Ying Song

Publication Date


Document Type


First Advisor

Erman, James E.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Chemistry


Peroxidase; Cytochromes; Cytochrome c; Chemical kinetics; Yeast


The reduction of yeast cytochrome c peroxidase compound I (CcP-I) by horse heart ferrocytochrome c (C2+) at 10 mM ionic strength is investigated by the stopped-flow kinetic analysis method. Two important instrument parameters of the APL Stopped-flow Spectroflurometer used in this study, the dead time of the instrument, td, and the reaction time zero on data acquisition scale, to, are determined. Due to the dead time of the instrument, 100% observation of a reaction is not possible. At a given td, the percent reaction observable decreases exponentially with increasing first order rate constant. Various absorbance change terms are defined as a measure of the reaction amplitude. Using these definitions, the stopped-flow instrument is calibrated and the accuracy of its absorbance measurement is validated against published extinction coefficient data. A series of kinetic experiments are studied using kinetic scan and kinetic titration techniques. The rate constants and reaction amplitudes are investigated. The rate constants of the two observed kinetic phases are determined using Global Integration (Glint) analysis for the pseudo first-order reactions with limiting [CcP I] and ten times excess [C2+]; reaction amplitudes are analyzed for kinetic titration experiments in which the [C2+] is decreased from ten times of [CcP-I] to one fourth of [CcP-I], Previous stopped-flow studies in our group have suggested that at low ionic strength, the fast kinetic phase observed in the reaction between CcP-I and C2+ corresponds to the reduction of the radical site in CcP-I to generate CcP-IIr and the slow kinetic phase observed is the reduction of the Fe(IV) site in CcP-IIr to generate the native enzyme. The concentration dependence of the fast and slow kinetic phases observed in this study agrees with the correlations previously found. The studies of reaction amplitudes in this work have revealed that the percentage of enzyme that reacts via the pathway of the intermediate CcP-IIr decreases with increasing reactant concentration and reduction of the first oxidizing equivalent in CcP-I is too fast to observe. This suggests that the previous mechanism is not complete, especially when the substrate concentration is high. A minimal partitioning mechanism, which allows the partitioning of CcP-I into two different reaction pathways via the enzyme intermediate CcP-IIr and CcP-IIr, is investigated by detailed kinetic analysis and demonstrates improvement in our understanding of the reaction at low ionic strength. An improved partitioning mechanism, which postulates two different reacting forms of CcP-I (or the CcP-I:C2+ complex), is further studied. It is found that this improved partitioning mechanism provides better understanding of the complicated observations of the reaction at low ionic strength and it is consistent with all kinetic data obtained to date.


Includes bibliographical references (pages [106]-108)


viii, 117 pages




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