JBC, Vol. 251, Issue 2, 270-276, Jan, 1976
Studies on cytochrome oxidase. Preliminary characterization of an enzyme containing only four subunits
S. H. Phan and H. R. Mahler
The preparation of a four-subunit enzyme from yeast, capable of catalyzing
the oxidation of ferrocytochrome c, is described. It is derived from
proteins containing seven or five subunits by means of recycling exclusion
chromatography in the presence of 0.1% sodium dodecyl sulfate. Its
catalytic properties are similar to those of the parent enzyme. Gel
electrophoresis of this preparation in the presence of sodium dodecyl
sulfate reveals three bands, migrating with RF values that correspond to
molecular weights of 14.6, 12.3, and 10.6 X 10(3), with the largest
exhibiting an apparent 2:1 stoichiometry relative to the other two. Visible
spectra in the region of 390 to 630 nm do not show any detectable
difference from that of the parent cytochrome oxidase, while its heme a and
copper content are raised to values around 20 nmol or ng atoms/mg of
protein, respectively, corresponding to minimal molecular weights of 50 X
10(3). The molecular weight determined by physical means equals 107 X
10(3). Thus the enzyme probably contains two copies of each subunit. After
extensive dialysis to remove as much as possible of the sodium dodecyl
sulfate used in its preparation, this enzyme remains in solution in
phosphate buffer in the absence of any added detergent, while under similar
conditions the seven-subunit complex precipitates completely. A similar
preparation can also be obtained from beef heart. The significance of these
findings is discussed with respect to the role of the large subunits in the
function as well as the biogenesis of the mitochondrial cytochrome oxidase
complex.
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