Identification of residues in the aromatic substrate binding site of horseradish peroxidase by 1H NMR studies on isozymes.

The cyanide-inhibited complexes of two horseradish peroxidase acidic isozymes, A1 (HRPA1, unsequenced) and A2 (HRPA2, sequenced), have been examined by solution two-dimensional 1H NMR methods, and the active site molecular and electronic structure compared to that of the well-characterized isozyme C (HRPC) (Chen, Z., de Ropp, J.S., Hernández, G., & La Mar, G.N. (1994) J. Am. Chem. Soc. 116, 8772-8783), as well as to that of cytochrome c peroxidase. The identity and alignment of catalytically relevant residues near the active site for HRPA1-CN and HRPA2-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, confirm 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 NH exchange with solvent, is also conserved for the three isozymes. An additional heme contact, Ile 148, is identified in HRPC-CN. Four residues in contact with the heme in HRPC-CN are replaced in HRPA2-CN, two of which are likely functionally neutral, Gly 169-->Ala and Ile 148-->Leu. However, two substitutions in the acidic isozymes in the aromatic substrate binding pocket on the heme edge, Ile 244-->Leu and Phe 179 or 221-->aliphatic residue, could well account for the dramatic decrease (approximately 10(3) in aromatic substrate binding in the A1 and A2 isozymes vs the C isozyme of HRP.(ABSTRACT TRUNCATED AT 250 WORDS)