JBC, Vol. 250, Issue 16, 6218-6221, Aug, 1975
Photoreduction of cytochrome c1
C. A. Yu, Y. L. Chiang, L. Yu and T. E. King
1. Ferricytochrome c1 solution was reduced completely between pH 7 and 10
by illumination under anaerobic conditions. Photoreduction was not affected
by the ionic strength of the medium. However, it did not take place at pH
lower than 6 or higher than 10, or in the presence of p-hydroxymercuric
benzoate. The ferricyanide-reoxidized photoreduced c1 was not further
reduced upon illumination. The reductant was most probably a specific
sulfhydryl group in the subunit containing the heme of the cytochrome since
this subunit contained one less p-HMB-titratable group in the photoreduced
sample than in the untreated preparation. 2. The photoreduced cytochrome c1
showed the same spectra as the native cytochrome, and was not reactive with
carbon monoxide. The equilibrium constant of the reaction c12+ + c3+
equilibrium c13+ + c2+ for the photoreduced c1 was found to be slightly
lower (Keq = 2.6) than that for the native c1 (Keq = 3.5). The antimycin
A-sensitive electron acceptor activity of ferricyanide-reoxidized
photoreduced c13+ catalyzed by succinate-cytochrome c reductase was about
80% of that of the native c1. 3. A somewhat simplified method for isolation
of cytochrome c1 was developed. Anaerobic ammonium sulfate fractionation
and calcium phosphate gel chromatography were still used in order to
achieve the purity level of about 25 nmol of heme/mg of protein. The
cytochrome c1 prepared by this procedure showed the same properties tested
as that by the beta-mercaptoethanol method (Yu, C.A., Yu, L., and King,
T.E. (1972) J. Biol. Chem. 247, 1012-1019).
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