Effects of surface amino acid replacements in cytochrome c peroxidase on complex formation with cytochrome c.
Site-directed mutagenesis was employed to examine the role played by specific surface residues in the activity of cytochrome c peroxidase. The double charge, aspartic acid to lysine, point mutations were constructed at positions 37, 79, and 217 on the surface of cytochrome c peroxidase, sites purported to be within or proximal to the recognition site for cytochrome c in an electron-transfer productive complex formed by the two proteins. The resulting mutant peroxidases were examined for catalytic activity by steady-state measurements and binding affinity by two methods, fluorescence binding titration and cytochrome c affinity chromatography. The cloned peroxidases exhibit similar UV-visible spectra to the wild-type yeast protein, indicating that there are no major structural differences between the cloned peroxidases and the wild-type enzyme. The aspartic acid to lysine mutations at positions 79 and 217 exhibited similar turnover numbers and binding affinities to that seen for the "wild type-like" cloned peroxidase. The same change at position 37 caused more than a 10-fold decrease in both turnover of and binding affinity for cytochrome c. This empirical finding localizes a primary recognition region critical to the dynamic complex. Models from the literature proposing structures for the complex between peroxidase and cytochrome c are discussed in light of these findings.
Corin
A F
AF
NSF Center for Photoinduced Charge Transfer, University of Rochester, New York 14627.
McLendon
G
G
Zhang
Q
Q
Hake
R A
RA
Falvo
J
J
Lu
K S
KS
Ciccarelli
R B
RB
Holzschu
D
D
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed
D000596:Amino Acids; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D003578:Cytochrome-c Peroxidase