SOLUTION NMR-STUDY OF THE ELECTRONIC AND MOLECULAR-STRUCTURE OF THE HEME CAVITY IN HIGH-SPIN, RESTING STATE HORSERADISH-PEROXIDASE

Three resting state horseradish peroxidase isozymes (HRPC, HRPA1, and HRPA2) have been investigated by solution 1D and 2D NMR to determine t he scope and limitation of these methods for large (similar to 44 kDa) , high-spin ferric heme enzymes and to develop an interpretive basis o f the hyperfine shifts in terms of the molecular and electronic struct ure of the active site. Definitive assignments are attained for the re solved heme and axial His resonances, as well as several residues more than 7 Angstrom from the iron. Four Phe side chains located in HRPC b y scalar correlation and characteristic NOEs to the heme are identifie d as Phe 152, Phe 172, and two unassigned Phes X and W, in contact wit h pyrrole D. The temperature dependence of the hyperfine shifted aroma tic 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 combi ned use of steady-state NOEs, paramagnetic relaxation, and the predict ed 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 shift ed, precluding assignment of other protons <7 Angstrom from the iron, While the 1D/2D NMR approaches are not as effective in high-spin resti ng state HRP as for low-spin cyanide-inhibited HRP. several residues c ould be assigned in the former that were not located in the latter bec ause both the residue and heme contact signals are lost under the diam agnetic envelope. With all heme signals resolved, HRP allows probing o f the peripheral environment for all four pyrroles, Comparison of the hyperfine shift pattern among natural HRP isozymes reveals that the di fferent 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 cont rast 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 subs trate binding site in HRP.