title: Importance of a conserved phenylalanine-35 of cytochrome b5 to the protein's stability and redox potential.
abstract: Phenylalanine-35, which is a residue of the hydrophobic patch on the surface of cytochrome b5, has been mutated into Tyr35, His35 and Leu35 to elucidate the functions of the Phe35 and give further insight into the roles of the hydrophobic patch and/or aromatic network. The effects of these mutations on the heme environment, denaturation towards heating and the denaturant urea, redox potential and stability of protein were studied. The relative stability of cytochrome b5 and its mutants towards heating has the order Phe35Tyr > wild type > Phe35Leu > Phe35His in the oxidized state and wild type > Phe35Tyr > Phe35Leu > Phe35His in the reduced state. All the mutants exhibit decreased reduction potentials: Phe35Tyr -66 mV, Phe35His -51 mV and Phe35Leu -28 mV, which are more negative than that of the wild type. The order of redox potential reflects the relative stability in the oxidized and reduced states. A method of producing multiple mutants at a single site of a gene is also described for the first time.
authors.affiliation: Department of Chemistry, Fudan University, Shanghai, China.
authors.forename: P
authors.initials: P
authors.lastname: Yao
authors.forename: Y
authors.initials: Y
authors.lastname: Xie
authors.forename: Y H
authors.initials: YH
authors.lastname: Wang
authors.forename: Y L
authors.initials: YL
authors.lastname: Sun
authors.forename: Z X
authors.initials: ZX
authors.lastname: Huang
authors.forename: G T
authors.initials: GT
authors.lastname: Xiao
authors.forename: S D
authors.initials: SD
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D015786:Cytochromes b5 / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism*; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D014508:Urea
chemical_list: D010649:Phenylalanine; D014508:Urea; D015786:Cytochromes b5