Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin

Abstract

Cytochrome P450s are diverse and powerful catalysts that can activate molecular oxygen to oxidize a wide variety of substrates. Catalysis relies on effective uptake of two electrons and two protons. For cytochrome P450cam, an archetypal member of the superfamily, the second electron must be supplied by the redox partner putidaredoxin (Pdx). Pdx also plays an effector role beyond electron transfer, but after decades the mechanism remains under investigation. We applied infrared spectroscopy to heme-ligated CN^- to examine the influence of Pdx binding. The results indicate that Pdx induces the population of a conformation wherein the CN^- ligand forms a strong hydrogen bond to a solvent water molecule, experimentally corroborating the formation of a proposed proton delivery network. Further, characterization of T252A P450cam implicates the side chain of Thr252 in regulating the population equilibrium of hydrogen-bonded states within the P450cam/Pdx complex, which could underlie its role in directing activated oxygen toward product formation and preventing reaction uncoupling through peroxide release.

Figure 1.

Schematic of the catalytic cycle of P450cam, illustrating camphor (purple) binding to P450cam (blue). Effector Pdx is shown in yellow.

Figure 2.

Structural model of CN⁻-ligated P450cam showing a hydrogen-bonding network of a solvent water molecule (red), Thr252 (pink), and the heme-ligated CN⁻ (green); also shown are camphor (aqua) and Asp251 (pink) (PDB: 1O76).

Figure 3.

FTIR spectra (corresponding second derivative spectra shown below) of heme-ligated CN⁻ for wt P450cam: (A) substrate-free, (B) substrate-free with sucrose, (C) camphor complex, (D) camphor complex in D₂O solvent, (E) complex with camphor and Pdx, (F) complex with camphor and Pdx in D₂O solvent. Vertical lines mark the maximum of each absorption or minimum of each second derivative spectrum.

Figure 4.

FTIR spectra (corresponding second derivative spectra shown below) of heme-ligated CN⁻ for L358P P450cam camphor complex in (A) H₂O and (B) D₂O solvent.

Figure 5.

FTIR spectra (corresponding second derivative spectra shown below) of heme-ligated CN⁻ for T252A P450cam: (A) substrate-free, (B) camphor complex, (C) camphor complex in D₂O solvent, (D) complex with camphor and Pdx, (E) complex with camphor and Pdx in D₂O solvent. Vertical lines mark the maximum of each absorption or minimum of each second derivative spectrum.