Insights into the role of substrates on the interaction between cytochrome b₅ and cytochrome P450 2B4 by NMR

Mammalian cytochrome b₅ (cyt b₅) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b₅ and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b₅-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b₅ and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b₅-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b₅-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b₅ and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b₅-P450 complexation process.