SPECTROELECTROCHEMICAL STUDIES OF CYTOCHROME B(5) PHE35 MUTANTS

Reduction potentials of wild type and Phe35Tyr, Phe35His, Phe35Leu mut ants of cytochrome b(5) were studied using a spectroelectrochemical me thod, and a 2D H-1 NMR study of a Phe35Tyr mutant was also performed t o investigate the structural influence of Phe35 on the redox potential of cytochrome b(5). All the Phe35Tyr, Phe35Leu and Phe35His mutant pr oteins exhibit a decreased redox potential in the order Phe35Tyr < Phe 35His < Phe35Leu < wild type. The difference in E-o' is about 70 mV be tween Phe35Tyr and the wild type protein. Spectroelectrochemistry and structural studies demonstrated that the mutation at the Phe35 site pe rturbed the hydrophobicity of the heme pocket of cytochrome b(5) serio usly, 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 sig nificantly with different mediators such as [Ru(NH3)(6)]Cl-3, K-3[Fe(C N)(6)], [Ru(NH3)(5)BzIm]Cl-3 or cytochrome c present in the solution. (C) 1998 Elsevier Science S.A. All rights reserved.