Heme peroxidases catalyze the H2O2-dependent oxidation of a variety of substrates, most of which are organic. Mechanistically, these enzymes are well characterized: they share a common catalytic cycle that involves formation of a two-electron, oxidized Compound I intermediate followed by two single-electron reduction steps by substrate. The substrate specificity is more diverse--most peroxidases oxidize small organic substrates, but there are prominent exceptions--and there is a notable absence of structural information for a representative peroxidase-substrate complex. Thus, the features that control substrate specificity remain undefined. We present the structure of the complex of ascorbate peroxidase-ascorbate. The structure defines the ascorbate-binding interaction for the first time and provides new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase catalysis for more than 20 years. A new mechanism for electron transfer is proposed that challenges existing views of substrate oxidation in other peroxidases.

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The heme prosthetic group is vital to many cellular processes and is therefore widespread throughout organisms of different phylogenetic origin. Heme is used in proteins involved in cellular respiration, acts as a chemical mediator in ligand binding and signalling proteins, and is the key co-factor in many enzymes. Strikingly, there are over 20 different folding topologies of b-type heme proteins that are able to incorporate the same, chemically identical heme ligand. Comparisons of structures show that heme-protein interactions are generally diverse, though a degree of conservation exists at contacts with the pyrrole rings, the propionate groups and the proximal ligand. These interaction "hot spots" presumably define major determinants for binding heme and provide guidelines for the future design of novel heme proteins. 1 Introduction 2 What makes a heme binding site? 3 Comparative analysis of heme environments in distinct structures 4 Diversity of heme-protein packing contacts 5 Re-occurring contacts: protein-heme interaction "hot spots" 6 Anchoring of heme propionates by arginine residues 7 Conserved interactions at the "heme face" involving leucine 8 Conserved interactions at the "heme face" involving phenylalanine/tyrosine side chains 9 Contacts at the heme edge 10 Interactions with the proximal ligand 11 Heme orientation/"flipping" relative to the proximal ligand 12 Perspectives for the design of novel heme proteins 13 Conclusions 14 Acknowledgements 15 References

Ascorbate peroxidase is a bifunctional peroxidase that catalyzes the H(2)O(2)-dependent oxidation of both ascorbate and various aromatic substrates. The ascorbate binding site was recently identified as being close to the gamma-heme edge [Sharp, K. H., Mewies, M., Moody, P. C. E., and Raven, E. L. (2003)Nat. Struct. Biol. 10, 303-307]. In this work, the X-ray crystal structure of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) in complex with salicylhydroxamic acid (SHA) has been determined to 1.46 A. The SHA molecule is bound close to the delta-heme edge in a cavity that connects the distal side of the heme to the surface of the protein. There are hydrogen bonds between the phenolic hydroxide of the SHA and the main chain carbonyl of Pro132, between the carbonyl oxygen of SHA and the side chain guanadinium group of Arg38, and between the hydroxamic acid group and the indole nitrogen of Trp41. The structure provides the first information about the location of the aromatic binding site in ascorbate peroxidase and, together with our previous data [Sharp, K. H., et al. (2003) Nat. Struct. Biol. 10, 303-307], completes the structural description of the binding properties of ascorbate peroxidase. The mechanistic implications of the results are discussed in terms of our current understanding of how APX catalyzes oxidation of different types of substrates bound at different locations.

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide-and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.