Chemical shift-based constraints for solution structure determination of paramagnetic low-spin heme proteins with bis-His and His-CN axial ligands: the cases of oxidized cytochrome b(5) and Met80Ala cyano-cytochrome c

The chemical shifts of the methyl protons of protoporphyrin IX, which are readily assigned, are related to the structural features of the axial histidine ligands in heme proteins with bis-His or His-CN axial coordination (Bertini I, Luchinat C, Parigi G, Walker FA (1999) JBIC 4:515-519). In the present paper, a module is developed which transforms the chemical shifts into a pseudo-potential energy that is a function of the dihedral angles defining the orientation of the axial ligand planes. Minimization of this pseudo-potential energy, together with the energetic contributions provided by the other constraints, yields structures consistent with the heme methyl chemical shifts. Oxidized cytochrome b5 from rat and the cyanide derivative of the M80A mutant of yeast cytochrome c are used for test calculations. In the case of scarcity of NOEs for the axial ligands, owing to the presence of the paramagnetic center, the above structural constraints are shown to be quite precious. The newly refined structures are deposited in the PDB.