Circular dichroic studies on the interaction between reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase and adrenodoxin.
Circular dichroic spectra of NADPH-adrenodoxin reductase, adrenodoxin and the complex between oxidized adrenodoxin reductase and oxidized adrenodoxin were studied. A 1 : 1 complex formation between adrenodoxin reductase and adrenodoxin resulted in the appearance of a distinct difference CD spectrum, which was obtained by subtraction of the sum of their CD spectra from the CD spectrum of the mixture of the two proteins. The difference CD spectrum showed a broad trough between 500 and 600 nm and CD extrema at 452, 383, and 350 nm. This spectrum may reflect a change in the environment of either or both of the protein chromophores. By applying the curve-fitting method to analyze the ellipticity change at 452 nm of the difference CD spectrum obtained on titration of a fixed amount of adrenodoxin reductase with various amounts of adrenodoxin, we directly obtained a value of 4.56 x 10(-7) M (S.D. 9.58 x 10(-8) M) as the dissociation constant of the oxidized complex. We also studied the change of the difference ellipticity induced by the addition of salt and obtained dissociation constants for the oxidized complex at various concentrations of NaCl. These values were larger than those obtained previously by kinetic methods, measurements of NADPH-cytochrome c reductase and NADPH-DCIP reductase activities. Further, we examined the relationship between the change of these activities on the addition of salt and the complex concentration calculated using the dissociation constant obtained in this study and found that the change of these activities could not be attributed simply to dissociation of the oxidized complex.
D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*
D000325:Adrenodoxin; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases