Identification and Characterization of the ctac (Coxb) Gene as Part of an Operon Encoding Subunits I, II, and III of the Cytochrome c Oxidase (Cytochrome aa3) in the Cyanobacterium SynechocystisPCC 6803

The gene (coxII = coxB = ctaC) encoding subunit II of SynechocystisPCC 6803 cytochrome c oxidase has been isolated by screening a genomic DNA library in pUC18 with a 17-bp oligonucleotide probe (probe C) derived from coxI of Paracoccus denitrificansafter Southern blots with a 19-kb oligonucleotide (probe A) derived from coxII of P. denitrificanshad given equivocal results. A 2.2 kb PstI-KpnI restriction fragment was subcloned into pUC 18 and the resulting plasmid pDAUV26, which contained the probe C-binding site near the downstream end was found also to contain the whole coxII gene upstream of this site. The novel plasmid pDAUV 26 was used to transform competent E. coilcells, propagated therein, and the sequence determined. The 2.2 kb insert contained the entire coding region for the coxII gene together with a GAG start codon, a TAA stop codon, and a putative Shine-Dalgarno sequence. The deduced COII polypeptide is composed of 319 aa (calculated molecular mass of 32,800) plus a N-terminal leader sequence of 20 aa. The hydropathy plot suggests two lipophilic transmembrane domains near the N-terminus connected with an extremely hydrophilic aa stretch on the cytosolic side, while an unusually long (>50 aa) aa stretch on the periplasmic (= intrathylakoidal) side leads to a typical cyanobacterial threonine in place of the first conserved glutamate of the cytochrome c-binding region in all other COII proteins. Together with a considerably shortened and interrupted aromatic aa stretch in this region, these differences are discussed in terms of the peculiar affinity of cyanobacterial cytochrome oxidases for acidic c-type cytochromes. Other invariant features such as the strictly conserved CuA-binding aa, however, are found in correct positions.