Reduction potentials of wild type and Phe35Tyr, Phe35His, Phe35Leu mutants of cytochrome b(5) were studied using a spectroelectrochemical method, and a 2D H-1 NMR study of a Phe35Tyr mutant was also performed to investigate the structural influence of Phe35 on the redox potential of cytochrome b(5). All the Phe35Tyr, Phe35Leu and Phe35His mutant proteins exhibit a decreased redox potential in the order Phe35Tyr < Phe35His < Phe35Leu < wild type. The difference in E-o＇ is about 70 mV between Phe35Tyr and the wild type protein. Spectroelectrochemistry and structural studies demonstrated that the mutation at the Phe35 site perturbed the hydrophobicity of the heme pocket of cytochrome b(5) seriously, leading to a considerable shift of the redox potential. The wild type and mutant cytochrome b(5) showed increased odor potentials with increase of ionic strength, but the redox potential did not alter significantly with different mediators such as [Ru(NH3)(6)]Cl-3, K-3[Fe(CN)(6)], [Ru(NH3)(5)BzIm]Cl-3 or cytochrome c present in the solution.