The ferric-hydroperoxo complex of chloroperoxidase

Abstract

The hydroperoxo-ferric complex, or Compound 0 (Cpd 0), is an unstable transient intermediate common for oxygen activating heme enzymes such as the cytochromes P450, nitric oxide synthases, and heme oxygenases, as well as the peroxidases and catalases which utilize hydrogen peroxide as a source of oxygen and reducing equivalents. Detailed understanding of the mechanism of oxygen activation and formation of the higher valent catalytically active intermediates in heme enzyme catalysis requires the structural and spectroscopic characterization of this immediate precursor, Cpd 0. Using the method of cryoradiolytic reduction of the oxy-ferrous heme complex, we have prepared and characterized hydroperoxo-ferric complex in chloroperoxidase (CPO) and compared this to the same intermediate generated in cytochrome P450 CYP101. Optical absorption spectrum of Cpd 0 in CPO has a Soret band at 449 nm and poorly resolved alpha, beta bands at 576 and 546 nm.

Figure 1

Absorption spectra of ferric CPO at varying temperatures in the absence (A) and in the presence (B) of the substrate thioanisole.

Figure 2

Absorption spectra of oxyferrous CPO at different temperatures in the absence (A) and in the presence (B) of thioanisole

Figure 3

Annealing of hydroperoxo-ferric intermediate (Compound 0) in CPO at different temperatures. Shown are absorption spectra of oxy-ferrous CPO (Compound III) at 80 K (1), and hydroperoxo-ferric CPO (Compound 0) at 80 K (2), 182 K (3), 200 K (4), 208 K (5), and 214 K (6). Compound 0 is stable in the frozen solution below 170 K, and decomposes at higher temperatures. Spectra of Compound 0 are calculated based on 50% yield as described in the text. .

Scheme I

The reaction cycles of chloroperoxidase (shown in the left cycle with the pathway from the resting state to Cpd0 utilizing hydrogen peroxide as a source of electrons and oxygen) and cytochrome P450 (the right pathway, utilizing dioxygen and two external electrons from redox partners). Compound 0 and Compound I are common intermediates for peroxidases and cytochromes P450.