Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding

Abstract

The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdxbinding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with nonphysiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.Cytochrome P450cam (P450cam) 1 (1, 2) catalyzes the regioand stereo-specific hydroxylation of D-camphor. To activate molecular oxygen, P450cam requires two protons and two electrons. Protons are provided from bulk water through a proton shuttle system that includes Asp-251, Thr-252, and water molecules (3-6). Electrons are provided by an iron-sulfur protein, putidaredoxin (Pdx). In the electron transfer steps, P450cam forms a complex with reduced Pdx, and the P450cam active site is perturbed as indicated by various spectroscopic studies including NMR (7, 8), EPR (9, 10), resonance Raman (11-13), and infrared (14, 15). Because nonphysiological redox proteins such as ferredoxin and adrenodoxin cannot support the monooxygenation reaction or induce the conformational changes (7, 16), the Pdxinduced changes in P450cam are thought to be important for the "effector" function of Pdx in addition to its reductase activity. In support of this view, a recent infrared and mutational study (15) has shown that Pdx-induced conformational changes promote the camphor monooxygenation reaction.Recent NMR studies found that the CO complex of the L358P mutant has very similar spectroscopic property to the WT P450cam-CO in a complex with Pdx (8,17). This suggests that the L358P mutant is a good model for Pdx-bound P450cam. However, our knowledge on the conformational change from the NMR studies is limited to only two residues, the axial cysteine ligand (Cys-357) and Thr-252 and the substrate. To overcome this limitation and to complement the NMR work, we have solved the crystal structures of the ferrous and ferrous-CO complex of the L358P mutant. EXPERIMENTAL PROCEDURESProtein Purification and Crystallization-WT P450cam and the L358P 2 mutant were overexpressed in Escherichia coli and purified as described elsewhere (18). The CO-bound P450cam structure was first solved by Raag and Poulos (19) by using the initial P2 1 2 1 2 1 crystal form used to solve the P450cam structure. For a more accurate...

Crystal Structures of the Ferrous Dioxygen Complex of Wild-type Cytochrome P450eryF and Its Mutants, A245S and A245T

“…Although the substrate slightly rotates about C-13, O 2 binding does not change the substrate-heme distances significantly, probably because of restrictions imposed by the Hbond between 5-OH and O 2 . This is different than in P450cam where diatomic ligand binding, CO and O 2 , displaces the substrate by ϳ1 Å further from the heme (15,22,23).…”

L358P Mutation on Cytochrome P450cam Simulates Structural Changes upon Putidaredoxin Binding

“…These observations support the view that the L358P mutant can serve as a structural and functional model for Pdx-bound P450cam. The following paper on the x-ray structural analysis of the L358P mutant (18) provides further information on the Pdx-induced structural changes in P450cam and the effector function of Pdx.…”

“…Similar enhancement of -electron donation from the axial thiolate to the heme iron is also induced by the binding of Pdx to wild type P450cam (5,15), raising the possibility that the ferrous-CO form of L358P in the absence of Pdx is similar to wild type P450cam in complex with Pdx. This study and the accompanying paper (18) provide detailed structural and functional analyses of the L358P mutant as a model for Pdx-bound wild type P450cam in order to understand the functional significance of the Pdx-induced structural changes in P450cam.…”

Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM

“…This observation is consistent with our proposal that the blue shift in the Soret peak position of the oxyferrous state correlates with the ease of reduction and, therefore, the rate of turnover of the enzyme. In addition, Nagano et al (49) and Tosha et al (50) have prepared and structurally characterized the L358P mutant of P450-CAM, which has altered hydrogen bonding to the proximal thiolate sulfur. The spectral properties of this mutant are similar to those induced by Pdx binding.…”