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MOLECULAR TOPOGRAPHY AND FUNCTION OF PERIDININ IN THE PERIDININ-CHLOROPHYLL a-PROTEIN COMPLEX by SIVARAMAKRISHNA PRASAD KOKA, B.S., M.A. A DISSERTATION IN CHEMISTRY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved _ Accepted December, 1977 Ho, 7^ ACKNOWLEDGMENTS I am extremely grateful and deeply indebted to my professor. Dr. Pill-Soon Song, for his constant guidance, patience and encouragement throughout my studies for the Ph.D. degree. I am thankful to my fellow graduate stu dents for their help and cooperation. The efficient typing work of Mrs. Evelyn Gaffga is well acknowledged. I continue to be grateful to my former professors, Dr. Robert R. Kuntz (University of Missouri) and Dr. Richard D. Doepker (University of Miami), without whose encourage ment I would not be where I am now. Last but not the least, my indebtedness persists forever to my parents and family, without whose help I would not have been able to pursue higher studies. XI TABLE OF CONTENTS ACKNOWLEDGrffiNTS ii LIST OF TABLES vi LIST OF ILLUSTRATIONS vii I. INTRODUCTION AND STATEI/IENT OF PROBLEM. ... 1 Introduction 1 Statement of Problem 2 II. LITERATURE REVIEW 3 Carotenoid-Chlorophyll Complexes . 3 Energy Transfer from Carotenoid to Chlorophyll 9 Exciton Interactions 12 Role of Carotenoid As A Photoreceptor 13 Singlet Oxygen-Induced Photo- oxidation and Its Inhibition ... 14 Singlet-Triplet Transitions of Photosensitizer Dyes 21 III. MATERIALS AND METHODS 22 Materials 22 Methods 24 Theoretical Section 24 Critical Distance Calculation. 24 iii IV Intermolecular Distance Cal culation 25 Polarization by Photoselection. 26 Experimental Section 28 Corrected Emission and Excita tion Measurements 28 Fluorescence Quenching 34 High Resolution Emission and Excitation Measurements 34 Low Temperature Measurements. 37 Circular Dichroism 38 Absorption Spectra 39 Fluorescence Lifetimes 39 Temperature Dependence of PCP and Chlorophyll a Fluorescence. 40 Dynamic Depolarization 40 Photo-irradiation 44 Actinometry 45 IV. RESULTS 50 PCP (Glenodinium sp.) - Absorption and Polarization ... 50 PCP (Glenodinium sp.) - Fluor- escence Excitation 50 PCP (Glenodinium sp.) - Circular Dichroism 51 PCP (Glenodinium sp.) - Fluorescence Emission 60 PCP (G. polyedra) - Absorp tion and Polarization 71 PCP (G. polyedra) - Fluores cence Excitation 71 PCP (G. polyedra) - Circular Dichroism 78 PCP (A. rhyncocephaleum) - Absorption and Polarization. ... 78 PCP (A. rhyncocephaleum) - Fluorescence Excitation 79 PCP (A. rhyncocephaleum) - Circular Dichroism 84 PCP (A. carterae) - ..Absorp tion, Polarization and Circular Dichroism 84 PCP (A. carterae) - Fluor escence Excitation 89 PCP (A. carterae) - Fluor escence Emission and .Fluor escence Lifetimes 89 Dynamic Depolarization 98 Fluorescence Quenching 98 "Reconstitution" of PCP 105 Temperature Dependence of PCP Fluorescence 110 Photobleaching of Chlorophyll a^ . 113 Rose Bengal Sensitization and Singlet Oxygen Quenching 122 V. DISCUSSION 157 VI. CONCLUSIONS 185 LIST OF REFERENCES 189 LIST OF TABLES Table Page 1. Intensity of the Bausch & Lomb monochromator Xenon lamp at 557 nm 47 2. The fluorescence maxima and lifetimes of PCPs. 96 3. The calculated values of In ^j~V.^^ with re ciprocal of absolute temperature^'^for PCP (Glenodinium sp.) 118 4. Photobleaching of chlorophyll a in solution and in PCP complex 123 5. Photobleaching of PCP in D20 125 6. Quantum yields of photooxidation of chloro phyll a in the absence of singlet oxygen quencher 129 7. Quantum yields of photooxidation of chloro phyll a in the presence of B-carotene. 133 8. Quantum yields of photooxidation of chloro phyll a in the presence of benzoquinone (in ethanol-benzene) 137 9. Quantum yields of photooxidation of chloro phyll a in the presence of menadione 141 10. Quantum yields of photooxidation of chloro phyll a_ in the presence of a-tocopherol. 145 11. Quantiom yields of photooxidation of chloro phyll a^ in the presence of benzoquinone (in ethanol) 149 12. Quantum yields of photooxidation of chloro phyll a in the presence of DABCO 153 13. Photooxidation of chlorophyll a-singlet oxygen quenching rate constants. • 179 VI LIST OF ILLUSTRATIONS Figure Page 1. Structure of peridinin 4 2. Isolation and purification of PCP complexes 7 3. The state of oxygen molecule and their electronic configurations 16 4. Optical layout of the Perkin-Elmer MPF-3 spectrophotometer. .... 30 5. Block diagram of Perkin-Elmer spectro photometer with Hitachi corrected spectra accessory 32 6. Block diagram with optical layout for high resolution spectrometer 35 7. Block diagram of the Phase-Modulation cross-correlation spectrofluorometer. ... 41 8. Absorption spectra of native and de natured PCP from Glenodinium sp 52 9. Fluorescence excitation polarization and absorption spectra of PCP (Glenodinium sp.) 54 10. Corrected fluorescence excitation spec trum of PCP (Glenodinium sp.) at room temperature recorded on Perkin-Elmer spectrof luorometer 56 11. Fluorescence excitation spectra of de natured and native PCP (Glenodinium sp.) recorded on high-resolution spectrofluor ometer 58 Vll Vlll Figure Page 12. Absorption and fluorescence excitation polarization spectra of chlorophyll a in ethanol 61 13. Circular dichroism spectrum of PCP (Glenodinium sp.) 63 14. Circular dichroism spectra of de natured PCP (Glenodinium sp.) and chloro phyll a 65 15. Polarized fluorescence excitation and absorption spectra of denatured PCP (Glenodinium sp.) 67 16. Fluorescence emission spectrum of PCP (Glenodinium sp .) 69 17. Absorption and polarized fluorescence ex citation spectra of PCP (G. polyedra) ... 72 18. Absorption and polarized fluorescence ex citation spectra of PCP (A. rhyncocephaleum) 74 19. Corrected fluorescence excitation spectra of (a) PCP from G. polyedra and (b) A. rhyncocephaleum. 76 20. Fluorescence excitation spectra of PCPs from Glenodinium sp., G. polyedra and A. rhyncocephaleum 80 21. Circular dichroism spectra of PCP from (a) G. polyedra and (b) A. rhyncocephaleum 82 22. Absorption spectra of PCPs from A. carterae and G. polyedra and fluorescence excitation polaFization spectrum of PCP from A. carterae 85 23. Absorption spectra of peridinin in ethanol at room temperature and 77K 87 IX Figure Page 24. Circular dichroism spectra of PCP from (a) A. carterae and (b) A. rhyncocephaleum. 90 25. Fluorescence excitation spectra of PCPs from A. carterae, A. rhyncocephaleum and G. polyedra 92 26. Fluorescence emission spectra of PCPs from A. carterae, A. rhyncocephaleum and Glenodinium sp 94 27. Fluorescence spectra of chlorophyll a_ as a function of potassium, iodide 99 28. Stern-Volmer plot of fluorescence quench ing of chlorophyll a—fluorescence inten sities vs. KI concentration 101 29. Stern-Volmer plot of fluorescence quench ing of chlorophyll a—fluorescence lifetimes vs. KI concentration 103 30. Absorption spectra of PCP as a function of acetone concentration 106 31. Effects of acetone on the CD spectrum of PCP 108 32. CD spectrum of PCP after "reconstitution" from 30 per cent acetone Ill 33. Fluorescence emission of PCP as a function of temperature 114 34. Plot of In A{1/TV against reciprocal temperature for PCP 116 35. CD spectra of PCP (Glenodinium sp.) as a function of temperature 120 36. Stern-Volmer plot of photooxidation of chloro phyll a in the absence of singlet oxygen quencher 131 X Figure Page 37. Stern-Volmer plot of inhibition o-f chlorophyll a^ photooxidation by B~ carotene 135 38. Stern-Volmer plot of inhibition of chlorophyll a photooxidation by benzoquinone (in ethanol-benzene) 139 39. Stern-Volmer plot of inhibition of chlorophyll a photooxidation by menadione . 143 40. Stern-Volmer plot of inhibition of chlorophyll a^ photooxidation by a- tocopherol 147 41. Stern-Volmer plot of inhibitions of chlorophyll a photooxidation by benzoquine (in ethanol) 151 42. Stern-Volmer plot of inhibition of chlorophyll a photooxidation by DABCO .... 155 43. Absorption and fluorescence (hypothetical) spectra of peridinin in ethanol 161 44. Absorption spectrum of chlorophyll a nor malized with respect to the peridinin fluorescence spectrum 163 45. Probable molecular arrangement of PCP complex 167 46. Corrected fluorescence spectrum of PCP (Glenodinium sp.) protein 172 CHAPTER I INTRODUCTION AND STATEMENT OF PROBLEM Introduction The isolation of tiie photosynthetic light-harvesting pigment complex, peridinin-chlorophyll a-protein (PCP) from marine dinoflagellate algae (1-5) opened the doors to under standing how the light-harvesting carotenoid, peridinin, transfers its energy to chlorophyll a in the complex. Up to now such an understanding has not been possible due to the non-fluorescent nature of the carotenoids and their extremely short excited state lifetimes (6). Carotenoids absorb light in the wavelength region where chlorophyll does not absorb, thus efficiently utilizing the light in the entire visible range for photosynthesis by the energy transfer. However in the absence of protein no energy is transferred from caro tenoid to chlorophyll such as in solution (6), and the PCP complex, unlike other carotenoid-chlorophyll-protein com plexes, containing a highly water-soluble protein, provides an excellent model to study the energy transfer mechanism. The carotenoid, in addition to its role as light- harvesting pigment is thought to be the protector of chloro phyll a from singlet oxygen induced damage. The low-lying 1 triplet level of carotenoid quenches the singlet oxygen

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