The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase.
Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.
D000325:Adrenodoxin / Q000378:metabolism; D001120:Arginine; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D010450:Endopeptidases; D005022:Ethyldimethylaminopropyl Carbodiimide; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism*; D006639:Histidine; D008239:Lysine; D008666:Metalloendopeptidases; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014357:Trypsin
D005288:Ferredoxins; D010446:Peptide Fragments; D000325:Adrenodoxin; D006639:Histidine; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D008239:Lysine; D005022:Ethyldimethylaminopropyl Carbodiimide