AN INTERPRETIVE BASIS OF THE PROTON NUCLEAR-MAGNETIC-RESONANCE HYPERFINE SHIFTS FOR STRUCTURE DETERMINATION OF HIGH-SPIN FERRIC HEMOPROTEINS - IMPLICATIONS FOR THE REVERSIBLE THERMAL UNFOLDING OF FERRICYTOCHROME C' FROM RHODOPSEUDOMONAS-PALUSTRIS

An NMR approach to determining the solution molecular structure of a h igh-spin ferric hemoprotein, 13 kDa ferricytochrome c' from Rhodopseud omonas palustris (Rp), has been investigated. In parallel with the use of appropriately tailored 1D and 2D experiments to provide scalar and dipolar correlations for the strongly relaxed and hyperfine-shifted h eme cavity residues, we explore an interpretive basis of the large hyp erfine shifts for noncoordinated residues which could provide constrai nts in solution structure determination for high-spin ferric hemoprote ins. It is shown that the complete heme can be uniquely assigned in sp ite of the extreme relaxation properties (T(1)s 1-8 ms). Sufficient sc alar connectivities are detected for strongly relaxed protons (T-1, gr eater than or equal to 4 ms) to uniquely assign residues on both the p roximal and distal sides of the heme. The spatial correlations indicat e that the structure is homologous to the four-helix bundle observed f or other cytochromes c'. The pattern of large hyperfine shifts for non coordinated residues is shown to be qualitatively reproduced by the di polar shifts for a structural homolog based on an axial zero-field spl itting of similar to 12 cm(-1) It is concluded that, when this approac h is combined with more conventional 2D methods for the diamagnetic po rtion of the protein, a complete structure determination of a five-coo rdinate ferric hemoprotein should be readily attainable. It is shown t hat the ferricytochrome c' unfolds reversibly at high temperature and that there exists at least one equilibrium intermediate in this unfold ing that is suggested to involve helix separation from the heme.