Figure 1.
The catalytic cycle of P450cam and the formation of the products, 10, 11 and 12.
a) R-H represents the substrate, camphor. i, ii, iii and iv represent the peroxide shunt reaction, four-electron uncoupling, two-electron uncoupling, and the loss of superoxide. b) Under highly oxygenated conditions, P450cam hydroxylates camphor 9 to 5-exo-hydroxy camphor 10 and further to 5-ketocamphor 11, whereas under low oxygen conditions, P450cam reduces camphor to borneol 12.
Table 1.
Assays with recombinant proteins: Formation of borneol, 5-ketocamphor and 5-exo- hydroxy camphor under various conditions.
Figure 2.
2H NMR of the 2-D-borneol and 17O NMR in the detection of H217O2.
a) 2H NMR of the 2-D-borneol obtained from the recombinant proteins incubated in 50 mM deuterated phosphate buffer (pD = 7.4) with camphor and m-CPBA. The extracted product was backwashed with H2O. The peak at 7.26 ppm corresponds to CHCl3 in CDCl3. b) 17O NMR spectrum of the incubation mixture in 17O phosphate buffer (pH 6.3) containing: i) camphor, recombinant P450cam and m-CPBA, ii) camphor and recombinant P450cam (m-CPBA absent), iii) camphor and m-CPBA (enzyme absent), and iv) m-CPBA and recombinant P450cam (substrate absent). The peaks at 0 ppm and 178 ppm correspond to H217O and H217O2, respectively.
Figure 3.
The Kinetic Isotope Effects for borneol and H2O2 and the Michaelis-Menten kinetics in their formation.
a) Ratios vH/vD at different temperatures for borneol and b) for H2O2 formation. c) Michaelis-Menten kinetics for borneol and d) 5-ketocamphor formation, under shunt conditions (with m-CPBA). To ensure a constant high O2 concentration for the 5-ketocamphor formation kinetics, reactions were run in vials fitted with septa and pressurized with pure O2.
Figure 4.
The proposed reduction mechanism and the Born-Haber estimates in the mechanism.
a) Proposed reduction mechanism of P450cam that accounts for the simultaneous formation of borneol 12 and H2O2, in a 1∶1 stoichiometry. b) Born-Haber cycle estimates of the reduction mechanism.
Figure 5.
Superposition of P450cam and CYP3A4.
a) Top row: superimposed ribbon diagrams of P450cam (1DZ4) and CYP3A4 (1TQN). P450cam is shown with red helices and yellow sheets, whereas CYP3A4 is shown all in pink. The porphyrin for P450cam is shown in gray and the one for CYP3A4, in brown. The two views are orthogonal to each other. The substrate access channel (SAC) is marked, as is Helix I, the central pillar of the fold. b) Lower row: superimposed active sites of P450cam and CYP3A4. The porphyrin of P450cam is shown in gray, the one for CYP3A4 in brown. The camphor ligand of P450cam is shown in green. Residues from the two proteins are red (P450cam) and pink (CYP3A4). The two views are orthogonal to each other.
Figure 6.
Sites in P450cam and in CYP3A4 with camphor docked.
a) Oxygen binding site in P450cam (residues shown in red), with superimposed residues in CYP3A4 shown in pink. The porphyrin of P450cam is gray, and the one for CYP3A4 is brown. b) Water channel in P450cam (residues shown in red), with superimposed residues in CYP 3A4 shown in pink. The view in a) and b) are from a similar angle, to emphasize the closeness of the O2 binding site and the water channel in P450cam. c) and d) Camphor docked into the active site of CYP3A4 (orthogonal views). The H-bond from Arg 105 to the camphor ketone can be seen in the lower right portion of d).
Table 2.
Tests for involvement of free reactive oxygen species: formation of borneol, 5-ketocamphor and H2O2.*
Figure 7.
The effect of camphor, borneol and DMSO on the P450 expression.
a) Outline of the experiment used to determine the effect of camphor and borneol on P450cam, PdX and PdR expression. b) The effect of camphor, borneol and DMSO on the P450 expression by Pseudomonas putida (ATCC 17453). The concentration of P450cam was obtained from the Soret peak absorbances and was normalized against the number of colony forming units/mL. Points represent the average ± S. E. of three replicates.