Solution H-1 NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase

Solution two-dimensional H-1 NMR studies have been carried out on cyanide-i nhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope an d limitations of identifying residues in the heme pocket and substrate bind ing site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimen tal methods use a range of experimental conditions to obtain data on residu e protons with a wide range of paramagnetic relaxivity, The signal assignme nt strategy is guided by the recently reported crystal structure of recombi nant HRPC and the use of calculated magnetic axes. The goal of the assignme nt strategy is to identify signals from all residues in the heme, as well a s proximal and distal, environment and the benzhydroxamic acid (BHA) substr ate binding pocket. The detection and sequence specific assignment of aroma tic and aliphatic residues in the vicinity of the heme pocket confirm the v alidity of the NMR methodologies described herein. Nearly all residues in t he heme periphery are now assigned, and the first assignments of several "s econd sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The sh ift changes induced by substrate binding are shown to reflect primarily cha nges in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution H-1 NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or po int mutations of HRP.