NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

Abstract

We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the ␤-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 Å. 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 Å. 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 ␥-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by ϳ0.25 Å. 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.Cytochrome P450 (P450) 1 is a heme-containing monooxygenase, which catalyzes the hydroxylation reactions of a wide variety of natural and unnatural substrates such as steroids, fatty acids, hydrocarbons, and xenobiotics (1). The P450 catalysis reaction requires two electrons from NADH or NADPH through the redox-linked proteins. In microsomal P450s, the electron transfer (ET) from NADPH to P450s is mediated by NADPH-P450 reductase containing FMN and FAD as the redox centers. In contrast, the electron from NADH or NADPH is sequentially transferred to ferredoxin reductase, ferredoxin, and finally P450 in mitochondrial and bacterial systems. The ET reaction between P450 and its redox partner is essential for the subsequent activation of molecular oxygen and the monooxygenation reaction.To understand the mechanism of the ET reaction in P450 and its redox partner system, the ET reaction between cytochrome P450cam (P450cam) from Pseudomonas putida, one of the bacterial P450s, and its redox partner, putidaredoxin (Pdx), has intensively been investigated. P450cam catalyzes the regio-and stereo-specific hydroxylation of its substrate, d-camphor (1, 2) by accepting two electrons from NADH. The ET from NADH to P450cam is sequentially mediated by a flavin group of putidaredoxin reductase and a [2Fe-2S] center of putidaredoxin (Pdx). With the availability of the P450cam (3) and Pdx (4, 5) structures, many investigators have performed the kinetic (6 -9), mutational (10 -14), and theoretical (15, 16) studies to clarify the molecular mechanism for the ET reaction ...

L358P Mutation on Cytochrome P450cam Simulates Structural Changes upon Putidaredoxin Binding

“…For the wild type enzyme, the signal x at Ϫ1.29 ppm, which is derived from the 9-methyl group of D-camphor, was shifted to the upfield region with increasing concentrations of Pdx red (Fig. 5, left panel), as observed at 40°C (6). In sharp contrast to the wild type enzyme, a new signal at Ϫ1.45 ppm appeared and increased its intensity with a decrease in the intensity of the signal x at Ϫ1.35 ppm upon the addition of Pdx red to the L358P mutant (Fig.…”

“…It is likely that tighter binding to Pdx in the L358P mutant might be caused by the structural rearrangements at the Pdx-binding site, which optimize the interaction between the L358P mutant and Pdx. Because previous reports (6,15,31) indicated that Pdx induces the structural changes in P450cam through the hydrogen bond between one of the heme propionates and Arg-112 at the putative Pdx-binding site, the structural changes around the heme could also evoke the structural changes in the Pdx-binding site. Therefore, we suggest that the Pdx-binding site of the L358P mutant as well as the heme environment correspond to the Pdx-bound wild type enzyme.…”

“…No significant line broadening of the NMR signals, except for signal y, in the L358P mutant upon the binding of Pdx implies that the exchange rate between free and bound proteins is fast enough on the NMR time scale in the L358P-Pdx system, which is the same as that in the wild type enzyme (6). In the fast exchange process, we can use Equation 1 for determining the limiting shifts (24).…”

“…On the other hand, the ␥-methyl group of Thr-252 moves away from the heme iron by ϳ0.05 Å in the L358P mutant. In our previous work we found that Pdx binding to P450cam results in the movement of the axial Cys, 9-methyl group, and 5-exo proton of D-camphor closer to the heme iron by ϳ0.05-0.15, ϳ0.15, and 0.5-0.7 Å, respectively, whereas the ␥-methyl group of Thr-252 moves by ϳ0.25 Å away from the heme iron (6). Although the magnitude of the structural changes on the Leu-358 to Pro mutation is smaller than from Pdx binding, the same trend in the structural changes in the heme environment indicates that the heme environment of L358P-CO has similar property to the Pdx-bound wild type enzyme.…”

“…The Fe-CO and C-O stretching modes also shifted up and down, respectively, upon Pdx binding to P450cam-CO (5), supporting enhancement of the electronic donation. Recently, we applied NMR spectroscopy to the P450cam-Pdx system to characterize further the Pdx-induced conformational changes of P450cam in detail (6). Upfield shifts of the ring current-shifted signals from the ␤-proton of the axial Cys and D-camphor in the P450cam-CO upon the binding of Pdx showed that the axial ligand and D-camphor move toward the heme iron by Pdx binding (6).…”