The gene coding for subunit IV of the cytochrome c oxidase in Paracoccus denitrificans has been cloned and sequenced. The derived amino acid sequence shows no significant homology to any known protein. Gene deletion has no consequences for the integrity of the complex and its spectral and enzymatic properties. Complementation of the deletion mutant in trans results in expression of subunit IV; sequence analysis of the 5noncoding region leads to the identification of a putative promoter

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... Cytochrome c oxidase (cytochrome aa 3 ; E.C.1.9.3.1) is one of the terminal oxidases in Paracoccus denitrificans (for reviews, see Refs. 1-3). Subunit I contains three redox active centers, heme a and the binuclear site (heme a 3 ⅐Cu B ). Cu A , the entry point for electrons from cytochrome c, is located in subunit II which represents the predominant binding site for this electron donor. The electrons are transferred from Cu A to heme a and then to the binuclear site where the oxygen reduction takes place. The function of subunit III is still obscure. All three subunits show strong homologies to the mitochondrially coded subunits of the eukaryotic enzyme. Several years ago, Haltia discovered a small polypeptide copurifying with the oxidase, which was partially sequenced (4, 5). A fourth component was found in the crystalline oxidase as well (6), and this small subunit could be characterized from the x-ray structure (7) as a polypeptide consisting of a single transmembrane helix and an N terminus protruding into the cytoplasmic space. Here we present the isolation and analysis of the gene coding for this fourth subunit. In addition, the purified 3-subunit enzyme complex resulting from a deletion of the gene is characterized. Furthermore, evidence for a putative promoter sequence is shown. MATERIALS AND METHODS Synthesis of Oligonucleotide Probes- The N-terminal sequence of the putative subunit IV (4) was taken to synthesize two degenerate oligonucleotides: primer A4U (5Ј-GCRAASGCSGCYTGYTG-3Ј) and primer A4D (5Ј-CAYCAYGAWATCACSGA-3Ј), the latter one as the reverse complement. DNA Manipulations-General cloning techniques and DNA manipulations were performed essentially as described in Ref. 8. Genomic DNA was obtained from P. denitrificans 1222 as described earlier (9). Polymerase chain reaction was used to amplify P. denitrificans genomic DNA with the primers A4D and A4U with an annealing temperature of 55°C. The sequence of the 65-bp 1 fragment obtained by polymerase chain reaction was used to synthesize a 30-mer oligonucleotide which was labeled with a dCTP/Dig-(11)-dUTP-tail for Southern and colony hybridization, which were performed as described by the manufacturer (Boehringer Mannheim) with a hybridization temperature of 42°C. A 1.9-kbp EcoRI/SphI and later a 3.8-kbp SacII/SalI fragment were isolated from a partial gene library derived from genomic digests with the respective enzymes. The second fragment was then cloned into pUC18 as a HindIII/SalI fragment (pHW5; see Fig. 2 ). Subcloning of various fragments, exo/mung digestion, and synthesis of specific primers were applied for sequence determination, using double-stranded DNA and T7 polymerase with 7-deaza-dGTP as described by the manufacturer (Pharmacia), according to the dideoxynucleotide chain termination method of Sanger (10). Construction of Deletion Mutants and Complementation- The 815-bp EcoRI/AscI fragment from pHW5 (see Fig. 2 ) was replaced by the 1.1-kb kanamycin resistance gene from pHP45⍀ (11) resulting in pHW14. The HindIII/SalI fragment of pHW14 was then ligated blunt-end into the SmaI site of the suicide vector pRVS1 (12) leading to pHW15 which was mated into PD1222. Homologous recombinants were selected for kanamycin resistance and loss of ␤-galactosidase activity. Two representative strains HW⌬1/24 and 55 (see Fig. 3A ) were characterized; for all subsequent steps, HW⌬1/24 was used and referred to as HW⌬1. Complementation Constructs-The promoterless broad host range vector pRG 2 was used to complement HW⌬1 with different fragments for promoter probe studies. The 683-bp PvuII fragment and the 1.7-kb EcoRI/SalI fragment of pHW5 were ligated into pRG, resulting in pHW21 and pHW22, respectively (see Fig. 2 ). These were used to complement HW⌬1 leading to HWK21 and HWK22. Strains, Growth Conditions, and Enzyme Preparation-P. denitrificans strain PD1222 (13) and the deletion strain HW⌬1 (see above) were grown on TY medium for DNA isolation or on succinate medium (14) for enzyme isolation, including kanamycin (50 g/ml), where appropriate. Complemented strains were grown in the presence of streptomycin sulfate (25 g/ml). Membrane isolation, enzyme purification, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and cytochrome difference spectra were performed as described in Ref. 15. Western blots were performed with monoclonal antibodies directed against subunit IV (6). Ligand Binding Spectra with Cyanide and Carbon Monoxide-All spectra were recorded on an Uvikon 941 (Kontron Instruments) between 380 and 650 nm. Difference spectra of cytochrome a and cytochrome a 3 were performed as follows: 10 l of a 10 mM potassium ferricyanide solution were added to 700 l of a 3 M oxidase solution. The sample was divided between two cuvettes. The sample cuvette was incubated with 20 l of 350 mM KCN for 2 h. All volume changes were balanced with buffer in the reference cuvette, and absolute spectra recorded. In the next step, N,N,NЈ,NЈ-tetramethyl-1,4-phenylenediamine and ascorbate were added to the sample cuvette to a final concentration of 60 M and 2 mM, respectively. The reference was reduced with dithionite. Again absolute spectra were measured, and difference spectra calculated according to Ref. 16. Carbon monoxide difference spectra were obtained by subtracting the dithionite-reduced from the (reduced ϩ CO) sample spectrum. Catalytic Activity-The spectrophotometric assay was performed at 25°C with reduced horse heart cytochrome c at 20 M as described before (15).