2D NMR APPROACHES TO CHARACTERIZING THE MOLECULAR-STRUCTURE AND DYNAMIC STABILITY OF THE ACTIVE-SITE FOR CYANIDE-INHIBITED HORSERADISH-PEROXIDASE

Cyanide-inhibited horseradish peroxidase, HRP-CN, has been investigated by 2D NMR in order to assign the signals for the active site residues and to determine their positions relative to each other and the heme. Analysis of the phase properties of COSY cross peaks reveals that cross correlation rather than coherence dominates for spatially close protons and that such cross peaks have limited use in identifying spin systems. It is shown, however, that TOCSY, particularly the CLEAN-TOCSY variant, is fully capable of mapping the spin systems for active site residues in low-spin ferric peroxidases. The prospects for detecting weak cross peaks from hyperfine shifted and paramagnetically relaxed, but unresolved, protons are significantly improved by combining WEFT or DEFT pulse-sequences with NOESY over a range of temperatures. Moreover, the presence of several very slowly exchanging labile protons for active site residues allows the standard sequence specific assignment of the proximal helical segment Gly 169-Phe 172, as well as part of Ser 167, and an unidentified residue in contact with the proximal His. Distal side assignments for the highly conserved Arg 38 and His 42 are revised and extended and the signals for Phe 41 located. The inter-residue and heme-residue dipolar contacts show that the position of the distal Arg and His are essentially the same as those in the crystal structures for lignin and cytochrome c peroxidases and that Phe 41 has an orientation similar to the homologous Phe in LiP. Identification of six labile protons with exchange lifetimes of several years at pH 7.0 in the holoprotein reveals that both the proximal and distal helices exhibit remarkable dynamic stability. The relevance of this NMR approach to other heme peroxidases is discussed.