ABSTRACT Name: Naw May Pearl Department: Chemistry and Biochemistry Title: Cytochrome c Binding to Cytochrome c Peroxidase: Mutagenesis and Kinetic Studies Major: Chemistry Degree: Doctor of Philosophy Approved by: Date: Dissertation Director NORTHERN ILLINOIS UNIVERSITY Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT The interaction between cytochrome c peroxidase (CcP) and cytochrome c is investigated through the characterization of charge-reversal mutations on CcP. Forty- four point mutants involve converting an acidic residue to a lysine residue, while two mutants involve positive-to-negative charge reversal, R31E and K149D. The mutants are characterized by uv-visible spectroscopy. The ratios of the absorbance in the Soret band to that in the protein band near 280 nm, Asoret/A 28o, are comparable to those of wild-type enzyme, with few exceptions. Many of the charge- reversal mutations induce changes in the heme coordination, from predominantly penta-coordinate heme as found in wild-type CcP to predominantly hexa-coordinate heme in D37K, K149D, D241K, and E267K. At pH 7.5, the fraction of hexa- coordinated heme increases for all mutants, indicating a pH-dependent shift in the binding of the sixth ligand. Analyses of the circular dichroic spectra for representative samples indicate that neither single-site mutations nor changes in heme coordination produce significant changes in the secondary structure of the proteins. Determination of the rates of the hydrogen peroxide reactions from stopped- flow studies indicate that penta-coordinate mutants have bimolecular rate constants similar to the wild type enzyme, while increases in hexa-coordination produce species which are less reactive toward hydrogen peroxide. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Steady-state kinetic studies of the H 2O2 oxidation of yeast cytochrome c catalyzed by CcP and its mutants yield the maximum turnover number, Vmax/eo, and the Michaelis constant, KM. Values of Vmax/eo show a general correlation with the fraction of active enzyme, with turnover numbers decreasing with decreases in the fraction of active enzyme. The Michaelis constant, Km, correlates well with the equilibrium constant for protein-protein binding. Five mutants, R3 IE, D34K, D37K, El 18K, and E290K, show at least a twenty-four-fold increase in Km, indicating a decrease in affinity for cytochrome c in these mutants. These five mutants are clustered at or near the crystallographically determined binding site. These results indicate the presence of a single cytochrome c binding domain on the surface of CcP. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NORTHERN ILLINOIS UNIVERSITY CYTOCHROME c BINDING TO CYTOCHROME c PEROXIDASE: MUTAGENESIS AND KINETIC STUDIES A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY BY NAW MAY PEARL DEKALB, ILLINOIS DECEMBER 2007 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3301644 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3301644 Copyright 2008 by ProQuest LLC. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 E. Eisenhower Parkway PO Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Certification: In accordance with departmental and Graduate School policies, this dissertation is accepted in partial fulfillment of degree requirements. Dissertation Director Date ' a n y u s e o f m a t e r i a l c o n t a i n e d HEREIN MUST BE DULY ACKNOWLEDGED. THE AUTHOR'S PERMISSION MUST BE OBTAINED IF ANY PORTION IS TO BE PUBLISHED OR INCLUDED IN A PUBLICATION. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page LIST OF TABLES .................................................................................................................. vi LIST OF FIGURES................................................................................................................ vii LIST OF APPENDICES........................................................................................................ x Chapter I. INTRODUCTION .......................................................................................................... 1 Cytochrome c Peroxidase Catalysis, Structure, and Reactivity 4 Complex Formation between Cytochrome c and C c P ............................ 14 G o a ls.................................................................................................................. 25 R eferences......................................................................................................... 29 II. CONSTRUCTION, EXPRESSION, AND ISOLATION OF CcP CHARGE- REVERSAL MUTANTS............................................................................................. 35 O verview .......................................................................................................... 35 Experimental Procedures.............................................................................. 38 R esu lts.............................................................................................................. 42 Location of the Mutation Sites Relative to the Crystallographic Cytochrome c Binding Site..................................................................... 45 R eferences........................................................................................................ 51 III. CHARACTERIZATION OF THE CHARGE-REVERSAL MUTANTS- ELECTRONIC ABSORPTION SPECTROSCOPY ............................................. 53 Introduction...................................................................................................... 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. iv Chapter Page Experimental Methods................................................................................... 60 R esu lts................................................................................................................ 60 D iscussion........................................................................................................ 64 R eferences........................................................................................................ 76 IV. CHACTERIZATION OF THE CHARGE-REVERSAL MUTANTS- CIRCULAR DICHROISM SPECTROSCOPY ...................................................... 78 Introduction........................................................................................................ 78 Experimental Methods.................................................................................... 80 R esu lts................................................................................................................. 82 A n alysis ............................................................................................................... 85 D iscussion.......................................................................................................... 89 Conclusions........................................................................................................ 98 R eferences.......................................................................................................... 99 V. CHARACTERIZATION OF THE CHARGE-REVERSAL MUTANTS- HYDROGEN PEROXIDE REACTIVITY ................................................................ 102 Introduction......................................................................................................... 102 Experimental Methods......................................................................................104 R esu lts...................................................................................................................106 D iscussion............................................................................................................124 R eferences............................................................................................................133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V Chapter Page VI. STEADY-STATE OXIDATION OF FERROCYTOCHROME c ..................... 135 Introduction...................................................................................................... 135 Experimental Methods.................................................................................. 141 R esu lts............................................................................................................... 145 D iscussion.......................................................................................................