ASSIGNMENT STRATEGIES AND STRUCTURE DETERMINATION IN CYANIDE-INHIBITED HEME PEROXIDASES

摘要

A strategy is described for locating and assigning all of the hyperfine-shifted and/or relaxed resonances in the active site of the low-spin, cyanide-inhibited complex of horseradish peroxidase. The serious problems in spectral resolution and dynamic range due to the large size (44 kDa) of the protein can be overcome by taking advantage of the strong temperature dependence of the hyperfine shift, and by use of DEFT or "WEFT pulse sequences in conjunction with standard 2D experiments. Detailed analysis of COSY experiments reveals that cross-correlation contributes significantly to cross peak intensity and dominates for geminal protons. It is shown that many COSY cross peaks, in fact, are inter-residue cross correlation peaks. Hence COSY has limited use in mapping scalar connectivities, even in its phase-sensitive form. TOCSY, on the other hand, particularly when ROESY is suppressed, provides an effective method for mapping essentially all spin systems. The sequence-specific assignment of active site residues via standard backbone NOESY connectivities in -tH^O is greatly facilitated by the very slow exchange rates of the peptide protons for both the proximal and distal helices. The combination of 2D NMR methods lead to the complete assignment of the active site protons <6.5 A from the iron. The resulting dipolar shifts for non-coordinated residues are shown to be quantitatively described by the orientation of the magnetic axes which reflect a tilt of the Fe-CN unit away from the heme normal. It is likely that similar methods will allow detailed structural studies in a variety of comparably sized cyanide-inhibited heme peroxidases.