A complete mechanism for steady-state oxidation of yeast cytochrome c by yeast cytochrome c peroxidase.
Steady-state oxidation of yeast cytochrome c (yCc) was monitored as a function of ionic strength (mu) for mutants of a cloned cytochrome c peroxidase [CcP(MI)]. The data are best interpreted in the context of a two binding site model, where the affinity of the two sites for yCc differs by approximately 1000-fold and rapid intracomplex electron transfer (ET) occurs only at the high-affinity site identified in the crystal structure. At low mu, catalysis is apparently limited by the rate of yCc dissociation from the reactive high-affinity site (koff). Binding of yCc at the low-affinity site increases koff and therefore increases the rate of catalysis. Mutations at the high-affinity site also increase the rate of catalysis by the 1:1 CcP(MI):yCc complex by increasing koff. Mutations at residues that interact strongly with yCc at the high-affinity site (Asp 34, Glu 290, and Ala 193) cause the greatest increase in koff (25-38-fold at mu = 20 mM). Mutations at residues that interact less strongly with yCc (Glu 32 and Glu 291) cause smaller increases in koff (10- and 3-fold, respectively, at mu = 20 mM). The results provide additional evidence that the high-affinity site formed in solution is similar to the one identified in the crystal structure and that yCc dissociation from this site limits enzyme turnover at low ionic strength. Numerical integration simulations show that the model accurately predicts enzyme turnover rates at the high-affinity site, using published rate constants for the elementary reaction steps.
Miller
M A
MA
Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla 92093-0506, USA.
D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D004579:Electron Transport; D005656:Fungal Proteins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology* / Q000378:metabolism*
D003574:Cytochrome c Group; D005656:Fungal Proteins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase