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| - An Interpretive Basis of the Proton Nuclear MagneticResonance Hyperfine Shifts for Structure Determination ofHigh-Spin Ferric Hemoproteins. Implications for the ReversibleThermal Unfolding of Ferricytochrome c‘ fromRhodopseudomonas palustris
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| - An NMR approach to determining the solution molecular structure ofa high-spin ferric hemoprotein, 13kDa ferricytochrome c‘ from Rhodopseudomonaspalustris (Rp), has been investigated. In parallelwith the use ofappropriately tailored 1D and 2D experiments to provide scalar anddipolar correlations for the strongly relaxed andhyperfine-shifted heme cavity residues, we explore an interpretivebasis of the large hyperfine shifts for noncoordinatedresidues which could provide constraints in solution structuredetermination for high-spin ferric hemoproteins. Itisshown that the complete heme can be uniquely assigned in spite of theextreme relaxation properties (T1s 1−8ms).Sufficient scalar connectivities are detected for strongly relaxedprotons (T1 ≥ 4 ms) to uniquely assign residuesonboth the proximal and distal sides of the heme. The spatialcorrelations indicate that the structure is homologous tothe four-helix bundle observed for other cytochromes c‘.The pattern of large hyperfine shifts fornoncoordinatedresidues is shown to be qualitatively reproduced by the dipolar shiftsfor a structural homolog based on an axialzero-field splitting of ∼12 cm-1. Itis concluded that, when this approach is combined with moreconventional 2Dmethods for the diamagnetic portion of the protein, a completestructure determination of a five-coordinate ferrichemoprotein should be readily attainable. It is shown that theferricytochrome c‘ unfolds reversibly at high temperatureand that there exists at least one equilibrium intermediate in thisunfolding that is suggested to involve helix separationfrom the heme.
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