Abstract
Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase.
Highlights
Catalytic heme enzymes carry out a wide range of oxidations in biology
Ascorbate peroxidase (APX) has high-sequence identity to cytochrome c peroxidase (CcP), which has served as a benchmark for heme enzyme catalysis over many years
The experimental difficulty of isolating Compound II is simplified considerably by working with APX because Compound I in APX exists as a ferryl heme and a porphyrin p-cation radical[14,15], and is distinct from its Compound II, which contains only a ferryl species
Summary
Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. The family of heme-containing peroxidase enzymes is widespread in biology They catalyse the H2O2-dependent oxidation of a range of different substrates, and in doing so underpin a number of essential biological processes in bacterial, yeast, plant, fungal and mammalian systems[1,2]. A particular focus has been the nature of the ferryl group in Compounds I and II, and whether it is best described as an Fe(IV) 1⁄4 O or a Fe(IV)–OH species This is important because the bonding interactions and the protonation state of the ligand bound to the iron controls the reactivity—and the biological usefulness—of each intermediate. We have used the approach to examine the Compound II intermediate of a heme peroxidase
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