Resonance Raman studies of cytochrome P450 2B4 in its interactions with substrates and redox partners.
Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.
Mak
Piotr J
PJ
Chemistry Department, Marquette University, Milwaukee, Wisconsin 53233, USA.
Im
Sang-Choul
SC
Zhang
Haoming
H
Waskell
Lucy A
LA
Kincaid
James R
JR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry*; D001589:Benzphetamine / Q000378:metabolism*; D002084:Butylated Hydroxytoluene / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D015786:Cytochromes b5 / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D008345:Manganese / Q000737:chemistry; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D013059:Spectrum Analysis, Raman
D005290:Ferric Compounds; D001589:Benzphetamine; D002084:Butylated Hydroxytoluene; D008345:Manganese; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D009251:NADPH-Ferrihemoprotein Reductase