NMR study on the structural changes of cytochrome P450cam upon the complex formation with putidaredoxin. Functional significance of the putidaredoxin-induced structural changes.

We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the beta-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 A. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo- and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 A. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the gamma-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by approximately 0.25 A. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.