Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues.
Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004579:Electron Transport; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010449:Peptide Mapping; D011817:Rabbits; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000032:analysis*
D014443:Tyrosine; D003577:Cytochrome P-450 Enzyme System; C031958:4-nitrophenetole-O-dealkylase; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D013774:Tetranitromethane