Primary structure of a novel subunit in ba 3 ‐cytochrome oxidase from thermus thermophilus

Abstract

The ba 3 ‐type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix “subunit IIa” spanning the membrane. The hydrophobic N‐terminally blocked protein was isolated in high yield using high‐performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C‐terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI‐MS and by ESI requires the N‐terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5‐Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart , however, with opposite polarity. It is 35% identical to subunit IV of the ba 3 ‐cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa ( cbaD ) is located upstream of cbaB in the same operon as the genes for subunit I ( cbaA ) and subunit II ( cbaB ).