Comparative studies on the accessibility and functional importance of tyrosine residues in cytochrome P-450 isozymes.

Cytochromes P-450 LM2 and P-450 LM4 from rabbit liver microsomes were chemically modified with tetranitromethane. Nitration of two tyrosine residues of both isozymes inhibits the benzphetamine N-demethylase activity of P-450 LM2 as well as the p-nitrophenetole O-deethylase activity of P-450 LM4 by about 80%. For identification of the modified tyrosine residues the inactivated enzymes were digested with trypsin, and the labeled peptides were separated by HPLC. Sequencing of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM2 showed that the tyrosine residues at positions 235 and 380 were nearly fully nitrated, while Tyr-348, Tyr-484 and Tyr-111 were only partially labeled (about 40-50%). In the presence of the heme-binding inhibitor metyrapone, Tyr-380 is partially protected against modification, and the extent of inactivation is diminished as well. These results suggest that Tyr-380 of cytochrome P-450 LM2 presents a catalytically essential amino acid residue at its active center. Sequence analyses of the 3-nitrotyrosine-containing peptides from cytochrome P-450 LM4 revealed that mainly Tyr-243 and Tyr-271 were labeled, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent (about 30-45%). Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are suggested to be functionally involved in the interaction with NADPH-cytochrome P-450-reductase.