Characterization of a covalently linked yeast cytochrome c-cytochrome c peroxidase complex: evidence for a single, catalytically active cytochrome c binding site on cytochrome c peroxidase.
A covalent complex between recombinant yeast iso-1-cytochrome c and recombinant yeast cytochrome c peroxidase (rCcP), in which the crystallographically defined cytochrome c binding site [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] is blocked, was synthesized via disulfide bond formation using specifically engineered cysteine residues in both yeast iso-1-cytochrome c and yeast cytochrome c peroxidase [Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580]. Previous studies on similar covalent complexes, those that block the Pelletier-Kraut crystallographic site, have demonstrated that samples of the covalent complexes have detectable activities that are significantly lower than those of wild-type yCcP, usually in the range of approximately 1-7% of that of the wild-type enzyme. Using gradient elution procedures in the purification of the engineered peroxidase, cytochrome c, and covalent complex, along with activity measurements during the purification steps, we demonstrate that the residual activity associated with the purified covalent complex is due to unreacted CcP that copurifies with the covalent complex. Within experimental error, the covalent complex that blocks the Pelletier-Kraut site has zero catalytic activity in the steady-state oxidation of exogenous yeast iso-1-ferrocytochrome c by hydrogen peroxide, demonstrating that only ferrocytochrome c bound at the Pelletier-Kraut site is oxidized during catalytic turnover.
Nakani
Siddartha
S
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA.
Viriyakul
Thanarat
T
Mitchell
Robert
R
Vitello
Lidia B
LB
Erman
James E
JE
D001665:Binding Sites; D002384:Catalysis; D003578:Cytochrome-c Peroxidase / Q000302:isolation & purification / Q000378:metabolism*; D045304:Cytochromes c / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase