Comparison of the complexes formed by cytochrome P450cam with cytochrome b5 and putidaredoxin two effectors of camphor hydroxylase activity

摘要

Structural perturbations in cytochrome P450cam (CYP101) induced by the soluble fragment of rate cytochrome b5, a non-physiological 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-¹⁵N] CYP101 backbone amide (NH) resonances were monitored as a function of cytochrome b5 concentration by ¹H, ¹⁵N TROSY-HSQC experiments. The association of cytochrome b5 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 (Kd = 13 ± 3 mM, for reduced cytochrome b5-CYP-S-CO complex compared to Kd = 26 ± 12 μM 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 et al. Biochemistry 42, 5649-5656 (2003)]. A titratable perturbation is observed at the ¹H resonance of the 8-CH3 of CYP101-bound camphor upon addition of cytochrome b5, a phenomenon also associated with the formation of CYP101·Pdx complex albeit with larger perturbations [Wei et al., J. Am. Chem. Soc. 127, 6974-6 976 (2005)]. The effector activity of the particular rat cytochrome b5 construct used for NMR studies was confirmed by monitoring the enzymatic turnover to yield 5-exo-hydroxy camphor using gas chromatography/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 considered.