Influence of site-directed mutation of cytochrome b5 Phe35 on the protein's structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b(5) indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded wit a different mechanism compared with wild type cytochrome b(5) and Phe35Tyr mutant protein. Because of the decrease of side chain volume Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydropholic pocket. When urea concentration is larger that 5 mol/L, the urea molecule may compare to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b(5) and cytochrome c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b(5) with cytochrome c are: wild type K-A = 4.2 (+/-0.01) X 10(6)(mol/L)(-1), Phe35Tyr K-A = 3.7 (+/-0.01) X 10(6) (mol/L)(-1) and Phe35Leu K-A = 4.7(+/-0.01) X 10(6) (mol/L)(-1) respectively (I=1 m mol/L, pH 7.0 soldium phosphate buffer, 25 degrees C). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b(5) with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b(5) and cytochrome c.