Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system.
The mechanism of the cytochrome P450 2B4 modification by hydrogen peroxide (H2O2) formed as a result of partial coupling of NADPH-dependent monooxygenase reactions has been studied in the monooxygenase system reconstituted from the highly purified microsomal proteins: cytochrome P450 2B4 (P450) and NADPH-cytochrome P450 reductase in the presence of detergent Emulgen 913. It was found, that H2O2-mediated P450 self-inactivation during benzphetamine oxidation is accompanied by heme degradation and apoenzyme modification. The P450 heme modification involves the heme release from the enzyme under the action of H2O2 formed within P450s active center via the peroxycomplex decay. Additionally, the heme lost is destroyed by H2O2 localized outside of enzyme's active center. The modification of P450 apoenzyme includes protein aggregation that may be due to the change in the physico-chemical properties of the inactivated enzyme. The modified P450 changes the surface charge that is confirmed by the increasing retention time on the DEAE column. Oxidation of amino acid residues (at least cysteine) may lead to the alteration into the protein hydrophobicity. The appearance of the additional ionic and hydrophobic attractions may lead to the increase of the protein aggregation. Hydrogen peroxide can initiate formation of crosslinked P450 dimers, trimers, and even polymers, but the main role in this process plays nonspecific radical reactions. Evidence for the involvement of hydroxyl radical into the P450 crosslinking is carbonyl groups formation.
Karuzina
I I
II
Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow.
Zgoda
V G
VG
Kuznetsova
G P
GP
Samenkova
N F
NF
Archakov
A I
AI
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D001589:Benzphetamine / Q000378:metabolism; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003902:Detergents; D017886:Durapatite; D006418:Heme / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D001051:Apoenzymes; D003902:Detergents; D001589:Benzphetamine; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D006861:Hydrogen Peroxide; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase