Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2

Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available hors eradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 Angstrom resolution at 100 K showed a water molecule only 2.1 Angstrom from heme ir on [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas sp ectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme i ron, which is common in peroxidases. Presented here, the X-ray crystallogra phic, single-crystal, and solution resonance Raman studies at room temperat ure confirmed that the sixth coordination position of heme iron of ATP A2 i s essentially vacant. Furthermore, electronic absorption and resonance Rama n spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and dockin g of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, t he additional methoxy group of the sinapyl moiety gave rise to steric hindr ance, not only in A2 type peroxidases but also in all peroxidases. We confi rm these predictions for ATP A2, HRP A2, and HRP C. The specific activity o f ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady state st udy at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 muM(-1) s(-1)). The oxidation of coniferyl al cohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, howeve r, gave modest but significantly different k(3) rate constants of 8.7 +/- 0 .3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 muM(-1) s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 0.004 m uM(-1) s(-1) for ATP A2, respectively. The structural origin of the differe ntial reactivity is discussed in relation to glycosylation and amino acid s ubstitutions. The results are of general importance to the use of homologou s models and structure determination at low temperatures.