Structures of Cytochrome b5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c

Glu44, Glu48, Glu56 and Asp60 are the negatively charged residues located at the molecular surface of cytochrome b 5. Two mutants of cytochrome b 5 were prepared, in which two or all of these four residues were mutated to alanines. The mutations give rise to slightly positive shifts of the redox potentials of cytochrome b 5 and obvious decrease of the cytochrome b 5-cytochrome c binding constants and electron transfer rates. The crystal structures of the two mutants were determined at 0.18 nm resolution, showing no alteration in overall structures and exhibiting slight changes in the local conformations around the mutation sites as compared with the wild-type protein. Based on the crystal structure of the quadruple-site mutant, a model for the binding of this mutant with cytochrome c is proposed, which involves the salt bridges from Glu37, Glu38 and heme propionate of cytochrome b 5 to three lysines of cytochrome c and can well account for the properties and behaviors of this mutant.