Kinetics and mechanism of CO binding to cytochromes P-450LM2 and P-450LM4. Effect of phospholipid, nonionic detergent, and substrate binding.

The kinetics of carbon monoxide binding to purified, soluble cytochrome P-450LM2 from liver microsomes of phenobarbital-induced rabbits and P-450LM4 from liver microsomes of 5,6-benzoflavone-induced rabbits was determined in the presence of catalytically essential phospholipid, d-benzphetamine or nonionic detergent (0.1% Tween 80, 0.1% Triton N-101, or 0.3% n-octylglucoside). The kinetics under these conditions was consistent with a two-step binding mechanism consisting of bimolecular association followed by a unimolecular rearrangement step. However, when d-benzphetamine was added to P-450LM2 in the presence of sonicated dilauroylglyceryl-3-phosphorylcholine (0.15 mM) or any of the above detergents, CO binding followed simple second order kinetics with a bimolecular rate constant of approximately 3 x 10 M-1 s-1 at 20 degrees C. P-450LM4-CO binding kinetics was not affected by concurrent addition of phospholipid and d-benzphetamine. These results imply that simultaneous binding of amphiphile and substrate induces a conformation of reduced P-450LM2 which is different from that found when either substance is bound alone.