In order to explore the nature of proposed ligands to
the CuA center in cytochrome c oxidase, site directed
mutagenesis has been initiated in subunit II of the
enzyme. Mutations were introduced into the mitochondrial
gene from the yeast Saccharomyces cerevisiae by high
velocity microprojectile bombardment. A variety of
single amino acid substitutions at each of the proposed
cysteine and histidine ligands (His-161, Cys-196, Cys-200
and His-204 in the bovine numbering scheme), as well
as at the conserved Met-207, all result in yeast which
fail to grow on ethanol/glycerol medium. Similarly,
all possible paired exchange Cys,His and Cys,Met mutants
show the same phenotype. Furthermore, protein stability
is severely reduced as evidenced by both the absence
of an absorbance maximum at 600 nm in the spectra of
mutant cells and the under-accumulation of subunit
II, as observed by immuno-labeling of mitochondrial
extracts. In the same area of the protein, a variety
of amino acid substitutions at one of the carboxylates
previously implicated in binding cytochrome c, Glu-198,
allow (reduced) growth on ethanol/glycerol medium,
with normal intracellular levels of protein. These
results suggest that a precise folding environment
of the CuA site within subunit II is essential for
assembly or stable accumulation of cytochrome c oxidase
in yeast.Return to publication list
