The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1 : 1 stoichiometry and is characterised by an equilibrium dissociation binding constant, Kd, of 11.6 t 0.4 pM (pH 7.002, p = 0.10 M, 25.0OC). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60 %) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular-modelling studies, using the NNM- Keywords: heme; peroxidase; ascorbate peroxidase; binding site.Haem peroxidases comprise a family of metalloenzymes that catalyse the H,O,-dependent oxidation of a wide range of organic and inorganic substrates (Frew and Jones, 1984;Everse et al., 1991 ;Welinder, 1992a;Poulos, 1993;English, 1994; English and Tsaprailis, 1995;Traylor and Traylor, 1995). The first published crystal structure for a haem peroxidase was that of cytochrome-c peroxidase (CcP) (Finzel and Poulos, 1984). More recently, structural information has been published for other peroxidases, namely manganese peroxidase (MnP), lignin peroxidase, peanut peroxidase, myeloperoxidase and Arthromyces ra-~O S U S peroxidase (which is identical to Coprinus cinereus peroxidase) (Sundaramoorthy et al., 1994;Poulos et al., 1993;Schuller et al., 1996;Zeng and Fenna, 1992;Petersen et al., 1994; Kunishima et al., 1994). Detailed comparison of the active-site structures of these enzymes has revealed a high level of sequence similarity around the haem, with five of the nine invariant residues in the plant peroxidase superfamily (Welinder, 1992b) forming part of the active site. Furthermore, site-directed-mutagenesis studies involving active-site residues, predominantly with CcP (Mauro et al., 1989;Erman et al., 1992Erman et al., , 1993Vitello et al., 1992Vitello et al., , 1993 Ferrer et al., 1994 (Smith et al., 1992a(Smith et al., , b, 1993Sanders et al., 1994; Miller et al., 1995;Newmeyer and Ortiz de Montellano, 1995, 1996; Ozaki and Ortiz de Montellano, 1995 ;Neptuno Rodriguez-Lopez et al., 1996), have helped to provide comprehensive understanding of the catalysis at the amino acid level.However, despite an increasingly detailed understanding of the mechanistic aspects of peroxidase catalysis at the active site, there is a very limited understanding of the way in which reducing substrates, in particular small organic substrates, are bound and oxidised by the enzyme. The deficiency arises, at least in part, from the absence of structural information for p...

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