J. Biol. Chem., Vol. 266, Issue 29, 19212-19216, 10, 1991
Modification of histidine 56 in adrenodoxin with diethyl pyrocarbonate inhibited the interaction with cytochrome P-450scc and adrenodoxin reductase
S Miura, S Tomita and Y Ichikawa
Department of Biochemistry, Kagawa Medical School, Japan.
Three histidine residues of bovine adrenodoxin, His-10, His-56, and His-
62, were modified with diethyl pyrocarbonate. The order of the modification
among the three histidines were monitored by measuring the proton NMR
spectra. The modified adrenodoxin exhibited reduced affinity for
adrenodoxin reductase as determined in cytochrome c reductase activity. In
the presence of cholesterol, the modified adrenodoxin induced a high spin
form of cytochrome P-450scc on complex formation in the same manner as
native adrenodoxin. The spectral titration showed that adrenodoxin modified
with diethyl pyrocarbonate exhibited a 5-fold higher Kd value than that of
native adrenodoxin. These effects of the modification of adrenodoxin on the
affinities for the redox partners were not proportional to the number of
modified histidines determined by the optical absorbance change at 240 nm.
Modification of adrenodoxin up to 2 histidine residues did not affect the
affinity for the redox partners, but further modification on the third one
resulted in an increase of apparent Km in cytochrome c reductase activity
by 2-fold and of Kd for cytochrome P-450scc by 5-fold. The 1H NMR spectra
of the modified adrenodoxin unequivocally demonstrated that histidine
residues at His-10 and His-62 reacted more readily with diethyl
pyrocarbonate than His-56 did, indicating that modification of His-56 was
responsible for the reduction of binding affinities of adrenodoxin for
redox partners. These results are consistent with the proposal that the
residue of His-56 in adrenodoxin has an essential role in the electron
transfer mechanism where adrenodoxin functions as a mobile shuttle.
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