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 (similar to 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 Angstrom 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 similar to 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 predicted dipolar shifts based on the homology model led to the assignment of parts of the distal Arg 38 and Phe 41. However, the remainder of the active site signals are strongly relaxed but only weakly dipolar shifted, precluding assignment of other protons <7 Angstrom from the iron, While the 1D/2D NMR approaches are not as effective in high-spin resting state HRP as for low-spin cyanide-inhibited HRP. several residues could be assigned in the former that were not located in the latter because both the residue and heme contact signals are lost under the diamagnetic envelope. With all heme signals resolved, HRP allows probing of the peripheral environment for all four pyrroles, Comparison of the hyperfine shift pattern among natural HRP isozymes reveals that the different shift magnitudes reflect variations in the extent of admixing of S < 5/2 spin states to the predominant high-spin ground state. The binding of the substrate benzhydroxamic acid to HRPC is shown to lead to altered hyperfine shifts that reflect an increase of the zero-field splitting and demonstrates that the binding of the substrate. in contrast to previous proposals. is not accompanied by ligation of a water at the sixth position. It is also concluded that the available methods are sufficient to allow definitive NMR studies of the peripheral substrate binding site in HRP.