Resonance Raman Investigations of Cytochrome P450_cam Complexed with Putidaredoxin

Abstract

We have performed resonance Raman and optical absorption studies on ferric cytochrome P450cam complexed with oxidized putidaredoxin. Optical absorption and resonance Raman measurements demonstrate that this complexation shifts the spin-state equilibrium of P450cam to the low-spin form. In the resonance Raman spectra, the ν3 heme skeletal mode characteristic of low-spin P450cam intensifies upon complexation with putidaredoxin. Its frequency is indistinguishable to that of the usual low-spin species, indicating that this low-spin form originates from a water-bound, hexacoordinate heme. This observation suggests that putidaredoxin binding affects the distal side of P450cam and allows water entry into the heme pocket. We also examined the effects of putidaredoxin binding on the heme axial ligand (Fe−S) stretching mode. The binding of putidaredoxin upshifts the mode by ∼3 cm-1. Investigations of the Fe−S mode for the Thr252 → Ala distal pocket mutant and P450cam bound to various camphor analogues demonstrate that the frequency of the νFe - S is sensitive to distal pocket water. The water lowers the Fe−S stretching frequency by ∼1 cm-1, demonstrating that the putidaredoxin-induced ∼3-cm-1 increase in the Fe−S stretching frequency is not simply caused by the increased hydration of the heme pocket. We also found that the Fe−S frequency is increased by ∼0.5 cm-1 in the presence of high salt concentrations. This result suggests that the shielding of positive charges at the proximal face of P450cam leads to an increased νFe - S frequency. The salt effect is consistent with the observation that the binding of negatively charged putidaredoxin at the proximal side of the enzyme also increases the νFe - S frequency. Taken together, these results strongly suggest that electrostatic shielding of charged groups on the proximal face of P450cam takes place when putidaredoxin binds and contributes to the observed upshift of the Fe−S mode.

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

“…Therefore, the ␤-proton of the axial Cys would move toward the heme-iron by ϳ0.05-0.15 Å upon the binding of Pdx. Such a structural change of the proximal side in P450cam is qualitatively consistent with the previous results from the resonance Raman spectroscopy (21). The complex formation between ferric P450cam and oxidized Pdx induces the upshift of the heme-axial ligand (Fe-S) stretching mode by 3 cm Ϫ1 (21), implying the movement of the axial thiolate to the heme-iron.…”

“…Other important feature of the P450cam⅐Pdx complex is that Pdx induces conformational changes of P450cam upon the complex formation (21)(22)(23)(24)(25)(26)(27)(28). EPR and resonance Raman studies clearly exhibited that the complex formation with Pdx converts the spin-state of ferric P450cam from the high to low spin state (21,24).…”

“…EPR and resonance Raman studies clearly exhibited that the complex formation with Pdx converts the spin-state of ferric P450cam from the high to low spin state (21,24). Associated with the change of the spin-state, the heme-iron axial ligand (Fe-S) stretching mode is upshifted by 3 cm Ϫ1 (21). In the ferrous-CO form, the Fe-CO and FeC-O stretching modes indicate 2 cm Ϫ1 upshift and 8 cm Ϫ1 downshift, respectively, by the binding of reduced Pdx, respectively (29).…”

Combined QM/MM calculations of active-site vibrations in binding process of P450cam to putidaredoxin

“…According to their findings, the change of the NMR spectrum of P450 after binding to pdx indicates that the binding process alters the electronic structure of the cyanide N atom, decreasing its electron spin density. Therefore the authors assume that the binding process of both proteins transfers an electron between iron and the CN ligand The similar electron delocalization between iron and the CO ligand upon binding of both proteins, is also proposed in another paper [37]. Therefore in order to reveal the proposed electron transfer in the active site of P450, we have performed calculations of the NBO populations of the Fe, S, C and O atomic orbitals in the triplet electronic state of the reduced iron form.…”

“…The calculated frequencies are scaled by a factor 0.92, which reflects a common characteristic of the DFT method [50]. Experimental data are taken from reference [37]. The properties of the Fe and S atoms calculated for the 6-coordinated heme active-site with a water molecule as the distal ligand in the oxidized form for the doublet, quartet and sextet electronic states.…”