Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups.
The role of cytochrome b5 heme propionate groups in the functional interactions between cytochromes P-450 RLM5 and b5 has been investigated by comparing the capacity of RLM5 to interact with both native b5 and a b5 derivative in which the native heme was replaced with ferric protoporphyrin IX dimethyl ester (DME-b5). Both forms of b5 interacted with RLM5 causing an increase in the RLM5 spin state from 28 to 68% high-spin RLM5 at saturation, as judged using uv-visible spectrophotometry. However, DME-b5 exhibited a 7-fold weaker affinity for RLM5. The apparent dissociation constant (Kd) for the interaction between RLM5 and b5 was also shown to be a strong function of ionic strength, in a manner consistent with the involvement of electrostatic attraction in complex formation. Reconstitution of b5 into an RLM5-dependent monooxygenase system stimulated the p-nitroanisole demethylase rate about 25-fold and 7-ethoxycoumarin deethylase about 6-fold. DME-b5, however, produced only 30% of the stimulation of RLM5-dependent turnover of p-nitroanisole observed at equivalent concentrations of native b5 without a change in Km. With 7-ethoxycoumarin, turnover was 50% diminished. The diminished capacity of DME-b5 to stimulate RLM5-dependent substrate turnover was shown not to be due to impairment of electron flow between NADPH-cytochrome P-450 reductase and DME-b5, since the Km of reductase for DME-b5 is 2.5-fold lower, and the Vmax is actually increased, but rather to an impairment of some aspect of functional interaction between the DME-b5 and RLM5. The data show that complex formation between cytochrome P-450 and b5 involves electrostatic attraction mediated in part by cytochrome b5 heme propionate groups.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D004563:Electrochemistry; D004951:Esterification; D006418:Heme / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009994:Osmolar Concentration; D011422:Propionates / Q000378:metabolism; D011485:Protein Binding; D011524:Protoporphyrins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry
D003573:Cytochrome b Group; D011422:Propionates; D011524:Protoporphyrins; D006418:Heme; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C041104:dimethyl protoporphyrin IX