CHARACTERIZATION OF A SITE-DIRECTED MUTANT OF CYTOCHROME B(5) DESIGNED TO ALTER AXIAL IMIDAZOLE LIGAND PLANE ORIENTATION

Mutants of cytochrome bs were designed to achieve reorientation of ind ividual axial imidazole ligands. The orientation of the axial ligand p lanes is thought to modulate the reduction potential of bis-(imidazole ) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residu e, in the sterically hindered hydrophobic heme binding pocket. Solutio n structures of mutant and wild-type proteins in the region of the mut ation were calculated using restraints obtained from H-1 and N-15 2D h omonuclear and heteronuclear NMR spectra and H-1-N-15 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the ref inement of both structures. Average local rmsd for 20 refined structur es was 0.30 Angstrom for the wild-type structure and 0.38 Angstrom for the A67V mutant. The transfer of amide proton resonance assignments f rom wild-type to the mutant protein was achieved through overlays of N -15-H-1 heteronuclear correlation spectra of the reduced proteins. Sid e chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z- component of the susceptibility tensor calculated for the mutant prote in differs by 17 degrees for the A-form and by 11 degrees for the B-fo rm from the orientation calculated for the wild-type protein. The rota tion of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.