Nuclear magnetic resonance studies of a variety of paramagnetic metalloproteins are proposed in order to extract unique electronic/molecular structural information from the hyperfine shifts as relevant to the formation of ligation channels in proteins, the control of redox potential of electron mediators, and the detection of intermediates in the unfolding of proteins. Central to all of these studies is the development and implementation of two-dimensional NMR strategies, that will provide the critical assignments for the paramagnetically relaxed, broadened and shifted resonances. Changes in labile proton exchange rates upon mutation/chemical modification in the distal heme pocket of cyano-metmyoblobin will be used to define the interactions that allows formation of a transient ligation channel to the heme pocket. The influence of heme orientation and hydrogen bonding by an axial histidine on heme electronic structure will be elucidated for cytochrome b5, and the origins of the pKs that modulate the redox potential of cytochrome b562 will be identified. The contact shifted resonance of bound cysteines in a variety of iron-sulfur cluster electron mediators will be assigned to specific residues in the protein sequence, and the valence states of individual irons in the cluster will be interpreted on the basis of available X-ray crystal structure to elucidate the mechanism of protein control of localized iron redox potential. The heme and axial histidine resonances of high-spin ferricytochromes c' will be assigned to interpret the nature of the equilibrium pre-transition structural changes detected during reversible thermal unfolding.
