Solution NMR Study of the Electronic and Molecular Structure of the Heme Cavity in High-Spin, Resting State Horseradish Peroxidase

Abstract

Three resting state horseradish peroxidase isozymes (HRPC, HRPA1, and HRPA2) have been investigated by solution 1D and 2D NMR to determine the scope and limitation of these methods for large (∼44 kDa), high-spin ferric heme enzymes and to develop an interpretive basis of the hyperfine shifts in terms of the molecular and electronic structure of the active site. Definitive assignments are attained for the resolved heme and axial His resonances, as well as several residues more than 7 A from the iron. Four Phe side chains located in HRPC by scalar correlation and characteristic NOEs to the heme are identified as Phe 152, Phe 172, and two unassigned Phes X and W, in contact with pyrrole D. The temperature dependence of the hyperfine shifted aromatic rings shows that dipolar shift arises from zero-field splitting; a value of D ∼ 7 cm-1 models the observed dipolar shift with use of a homology model constructed from peanut peroxidase. The combined use of steady-state NOEs, paramagnetic relaxation, and the predi...