Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 1992 Resonance Raman spectra of chloroperoxidase reaction intermediates Ann Marie G. Sullivan Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Chemistry Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/5610 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. College of Humanities and Sciences Virginia Commonwealth University This is to certify that the dissertation prepared by Ann Marie Sullivan entitled "Resonance Raman Spectra of Chloroperoxidase Reaction Intermediates" has been approved by her committee as satisfactory completion of the dissertation requirement for the degree of Doctor of Philosophy in Chemistry. !f-�-" • • I " I I I � •• • Dare @Ann Marie G. Sullivan 1992 All Rights Reserved RESONANCE RAMAN SPECTRA OF CHLOROPEROXIDASE REACTION INTERMEDIATES A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry at Virginia Commonwealth University. By Ann Marie G. Sullivan B.s. Chemistry, 1982 Virginia Polytechnic Institute and State University Director: Dr. James Terner Associate Professor Department of Chemistry Virginia Commonwealth University Richmond, Virginia December, 1992 11 To David and Bryan III Table of Contents Table of Tables ............................. ............................ ............................ ............... v Table of Figures ........ ............................ ................ .......................... ..................... vi Abstract............................... .......... .. .. ............ .................. ...................................... Xl CHAPTER I Historical Overview..................... .............................. .................. 1 A. Introduction ............................................................................................... 1 B. Chloroperoxidase Reaction Cycle .......... .................................. ................ 2 C. Structure of Chloroperoxidase ... .... ... .......... ...... ........ ..... .... .. .. .. .. .. .. ....... .... 4 1. Ferrous and Ferric Native Enzyme ..... .. .. ................................ .... .... ..... 7 2. Sixth Position Axial Ligands ............................................................... 14 a. Carbon-monoxide ...... .. ....... ................. ........... .... .. .. .. .. ...... .... .. .... .... 15 b. Halides and other Ligands ............................. ...... ........................... 17 c. Dioxygen .. .................. ................ ...................................................... 21 D. Reaction Intermediates of Chloroperoxidase ...... ... .. .. .... .. .. .. .... .... .. .. ..... .. 23 1. Compound I ..... ..... ...... .. ...................... ................................................. 24 2. Compound II.. .................. .................... ................................................ 26 E. Summary ................... .. .................. .................. .......................... ................. 26 CHAPTER II Raman and Resonance Raman Scattering ... ... ...... .. ... .... .. .... ... 28 A. Introduction ......... ........... .................. .................. ...... ...................... ...... ...... 28 B. Raman Scattering ...................................................................................... 29 C. Classical Raman Theory ......... ... .................... ........................................... 32 D. Quantum Mechanical Treatment of Raman Scattering .. .... .. ... ....... ......... 34 E. Resonance Raman Enhancement. .............. .......................................... .... 39 F. Depolarization Ratio...... ..... .. .. ..... .. .. ..... ...... ... ..... .......... .... .... .. .. ...... .. .. .. ... .. 42 G. Heme Scattering.... ... .. .. .. ..... .. ....... .. .. ... .. ..... .. .. .. ... .. .. .. .... ... ..... .... ... .. .. .. .... ... 46 CHAPTER ill Experimental.............................................................................. 58 A. Introduction .. .. .. ... .. .. .. ... ..... ... .. ....... .. ............ .. .. ..... .. .. .. ... .. .... .. .. .. .. ... ..... ..... 58 B. Production of Chloroperoxidase from Ca/dariomycesjumago . ....... .. .. .. 59 1. Slant Cultures ..... .................. .............. ........................ ........ ................. 59 2. Starter Flasks ........................ .. ..... .. ... .. .. .. ... .. ... .. .. .. .. .. .. .. ... ... ........... .... 59 3. Culture Flasks. ... ... .. ..... .. ..... ....... ..... .. ......... ... .. ... ...... .. .. .. .......... ....... .. ... 60 IV 4. Large Scale Production in Carboys ..................................................... 61 C. Assay of Enzyme Activity ....................................................................... 67 D. Isolation and Purification of Chloroperoxidase ..................................... .. 68 1. Ethanol Precipitation ........................................................................... 68 2. Chromatography ................................................................................. 70 E. Instrumentation ......................................................................................... 73 F. Sample preparation .................................................................................... 78 1. Complexes ........................................................................................... 78 2. Compound I ........................................................................................ 79 3. Compound II ........................................................................................ 82 CHAPTER IV Results and Discussion ............................................................ 86 A. Introduction .............................................................................................. 86 B. Region between BOO and 1700 cm·l ..................................................... .. 88 1. Ferric and Ferrous Enzyme ................................................................ 89 2. Reaction Intennediates ........................................................................ 99 a. Compound I ................................................................................... 99 b. Compound II .................................................................................. 109 C. Region between 600 and BOO cm·l ........................................................ 120 1. Compound I .......................................................................................... 120 2. Compound II ........................................................................................ 132 D. Complexes ................................................................................................. 141 1. Ferric Low Spin Complexes .............................................................. .. 141 2. Ferric High Spin Complexes .............................................................. 142 3. Ferrous Complexes ............................................................................. 168 CHAPTER V Conclusions and Future Directions ......................................... 181 BffiLIOGRAPHY ................................................................................................ 184 v Table of Tables Table ITLI. Growth medium for Ca/dariomyces jumago ............. ......... ..... .. 60 Table IV.1. Coordination numbers, spin states and oxidation states of various form of native, ferric and ferrous, reaction intermediates and complexes of chloroperoxidase ... .......... ................ ......................................... 87 Table IV.2. Resonance Raman band assignments given in cm·' for the region between 1000 and 1700 em·' for the ferric and fe rrous forms of chloroperoxidase compared to nickel octaethylporphyrin ............................ 100 Table IV.3. Resonance Raman band assignments given in cm·' for the region between 1000 and 1700 cm·' of ferrous enzyme, ferric enzyme, compounds I and II of chloroperoxidase ..... .................................. .... ............ 119 Table IV.4 Resonance Raman band assignments in cm·' for fe rric, ferrous, compounds I and IT of chloroperoxidase in the region between 500 and 1300 cm·' .......................................................................................... 140 Table IV.5. Resonance Raman band assignments for the ferric complexes of chloroperoxidase given in cm·' .......................................... ............... .. .... 143 Table IV.6. Resonance Raman band assignments for the ferrous complexes of chloroperoxidase given in cm·' .............. .............................. .. 170 Vl Table of Figures Figure 1. 1. Catalytic reaction cycles of chloroperoxidase and the postulated structures of the intennediates ......... ........................ ................ 5 Figure IT.l. Schematic representation of Rayleigh, Stokes, anti-Stokes and resonance Raman scattering processes ............ ......................................... 30 Figure IT.2. Spaced fixed coordinate system fo r the polarizationof Raman lines and the detennination of the depolarization ratio for Raman bands ..........................................................................................................