located in conserved positions indicate cytochrome ba 3 to be a c-type oxidase. Sequence comparison and phylogenetic analysis place the natronobacterial enzyme together with the archaeal quinol oxidase Sox-ABCD from Sulfolobus acidocaldarius and the eubacterial ba 3 -type oxidase from Thermus thermophilus into a distinct evolutionary group. All three members are missing residues which are functionally important for vectorial proton translocation. The four-subunit enzyme complex was also identified on the protein level using chromatographic buffers containing ethylene glycol for purification.Keywords : archaea ; cytochrome ba 3 ; Natronobacterium pharaonis ; proton pump ; terminal oxidase.Cytochrome oxidase, an integral membrane complex of the cytochrome c oxidases, one from Paracoccus denitrificans [9] and the other from bovine heart [10] has been a significant scirespiratory chain, is a key enzyme in aerobic metabolism. It catalyzes the reduction of oxygen to water and creates a proton entific accomplishment. Both structures not only confirmed the location and ligation pattern of redox centers within subunits I gradient by active redox-driven proton transfer, which is a major and II but also initiated proposals on the structure of protonprerequisite for energizing aerobic cells [1, 2]. The sequence conducting pathways located in subunit I. Based on the earlier information on numerous cytochrome oxidases and related ensuggested histidine cycle [11], Michel and coworkers developed zymes gained during the past several years led to the proposition a functional model for electron-transport-coupled proton transloof a superfamily of heme-copper oxidases. All its members have cation [9]. In this model two conserved carboxylic acid residues in common a binuclear center as active site, consisting of a heme function as proton donor/acceptor groups for the vectorial promolecule and a so-called Cu B atom. These two, as well as a tons. This proposal has recently been corroborated by Fouriersecond heme molecule, are coordinated via six highly conserved transform infrared spectroscopic analysis of the Paracoccus histidine residues located in the catalytic subunit I. These ligands enzyme [12]. are indicative of the membership of the superf...
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