Crystallographic and single-crystal spectral analysis of the peroxidase ferryl intermediate

Abstract

The ferryl [Fe(IV)O] intermediate is important in many heme enzymes, and thus, the precise nature of the Fe(IV)-O bond is critical in understanding enzymatic mechanisms. The 1.40 A crystal structure of cytochrome c peroxidase Compound I has been determined as a function of X-ray dose while the visible spectrum was being monitored. The Fe-O bond increases in length from 1.73 A in the low-X-ray dose structure to 1.90 A in the high-dose structure. The low-dose structure correlates well with an Fe(IV) horizontal lineO bond, while we postulate that the high-dose structure is the cryo-trapped Fe(III)-OH species previously thought to be an Fe(IV)-OH species.

Figure 1

Single crystal spectra of CCP Compound I as a function of x-ray dose. Prior to x-ray exposure the spectrum is identical to the solution spectrum of Compound I. The estimated percentage of Compound I remaining in the crystal as a function of x-ray dose in panel B was based on the decrease in the absorbance peak at 634 nm.

Figure 2

A) Superposition of the low dose structure (red) on the Fe(III) structure (cyan). Note that the iron is displaced below the plane of the heme in the Fe(III) structure and above the plane of the heme in the low dose structure; B) Fo(low dose)-Fo(high dose) electron density difference map using phases obtained from the low dose structure. The map is contoured at -5.0σ (green) and +5.0σ (blue); C and D) 2Fo-Fc electron density maps contoured at 4.0σ for the dose data set 1 (panel C) and high dose data set 15 (panel D). Oxygen and water molecules are represented by the small spheres.

Figure 3

Plot of the Fe-O distance as a function of x-ray dose. Each of the 13 structures was refined exactly the same way using the same starting structure and two different protocols. In the first the distances between the Fe and N atoms (4 pyrrole and 1 His closed circles) were restrained while in the second protocol no restraints were applied (open circles). At no time were restraints imposed on the Fe-O distance. The estimated error in the Fe-O bond distance is ≈0.017Å (see Supporting Information).