Cytochrome C (Fe2+) as a competitive inhibitor of NADPH-dependent reduction of cytochrome P450 LM2: locating protein-protein interaction sites in microsomal electron carriers.
The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed. Under these conditions ferrous cytochrome c was found to be a competitive inhibitor of the slow phase of the reduction process, which accounted for 80% of the total reduction amplitude. Inhibition experiments yield a dissociation constant for the LM2-reductase complex of 3.0 +/- 1.5 microM. This constant was the same both in the presence and in the absence of benzphetamine. Based on these data we conclude that cytochromes P450 and c bind to the same center on the NADPH-cytochrome P450 reductase molecule. Comparative analysis of the amino acid sequences reveals a detectable similarity between cytochrome c and cytochrome P450 LM2 at positions 68-87 and 121-145, respectively. In addition, a substantial similarity was shown for sequence fragments 204-224 of NADPH-cytochrome P450 reductase and 40-60 of cytochrome b5. Based on these findings a hypothesis for the location of the centers of intermolecular interactions on the molecules of cytochrome P450 LM2 and NADPH-cytochrome P450 reductase is proposed.
Davydov
D R
DR
Institute of Biological and Medical Chemistry, Academy of Medical Science, Moscow, Russia.
Darovsky
B V
BV
Dedinsky
I R
IR
Kanaeva
I P
IP
Bachmanova
G I
GI
Blinov
V M
VM
Archakov
A I
AI
D000595:Amino Acid Sequence; D000818:Animals; D001589:Benzphetamine / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003574:Cytochrome c Group / Q000494:pharmacology*; D015786:Cytochromes b5 / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
D003574:Cytochrome c Group; D001589:Benzphetamine; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase