M. Fukaya et al., CHARACTERIZATION OF A CYTOCHROME-A(1) THAT FUNCTIONS AS A UBIQUINOL OXIDASE IN ACETOBACTER-ACETI, Journal of bacteriology, 175(14), 1993, pp. 4307-4314
The terminal oxidase for ethanol oxidation in Acetobacter aceti was pu
rified as a complex consisting of four subunits (subunits I, II, Ill,
and IV) with molecular masses of 72, 34, 21, and 13 kDa, respectively.
Spectrophotometric analysis and catalytic properties determined with
the purified enzyme showed that it belonged to a family of cytochrome
al (ba)-type ubiquinol oxidases. A polymerase chain reaction with two
oligonucleotides designed for amino acid sequences that are conserved
in subunit I of the aa3-type cytochrome c oxidases from various origin
s and of an Escherichia coli o (bo)-type ubiquinol oxidase was used fo
r cloning the cytochrome a, gene. A 0.5-kb fragment thus amplified was
used as the probe to clone a 4.5-kb KpnI fragment that contained a pu
tative open reading frame for the whole subunit I gene. The molecular
weight and amino acid composition of the product of this open reading
frame (cyaA) were the same as those of the purified protein from A. ac
eti. The amino acid sequence of CyaA was homologous to that of subunit
I of the E. coli o-type ubiquinol oxidase. Nucleotide sequence analys
is of the region neighboring the cyaA gene revealed that the genes (cy
aB, cyaC, and cyaD) encoding the other three subunits (subunits II, II
I, and IV) were clustered upstream and downstream of the cyaA gene in
the order cyaB, cyaA, cyaC, and cyaD and with the same transcription p
olarity, forming an operon. As expected from the enzymatic properties,
CyaB, CyaC, and CyaD showed great similarity in amino acid sequence t
o the corresponding subunits of the E. coli o-type ubiquinol oxidase a
nd aa3-type cytochrome c oxidases.