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Abstract: Aerobic organisms have evolved to activate oxygen from the atmosphere, which allows them to catalyze the oxidation of different kinds of substrates. This activation of oxygen is achieved by a metal center (usually iron or copper) buried within a metalloprotein. In the case of iron-containing heme enzymes, the activation of oxygen is achieved by formation of transient iron-oxo (ferryl) intermediates; these intermediates are called Compound I and Compound II. The Compound I and II intermediates were first discovered in the 1930s in horseradish peroxidase, and it is now known that these same species are used across the family of heme enzymes, which include all of the peroxidases, the heme catalases, the P450s, cytochrome c oxidase, and NO synthase. Many years have passed since the first observations, but establishing the chemical nature of these transient ferryl species remains a fundamental question that is relevant to the reactivity, and therefore the usefulness, of these species in biology. This Account summarizes experiments that were conceived and conducted at Leicester and presents our ideas on the chemical nature, stability, and reactivity of these ferryl heme species. We begin by briefly summarizing the early milestones in the field, from the 1940s and 1950s. We present comparisons between the nature and reactivity of the ferryl species in horseradish peroxidase, cytochrome c peroxidase, and ascorbate peroxidase; and we consider different modes of electron delivery to ferryl heme, from different substrates in different peroxidases. We address the question of whether the ferryl heme is best formulated as an (unprotonated) FeIV═O or as a (protonated) FeIV–OH species. A range of spectroscopic approaches (EXAFS, resonance Raman, Mossbauer, and EPR) have been used over many decades to examine this question, and in the last ten years, X-ray crystallography has also been employed. We describe how information from all of these studies has blended together to create an overall picture, and how the recent application of neutron crystallography has directly identified protonation states and has helped to clarify the precise nature of the ferryl heme in cytochrome c peroxidase and ascorbate peroxidase. We draw comparisons between the Compound I and Compound II species that we have observed in peroxidases with those found in other heme systems, notably the P450s, highlighting possible commonality across these heme ferryl systems. The identification of proton locations from neutron structures of these ferryl species opens the door for understanding the proton translocations that need to occur during O–O bond cleavage.

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Abstract: We have previously demonstrated (Badyal et al., J. Biol. Chem., 2006, 281, 24512) that removal of the active site tryptophan (Trp41) in ascorbate peroxidase increases the conformational mobility of the distal histidine residue (His42) and that His42 coordinates to the iron in the oxidised W41A enzyme to give a 6-coordinate, low-spin peroxidase. In this work, we probe the conformational flexibility of the active site in more detail. We examine whether other residues (Cys, Tyr, Met) can also ligate to the heme at position 42; we find that introduction of other ligating amino acids created a cavity in the heme pocket, but that formation of 6-coordinate heme is not observed. In addition, we examine the role of Asn-71, which hydrogen bonds to His42 and tethers the distal histidine in the active site pocket; we find that removal of this hydrogen bond increases the proportion of low-spin heme. We suggest that, in addition to its well-known role in facilitating the reaction with peroxide, His42 also plays a role in defining the shape and folding of the active site pocket.

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Abstract: The interactions of heme peroxidase enzymes with their substrates have been studied for many years, but only in the last decade or so has structural information begun to appear. This review looks at crystal structures for a number of heme peroxidases in complex with a number of (mainly organic) substrates. It examines the nature and location of the binding interaction, and explores functional similarities and differences across the family.