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Cytochrome c oxidase contains an extra charged amino acid cluster in a new type of respiratory chain in the amino-acid-producing Gram-positive bacterium Corynebacterium glutamicum

Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu 680-4, Iizuka, Fukuoka-ken 820-8502, Japan¹
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753-0841, Japan²

Author for correspondence: Junshi Sakamoto. Tel: +81 948 297823. Fax: +81 948 297801. e-mail: sakamoto{at}bse.kyutech.ac.jp

The membranes from Corynebacterium glutamicum cells contain a hydrophobic di-haem C protein as the cytochrome c subunit of the new type of cytochrome bc complex (complex III in the respiratory chain) encoded by the qcrCAB operon [Sone, N., Nagata, K., Kojima, H., Tajima, J., Kodera, Y., Kanamaru, T., Noguchi, S. & Sakamoto, J. (2001). Biochim Biophys Acta 1503, 279–290]. To characterize complex IV, cytochrome c oxidase and its structural genes were isolated. The oxidase is of the cytochrome aa₃ type, but mass spectrometry indicated that the haem is haem As, which contains a geranylgeranyl side-chain instead of a farnesyl group. The enzyme is a SoxM-type haem–copper oxidase composed of three subunits. Edman degradation and mass spectrometry suggested that the N-terminal signal sequence of subunit II is cleaved and that the new N-terminal cysteine residue is diacylglycerated, while neither subunit I nor subunit III is significantly modified. The genes for subunits II (ctaC) and III (ctaE) are located upstream of the qcrCAB operon, while that for subunit I (ctaD) is located separately. The oxidase showed low enzyme activity with extrinsic substrates such as cytochromes c from horse heart or yeast, and has the Cu_A-binding motif in its subunit II. A prominent structural feature is the insertion of an extra charged amino acid cluster between the ß2 and ß4 strands in the substrate-binding domain of subunit II. The ß2–ß4 loop of this oxidase is about 30 residues longer than that of major cytochrome c oxidases from mitochondria and proteobacteria, and is rich in both acidic and basic residues. These findings suggest that the extra charged cluster may play a role in the interaction of the oxidase with the cytochrome c subunit of the new type of bc complex.

Keywords: cytochrome aa₃, dihaem cytochrome c, glutamate fermentation, high-G+C Gram-positive bacteria

Abbreviations: DG, n-decyl-D-glucoside; MALDI, matrix-assisted laser desorption ionization; MEGA 9, n-nonanoyl N-metylglucamide; MEGA 10, n-decanoyl N-methylglucamide; PTH, phenylthiohydantoin; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB052748 and AB052749.

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