The main objective of this thesis project was to gain a better understanding of the protein structural factors that modulate heme reduction potentials in b-type cytochromes. Rat liver microsomal cytochrome {dollar}b\sb5{dollar} was chosen as a model system because the availability of a synthetic gene coding for the protein permits mutagenic experiments to be performed. The synthetic gene encoding for cytochrome {dollar}b\sb5{dollar} was subcloned into a high yield bacterial overexpression system that enabled isotopic enrichment of proteins. Site directed mutants of cytochrome {dollar}b\sb5{dollar} were prepared by subcloning the synthetic gene into bacteriophage DNA m 13mp18. Mutants of cytochrome {dollar}b\sb5{dollar} were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V (alanine to valine) mutation resulted in a reorientation of the H63 imidazole ring and a shift in reduction potential by {dollar}-20{dollar} mV. Structural characterization of the A67V mutant protein was achieved using homonuclear and heteronuclear NMR methods. Calculation of the orientation of the components of paramagnetic susceptibility tensor showed that the wild type and mutant proteins differed only in the orientation of the z-component. The rotation of the z-component of the susceptibility tensor is in the same direction as the rotation of the H63 imidazole ring. EPR and Near-IR data suggest that the stability of iron d-orbital energy levels in the reduced proteins may be significant in determining the reduction potential of b-type heme proteins. Multidimensional double and triple resonance NMR methods have been applied to assign the backbone and side-chain {dollar}\sp{lcub}13{rcub}{dollar}C resonances for both equilibrium conformers of ferricytochrome {dollar}b\sb5{dollar} On the basis observed NOEs and backbone {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts, the solution secondary structure of cytochrome {dollar}b\sb5{dollar} has been determined. The {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts of backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts are observed, is evaluated through calculations of the magnitude of such shifts.