Comparison of the Complexes Formed by Cytochrome P450_(cam) with Cytochrome b_5 and Putidaredoxin,Two Effectors of Camphor Hydroxylase Activity

摘要

Structural perturbations in Cytochrome P450_(cam) (CYP101) induced by the soluble fragment of cytochrome b_5,a nonphysiological effector of CYP101,were investigated by NMR spectroscopy and compared with the perturbations induced by the physiological reductant and effector putidaredoxin (Pdx).Chemical shifts of perdeuterated [U-~(15)N]CYP101 backbone amide (NH) resonances were monitored as a function of cytochrome b_5 concentration by ~1H-~(15)N TROSY-HSQC experiments.The association of cytochrome b_5 with the reduced CYP101-camphor-carbon monoxide complex (CYP-S-CO) perturbs many of the same resonances that Pdx does,including regions of the CYP101 molecule implicated in substrate access and orientation.The perturbations are smaller in magnitude than those observed with Pdx~r due to a lower binding affinity (a K_d of 13 +- 3 mM,for the reduced cytochrome b_5-CYP-S-CO complex compared to a K_d of 26 +- 12 muM for the Pdx-CYP-S-CO complex).The results are in accord with our previous suggestion that the observed perturbations are related to effector activity and support the proposal that the primary role of the effector is to populate the active conformation of CYP101 to prevent uncoupling [Pochapsky,S.S.,et al.(2003) Biochemistry 42,5649-5656].A titratable perturbation is observed at the ~1H resonance of the 8-CH3 group of CYP101-bound camphor upon addition of cytochrome b_5,a phenomenon also associated with the formation of the CYP101 centre dot Pdx complex,albeit with larger perturbations [Wei,J.Y.,et al.(2005) J.Am.Chem.Soc.727,6974-6976].The effector activity of the particular rat cytochrome b_5 construct used for NMR studies was confirmed by monitoring the enzymatic turnover that yielded 5-exo-hydroxycamphor using gas chromatography and mass spectrometry.Finally,the common features of the perturbations observed in the NMR spectra of the two complexes are discussed,and their relevance to effector activity is considered.