Temperature dependence of cytochrome P-450 reduction. A model for NADPH-cytochrome P-450 reductase:cytochrome P-450 interaction.
The NADPH-dependent reduction of rat hepatic microsomal cytochrome P-450 has been studied as a function of temperature. In the temperature range 4-37 degrees the reduction reaction was found to be biphasic and composed of two concurrent first order processes. This phenomenon was observed with microsomes from untreated and phenobarbital-induced animals in the presence or absence of exogenous Type I substrates. The amount of cytochrome P-450 reduced in the fast phase comprised approximately 70% of the total cytochrome P-450 at temperatures above 20 degrees. The temperature dependence of the fast phase was unusual for a membrane-bound enzyme system in that it lacked a discontinuity in the Arrhenius plot at a presumed phase transition temperature for the microsomal membrane. The slow phase of reduction behaved in a normal fashion for a membrane-bound enzyme system with a break in the Arrhenius plot at about 20 degrees. The data presented here combined with previous observations which include (a) the ratio of cytochrome P-450 to NADPH cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) is 20:1, (b) the catalytic portion of the reductase molecule probably protrudes above the surface of the membrane, and (c) the cytochrome P-450 molecules are presumably embedded in the membrane support the hypothesis that the hepatic microsomal drug-metabolizing system exists as clusters with most of the cytochrome P-450 molecules arranged about a central reductase molecule. This central flavoprotein reductase is able to randomly reduce those cytochrome P-450 molecules within the cluster without translational motion through the microsomal membrane. The slow phase of reduction represents the reduction of those molecules not directly associated with the clusters.
D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003579:Cytochrome Reductases / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008961:Models, Structural; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin
D003577:Cytochrome P-450 Enzyme System; D003579:Cytochrome Reductases; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin