Nature of the ferryl heme in compounds I and II

Abstract

Heme enzymes are ubiquitous in biology and catalyze a vast array of biological redox processes. The formation of high valent ferryl intermediates of the heme iron (known as Compounds I and Compound II) is implicated for a number of catalytic heme enzymes, but these species are formed only transiently and thus have proved somewhat elusive. In consequence, there has been conflicting evidence as to the nature of these ferryl intermediates in a number of different heme enzymes, in particular the precise nature of the bond between the heme iron and the bound oxygen atom. In this work, we present high resolution crystal structures of both Compound I and Compound II intermediates in two different heme peroxidase enzymes, cytochrome c peroxidase and ascorbate peroxidase, allowing direct and accurate comparison of the bonding interactions in the different intermediates. A consistent picture emerges across all structures, showing lengthening of the ferryl oxygen bond (and presumed protonation) on reduction of Compound I to Compound II. These data clarify long standing inconsistencies on the nature of the ferryl heme species in these intermediates.

FIGURE 1.

Stereo images of the crystal structures of the ferryl heme intermediates of CcP and APX. A–E, CcP Compound I (A). CcP Compound II (B), APX Compound III (ferrous-oxy) (C), APX Compound I (D), APX Compound II (E), showing electron density maps calculated with coefficients 2F_o − F_c contoured at 2σ in blue and the F_o − F_c map (contoured at 4σ, shown in green) calculated after refinement omitting the oxygen. Oxygen atoms are shown as red spheres, the heme is in red, and the iron is shown as an orange sphere. Key residues are labeled.

FIGURE 2.

Comparison of UV-visible spectra in solution and from single crystal microspectrophotometry. A–D, comparisons for CcP (A and D) and for APX (B and C). A, the single crystal spectrum of CcP Compound I collected before x-ray exposure (solid line, maxima: 530, 560, and 632 nm) and after collection of a full dataset (dose > 0.35 Mgy, dash-dotted line, maxima: 542, 559, and 585^sh nm corresponding to reduced heme) when compared with the spectrum of CcP Compound I obtained in solution under the same conditions (dotted line, maxima: 530, 560, and 630^sh nm). Abs, absorbance. B, the spectrum of APX Compound II collected before x-ray exposure (solid line, maxima: 531, 558 nm) and after exposure to a dose of 0.3 MGy (dash-dotted line, maxima: 537^sh, 554, and 580^sh nm corresponding to reduced APX (compare 555 and 583 nm (29))) when compared with the solution spectrum of APX Compound II obtained under the same conditions (dotted line, maxima: 530, 559 nm (compare 529 and 560 nm (27))). C, spectra of APX Compound III (dash-dotted line, maxima: 540 and 575 nm) and APX Compound I (solid line, maxima 535, 573^sh, and 632 nm) obtained by photoreduction of APX Compound III after exposure to 0.15–0.2 MGy. In all cases, absorbance spectra in solution have been magnified 10 times for comparison. D, single crystal spectrum of CcP Compound II (solid line, maxima: 533, 560, 558^sh, and 633 nm) formed by photoreduction of CcP Compound I (dotted line) by exposure to a dose of ∼0.15–0.2 MGy. Appearance of the shoulder at 585 nm and slight red shift of the β band from 530 to 533 nm indicate the presence minor amounts of ferrous heme formed during photoreduction.

FIGURE 3.

Structures of ferrous heme species. A and B, ferrous derivative of CcP (A) and APX (B), showing electron density maps calculated with coefficients 2F_o − F_c contoured at 2σ in blue and the F_o − F_c map (contoured at 4σ shown in green) calculated after refinement omitting the oxygen. Oxygen atoms are shown as red spheres, the heme is in red, and the iron is shown as an orange sphere. Key residues are labeled. For CcP, the ESUs of the iron and oxygen atom positions calculated by full matrix inversion are 0.019 and 0.159 Å, respectively; for APX, the corresponding values are 0.023 and 0.170 Å, respectively.

SCHEME 1.

Different routes for oxygen activation by heme enzymes. The reaction using dioxygen (in the P450s and NO synthases) or hydrogen peroxide (in the peroxidases) as a route to the activated Compound I intermediate is shown. X^•+ represents either a porphyrin π-cation radical or a tryptophan radical.

SCHEME 2.

A summary of all structures obtained in this work. The structures of CcP Compound I and APX Compounds II and III were obtained by reaction with hydrogen peroxide; Compound II of CcP and Compound I of APX were obtained by photoreduction of Compound I and Compound III, respectively (as described under “Results”). The structures of the ferric CcP and APX enzymes are taken from the Protein Data Bank (2ZBY and 1OAG). X^•+ represents either a porphyrin π-cation radical or a tryptophan radical (APX or CcP, respectively).