Modification of histidine 56 in adrenodoxin with diethyl pyrocarbonate inhibited the interaction with cytochrome P-450scc and adrenodoxin reductase.
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.
Department of Biochemistry, Kagawa Medical School, Japan.
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Miura
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Tomita
Y
Y
Ichikawa
D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D009245:NADH Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D000325:Adrenodoxin; D006639:Histidine; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase; D009245:NADH Dehydrogenase; D004047:Diethyl Pyrocarbonate