IDENTIFICATION OF RESIDUES IN THE AROMATIC SUBSTRATE-BINDING SITE OF HORSERADISH-PEROXIDASE BY H-1-NMR STUDIES ON ISOZYMES

The cyanide-inhibited complexes of two horseradish peroxidase acidic i sozymes, A1 (HRP(A1), unsequenced) and A2 (HRP(A2), sequenced), have b een examined by solution two-dimensional H-1 NMR methods, and the acti ve site molecular and electronic structure compared to that of the wel l-characterized isozyme C (HRP(C)) (Chen, Z., de Ropp, J. S., Hernande z, G., & La Mar, G. N. (1994) J. Am. Chem. Sec. 116, 8772-8783), as we ll as to that of cytochrome c peroxidase. The identity and alignment o f catalytically relevant residues near the active site for HRP(A1)-CN and HRP(A2)-CN are determined, and key residue replacements implicated in the differential catalytic properties of the acidic vs C isozymes are identified. Heme and axial His contact shift patterns, as well as dipolar contacts of residues with the heme and with each other, confir m a highly conserved structure among the three isozymes, including for the distal pocket residues involved in the activation of the enzyme. The remarkable dynamic stability of the heme pocket, as reflected in N H exchange with solvent, is also conserved for the three isozymes. An additional heme contact, lie 148, is identified in HRP(C)-CN. Four res idues in contact with the heme in HRP(C)-CN are replaced in HRP(A2)-CN , two of which are likely functionally neutral, Gly 169 --> Ala and li e 148 --> Leu. However, two substitutions in the acidic isozymes in th e aromatic substrate binding pocket on the heme edge, lie 244 --> Leu and Phe 179 or 221 --> aliphatic residue, could well account for the d ramatic decrease (similar to 10(3)) in aromatic substrate binding in t he Al and A2 isozymes vs the C isozyme of HRP. The identification of h eme pocket substitutions provides important constraints on molecular h omology models of HRP. The present NMR approach is expected to have br oad applications for determination of active site structure both for g enetically engineered moderately large (34-48 kDa) point mutants of he me peroxidases and for heme oxygenases.