Studies on nitrotyrosine-82 and aminotyrosine-82 derivatives of adrenodoxin. Effects of chemical modification on the complex formation with adrenodoxin reductase.
The coordination structure of the iron-sulfur center of the nitrotyrosine and the aminotyrosine derivates of bovine adrenodoxin was investigated by electron paramagnetic resonance spectroscopy. The reduced form of both modified samples exhibited signals identical with those for the native protein at g= 1.94 and g=2.01. From these results together with optical absorption and chemical analyses, it was concluded that the coordination structure of the iron-sulfur chromophore for both the derivatives was identical with the binuclear tetrahedral structure of native adrenodoxin. The configuration of the iron-binding area in nitro- and amino-adrenodoxin was studied by ovserving the circular dichroism spectra between 350 and 600 nm. The maxima for the nitro or amino derivatives were all identical with those for the native protein but different in the magnitude of their molar ellipticity. The molar ellipticities at 440 nm were 45.8 X 10(3), 14.5 X 10(3), and 9.5 X 10(6) deg cm2 per mol of iron for native adrenodoxin, nitro or amino derivative, respectively. These results suggest that the chemical modification of the tyrosine residue causes a conformational change in the iron-binding area. We have previously reported that the enzymatic activities of these reconstituted nitro and amino derivatives toware cytochrome c reduction in the presence of adrenodoxin reductase and reduced nicotinamide adenine dinucleotide phosphate were 19 and 7% of native adrenodoxin, respectively. The cytochrome c reductase activities of nitro- and aminoadrenodixin were drastically affected by the ionic strength of the assay medium, as found in native adrenodoxin. Fluorometric titration of the reductase with aminoadrenodoxin revealed that aminoadrenodoxin forms a 1:1 molar complex with the reductase. These results suggest that both the nitro and amino derivatives form a complex with the reductase. The dissociation constants of nitro- and aminoadrenodoxin for the reductase were 6.1 X 10(-7)M and 3.3 X 10(-7) M at mu = 0.04 and 1.9 X 10(-6) M and 2.0 X 10(-6) M at mu = 0.20, respectively. Comparison of these values with those of native adrenodoxin (approximately 10(-9) M at mu = 0.04 and 2.2 X 10(-7) M at mu = 0.20) suggests that an increase in the dissociation constant for the reductase is responsible for the decreased electron transferring activity of the modified adrenodoxins.
D000325:Adrenodoxin / Q000378:metabolism; D000588:Amines; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D013455:Sulfur / Q000032:analysis; D014443:Tyrosine
D000588:Amines; D000325:Adrenodoxin; D014443:Tyrosine; D013455:Sulfur; D007501:Iron; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase