Roles of the Proximal Hydrogen Bonding Network in Cytochrome P450_cam-Catalyzed Oxygenation

Abstract

Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I. J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause approximately 45 and approximately 35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482 approximately 483 cm(-)(1) compared with that of wild-type P450(cam) at 484 cm(-)(1). The Q360 mutants also showed the upshift by 4 approximately 5 cm(-)(1) of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the Q360 mutants and were in contrast to the increased pi-back-donation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-)(1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.

L358P Mutation on Cytochrome P450cam Simulates Structural Changes upon Putidaredoxin Binding

“…Reactivity of Oxy-P450cam with Electron Donors-Besides the enhanced push effect in the L358P mutant (16,17), our current NMR data show that both D-camphor and the ␤-proton of the axial Cys approach closer to the heme-iron as observed in the Pdx-bound wild type enzyme. Therefore, we expected that the mutant should exhibit functional properties characteristic of Pdx-bound P450cam.…”

“…Here, in order to confirm that the Pdx-induced structural changes can be the trigger for the turnover reaction, we focused on a P450cam mutant, Leu-358 3 Pro mutant (L358P) (16,17), whose spectroscopic properties are similar to those of wild type P450cam in the presence of Pdx. Earlier work (16,17) showed that mutating Leu-358 to Pro in P450cam disrupts the hydrogen bond between the amide proton of the main chain and the axial thiolate (Fig. 1), and leads to enhanced -electron donation from the axial thiolate to the heme iron, which changes Fe-CO and C-O in the ferrous-CO form (Table I).…”

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

“…The enhancement of the electronic donation from the axial Cys, referred to as the enhanced "push effect," was believed to facilitate the heterolysis of molecular oxygen bound to the heme-iron (58). The P450cam mutant, Leu-358 3 Pro, whose Fe-CO and FeC-O Raman lines indicated up-shift and down-shift, respectively, as found for wild-type P450cam in the presence of Pdx, exhibited the high oxygen activation activity, confirming that the O-O bond scission is accelerated by the enhanced push effect (36,58,59). The Pdx-induced conformational changes at the proximal side of the heme, therefore, substantially contribute to the activation of molecular oxygen on the heme-iron in P450cam.…”

The bioinorganic chemistry of iron in oxygenases and supramolecular assemblies

“…This effect is also seen in model compounds with a thiolate ligand to iron (39). The protein-derived hydrogen bonds to the axial thiolate ligand to iron in P450cam have been shown to affect the OOO bond cleavage (40).…”