Structure and quantum chemical characterization of chloroperoxidase compound 0, a common reaction intermediate of diverse heme enzymes

Abstract

We have determined the crystal structure of the chloroperoxidase (CPO) hydroperoxo reaction intermediate (CPO compound 0) at 1.75-A resolution. The intermediate was generated through controlled photoreduction of the CPO oxygen complex during x-ray data collection, which was monitored by recording of the crystal absorption spectra. Initially, the peroxo-anion species was formed and then protonated to yield compound 0. Quantum chemical calculations indicate that the peroxo-anion species is not stable and collapses instantaneously to compound 0. Compound 0 is present in the ferric low-spin doublet ground state and is characterized by a long O O bond length of 1.5 A and a Fe O bond distance of 1.8 A, which is also observed in the crystal structure.

Fig. 1

Reaction scheme of CPO. The halogenating ferric-hypohalite intermediate is formed in vivo by the reaction of CPO with hydrogen peroxide and proceeds via compound 0 and compound I. Shown in red is the experimental strategy used in this study to generate the compound 0 species, using PAA instead of the natural substrate H₂O₂.

Fig. 2

Absorption spectra measured from CPO crystals, mounted in a loop and kept at 90 K. (A) Comparison of the ferric ground state (blue), compound III (red), and compound 0 (black) crystal absorption spectra. (B) Temporal evolution of absorption spectra recorded during x-ray exposure of CPO compound III crystals.

Fig. 3

Stereoview of the active site of CPO compound 0. The structure was refined against the medium-dose data. The final 1.75-Å resolution sigmaA-weighted 2m F_O − DF_C map (1σ contour level) is shown in gray and the mF_O − DF_C omit map (+3σ contour level) is superimposed in red.

Fig. 4

Characterization of the doublet and quartet states. (A) QM/MM (UB3LYP/B1:CHARMM) potential energy surfaces between the Fe-OOH⁻ and Fe-O₂^2- states. (B) The electronic configurations of the doublet and quartet states of Fe-OOH⁻.

Fig. 5

QM/MM-optimized structures corresponding to CPO compound 0 in the doublet (A) and quartet (B) spin states and of Fe-O₂²⁻ species C (doublet) and D (quartet spin state) identified during the energy profile shown in Fig. 4A.