The ba3-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 ba3-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).