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University Microfilms International 300 N. ZEEB RD„ ANN ARBOR. Ml 48106 8128972
Burkey, Kent Oliver
A CHEMICAL MODIFICATION STUDY OF THE MECHANISM FOR CATION REGULATION OF PHOTOSYSTEM I PARTICLE ACTIVITY
The Ohio State University PH.D. 1981
University Microfilms international 300 N. Zeeb Road, Ann Arbor, M I 48106
Copyright 1981 t>y Burkey, Kent Oliver All Rights Reserved A CHEMICAL MODIFICATION STUDY OF THE MECHANISM FOR
CATION REGULATION OF PHOTOSYSTEM I PARTICLE ACTIVITY
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Kent Oliver Burkey, B.A.
The Ohio State University
1981
Reading Committee:
Dr. Elizabeth L. Gross Dr. Robert T. Ross Adviser Dr. Garfield P. Royer Department of Biochemistry Acknowledgments
I would like to thank my committee members, and es
pecially Dr. Gross, for the leadership they have provided
the last four years. I also thank Mr. Dale Hershberger, Dr.
A.G. Marshall, and Dr. G.E. Means for their help with vari
ous aspects of this project. I would also like to ac
knowledge the financial support that has been generously
provided by the Department of Biochemistry and the National
Science Foundation.
I would like to express my appreciation for the many
friends I have met while at Ohio State. Their encouragement and support are the basis for many warm memories as well as enthusiasm toward future endeavors. Finally, I would like
to dedicate this work to Susan for her love and understand ing and for helping to keep life in the proper perspective.
ii VITA
November I, 1955 Born - Greeneville, Tennessee 1973 Graduate, West Greene High School, Mosheim, Tennessee 1977 B.A. (Physical Science), Warren Wilson College, Swannanoa, North Carolina. 1977-1981 Graduate Teaching and Research Associate, Department of Biochemistry, The Ohio State University.
Publications
1. Abdella, P.M., Burkey, K., and Gross, E.L. "Immobiliza tion and Chemical Modification of Photosystem I", Biophysical Journal 2J5, 146a (1979).
2. Gross, E.L., Abdella, P.M., and Burkey, K. "The Effects of Cations, Chemical Modification, and Immobilization on the Rate of P700+ Recovery Using Plastocyanin as an Electron Donor", Plant Physiol, (supplement) 63, 54 (1979).
3. Burkey, K.O., and Gross, E.L. "Chemical Modification of Spinach Plastocyanin" Federation Proceedings 39, 1799 (1980). —
4. Burkey, K.O., and Gross, E.L. "Chemical Modification of Spinach Plastocyanin" Plant Physiol, (supplement) 65, 11 (1980). —
5. Burkey, K.O, and Gross, E.L. "Use of Chemical Modifica tion to Study the Relationship between Activity and Net Protein Charge of The Photosystem I Core Complex", Bio chemistry 20, 2961-2967 (1981),
6. Burkey, K.O, and Gross E.L. "The Effect of Carboxyl Group Modification on the Redox Properties and Electron Donation Capability of Spinach Plastocyanin", Bio chemistry, in press. Fields of Study
Major Field: Biochemistry
Studies in Molecular Photobiology. Professor E.L. Gross Studies in Molecular Biophysics. Professor J.Y, Cassim Studies in Protein Structure and X-ray Crystallography. Professor L.J. Berliner Table of Contents
Page
Acknowledgments...... ii
Vi t a ...... iii
List of Tables...... viii
List of Figures...... ix
Abbreviations xi
Introduction 1 The Chloroplast...... 1 Electron Transport...... 5 Pigment-Protein Complexes...... 10 Plastocyanin...... 12 Cation Effects on Chloroplast Membranes...... 14 Cation Effects on Subchloroplast Particles...... 18 Statement of Problem...... 19
Materials and Methods...... 24 Isolation of PSI Particles...... 24 Chlorophyll Determination...... 26 isolation of Spinach Plastocyanin...... 27 Determination of Plastocyanin Concentrations...... 31 Diphenylcarbazone Disproportionation 3 2 Determination of P700 Content in PSI Particles...... 33 Kinetics of P700'*' Reduction...... 34 Kinetics of P700 Oxidation-...... 36 Kinetics of Cytochrome f Photooxidation...... 37 Absorption Spectra ...... 38 Discontinuous Polyacrylamide Gel Electrophoresis of Plastocyanin...... 39 Chemical Modification of PSI Particles...... 42 Gel Filtration Chromatography of PSI Particles...... 46 Determination of the Isoelectric Profile for Control and Modified PSI Particles...... 46 Chemical Modification of Plastocyanin...... 47 Determination of the Redox Midpoint Potential for Con trol and Modified Plastocyanin...... 49 Tryptic Digestion of Modified Plastocyanin...... 50
v Page
Elution of Plastocyanin Peptides from Whatman 3MM Paper...... 55 Identification of Plastocyanin Peptides by Amino Acid Analysis...... 55 Separation of Modified Plastocyanin Species by DEAE- Sephadex Chromatography...... 56 Proton NMR of Control and Modified Plastocyanin. 58 Materials...... 59
Results and Discussion...... 61 Isolation of PSI Particles...... 61 Chemical Modification of PSI Particles Using Ethylenediamine or Glycine Ethyl Ester as a Nucleophile...... 63 Determination of the Optimal Conditions for the Chemical Modification of PSI Particles by Ethylenediamine...... 66 Storage of EDA-PSI...... 74 The Effect of l-ethyl-3- (3-dimethylaminopropyl) Carbodiimide on PSI Particles...... 74 Comparison of the Visible Absorption Spectra of Isolated PSI Particles with EDA-PSI and GEE-PSI.. 82 The Effect of Chemical Modification on the Net Charge of PSI Particles...... 82 Evidence for Two Types of Monomeric Units in PSI Particles...... 88 The Effect of Chemical Modification on the Molecular Weight of the PSI Complex...... 90 The Light and Dark Processes of PSI Particles...... 95 The Effect of Mg+2 an vi Page The Effect of Chemical Modification on the Oxidation- Reduction Potential of Plastocyanin...... 141 The Effect of Chemically Modified Plastocyanin on Cytochrome f Photooxidation in PSI Particles...... 149 Identification of Modified Plastocyanin Peptides...... 156 The Physiological Role of the Modified Plastocyanin Amino Acids...... 164 NMR of Control and Modified Plastocyanin...... 172 Conclusions...... -...... -...... 180 References...... 189 List of Tables Table Page 1 The Protocol for Discontinuous Buffer SDS Poly acrylamide Gels 40 2 The Protocol for Discontinuous Buffer Poly acrylamide Gels ...... 43 3 Properties of the Isolated PSI Particles ...... 62 4 The Effect of Modification of PSI Particles by Various Nucleophiles ...... 81 5 Determination of Ethylenediamine Incorporations into PSI Particles...... 89 + 2 6 The Effect of Mg and Chemical Modification on DPCN Disproportionation by PSI Particles ...... 97 7 Electron Donation to F700+ in EDA-PSI ...... 104 8 Electron Donation to P700+ in GEE-PSI ...... 105 9 The Effect of Chemical Modification on the Cyto chrome Content of PSI Particles...... H 3 10 Incorporation of Ethylenediamine in the Modified Plastocyanin Species ...... 129 11 The Effect of MgCl2 and Chemical Modification on Plastocyanin Donation to P700 ...... 132 12 P700+ Reduction by The Separated Modified Plasto cyanin Species...... I3® 13 The Effect of Sodium Chloride on the Oxidation- Reduction Potential of Modified Plastocyanin .... 147 14 The Redox Midpoint Potential of the Separated Modified Plastocyanin Species ...... 148 T5 Identification of Peptides from Modified Plasto cyanin ...... 155 16 Comparison of Radioactivity Distribution in Modified Plastocyanin Peptides ...... 171 viii List of Figures Figure Page 1 A Schematic representation of a chloroplast ...... 3 2 A schematic representation of the Z-scheme for electron transport...... 7 3 Chemical modification of protein carboxyl groups with a water-soluble carbodiimide and ethylenediamine ...... 21 4 Comparison of the light-induced P700 assay for con trol PSI particles, EDA-PSI, and GEE-PSI ...... 64 5 The concentration dependence of ethylenediamine on the modification reaction ...... 67 6 The concentration dependence of EDC on the modifi cation of PSI particles .□...... 69 7 The pH profile for the modification of PSI p a r t i c l e s ...... 72 8 Time course of the modification of PSI particles.. 75 9 The effect of storage at 4°C on the activity of EDA-PSI ...... 77 10 Visible absorption spectra of PSI particles before and after chemical modification ...... 83 11 Determination of the isoelectric pH for PSI par ticles before and after chemical modification .... 85 12 Gel filtration of PSI particles before and after chemical modification ...... 93 13 The effect of chemical modification on P700 oxidation in PSI particles ...... 100 14 The effect of MgCl- on electron donation by plasto cyanin to P700+ in PSI particles ..... 109 15 Absorption spectrum of oxidized plastocyanin ..... 119 ix Page 16 Polyacrylamide gel electrophoresis of chemically modified and native spinach plastocyanin...... 123 17 Separation of modified plastocyanin species by ion exchange chromatography on DEAE-Sephadex...... 126 18 Comparison of MgCl^ and chemical modification on electron donation from plastocyanin to P700+__#^^ 133 19 The effect of MgCl^ on the ability of modified plastocyanin to donate electrons to P700+'...... 135 20 The effect of chemical modification on the redox midpoint potential of plastocyanin...... 142 21 Cytochrome f photooxidation in PSI particles...... 150 22 The primary structure of spinach plastocyanin...... 157 23 Removal of urea from modified plastocyanin pep tides after trypsin hydrolysis...... 160 24 Schematic representation of the peptide map of modified plastocyanin...... 162 25 The amino acid sequence of spinach plastocyanin projected onto the three dimensional structure of poplar plastocyanin •••.•»•••■•••■•••.«••••••••••••• 167 26 The aromatic region of the proton NMR spectra for reduced control and modified plastocyanin...... 174 27 The proton NMR spectra of the reduced forms of con trol and modified plastocyanin...... 177 x ABBREVIATIONS 1. 14c carbon-14 2. chi chlorophyll 3. cpm counts per minute 4. cyt cytochrome 5. DCIP 2,6-dichlorophenol indophenol 6. DEAE diethylaminoethyl 7. DPCN diphenylcarbazone 8. dpm decompositions per minute 9. EDA-PSI PSI particles modified with ethylenediamine. 10. EDC l-ethyl-3- (3-dimethylaminopropyl) carbodiimide 11. EDTA ethylenediamine-tetra-acetic acid 12. GEE-PSI PSI particles modified with glycine ethyl ester 13. I' light intensity 14 * KD rate constant for dark electron transport 15. K t rate constant for light processes J _ r 16. K m apparent binding constant 17. LHC light-harvesting chlorophyll protein 18. ox oxidized 19. P680 reaction center of photosystem II 20. P700 reaction center of photosystem I 21. P700+ oxidized P700 22. POPOP 1,4-bis-2-(5-phenyloxazolyl)-benzene XI 23. PPO 2,5-diphenyloxazole 24. PSI photosystem I 25. PSII photosystem II 26. red reduced 27. SDS sodium dodecyl sulfate 28. TEMED tetramethylethylenediamine 29. TMPD N ,N ,N 1, N1-tetramethy1-p- phenylenediamine 30. Tris tris-(hydroxymethyl) aminomethane extrapolated maximum rate 31. V,max xii Introduction Photosynthesis is the process by which green plants make carbohydrates from carbon dioxide and water using visible light as the energy source. Oxygen is evolved as a byproduct. The net result of the complicated photo synthetic process can be described by the following basic equation: H20 + C02 + Light ---- :------? (CH20) + 0 2 The many reactions of photosynthesis can be di vided into two general categories. The first category is comprised of a series of enzymatically catalyzed reactions in which'C02 is incorporated into stable or ganic compounds (10). The second category consists of a series of oxidation-reduction reactions which are driven by light. These light-dependent reactions result in the oxidation of water to molecular oxygen, which generates electrons that are used to produce NADPH and ATP. The Chloroplast The photosynthetic processes in higher plants take place within a subcellular organelle called a chloroplast (Figure 1)- The contents of a chloroplast are separated from the cytosol of the cell by two envelope membranes. These membranes control the transport of metabolites by either diffusion or by specific translocators (49) . The envelope membranes are also the site of enzymatic reac tions such as those involved in galactolipid biosynthesis (67). The envelope membranes may also function in the biosynthesis of the thylakoid membranes (23). Inside the envelope membranes, there exists a net work of thylakoid membrane vesicles which separate two aqueous phases. The large aqueous phase between the envelope and the thylakoid membranes is called the stroma. The stroma contains soluble enzymes and metabolites in cluding those required in CC^ fixation as well as RNA, DNA, and ribosomes (23). The stroma also contains dis crete particles such as starch granules or lipid-con- taining bodies called plastoglobuli, which consist of plastoquinones and pigment precursors (23). The second aqueous phase is called the loculus and is located in side the thylakoid membrane vesicles. The loculus may serve as a reservoir for the protons generated by the light dependent processes of photosynthesis (8). The thylakoid membranes consist of approximately half protein and half lipid (75). Chloroplast lipids Figure 1. A Schematic Representation of a Chloroplast THYLAKOID MEMBRANES LOCULUS STROMA GRANA LAMELLAE STACKS ENVELOPE MEMBRANE Figure 1 consist mainly of mono- and digalactosyldiglycerides (45%) and pigments (26%) with the remaining lipid con sisting of phospholipids, sulpholipids, and sterol glycosides (67). The proteins of the thylakoid mem brane are responsible for all of the light-dependent reactions of photosynthesis. These include proteins complexed with pigment which collect and utilize light energy, as well as other proteins which are involved in oxygen evolution, electron transport, ATP synthesis, and NADP reduction. Regions of the thylakoid membranes are known to be involved in cation regulated interactions which re sult in the formation of stacks of membrane vesicles called grana (23) . Membranes called stroma lamellae form a continuous connection between vesicles within a single grana stack as well as between separate grana stacks. The regulation of this membrane organization may affect the function of the membrane components, such as the distribution of excitation energy between the two photosystems (8). Electron Transport A large body of evidence supports the concept of the Z-scheme for electron transport in plant photo synthesis (35, 37, 82, 88). A schematic representation of linear electron transport through the Z-scheme is shown in Figure 2. This electron transport pathway consists of a series of components which undergo oxidation-reduction reactions. The net result of these reactions is the transfer of electrons from water to NADP+. The overall process lifts electrons from a potential of approximately +800mV to a potential of -300mV (57). The energy for movement of electrons against this electrochemical gradient is provided by the photochemical reactions of photosystem XI (PSII) and photosystem I (PSI), which are connected in series. The two photosystems are distinct chlorophyll-protein com plexes which contain both light-harvesting chlorophyll a molecules and a specialized pair of reaction center chloro phyll a molecules* Light energy is absorbed by the light-harvesting chlorophyll molecules of the two photosystems or the light-harvesting chlorophyll a/b protein (LHC). The light energy is transferred to the specialized reaction center chlorophyll molecules (P680 for PSII and P700 for PSI) which excites an electron in the respective reaction centers. A portion of the energy in the excited electron is trapped by the transfer of the electron to the acceptor molecules, Q and X for PSII and PSI, respectively. Figure 2. A Schematic Representation of the Z-Scheme for the Electron Transport. The components are described in the text. 7 Fd A NADP Fd-Red Q A Cytf h 2o PC ^ P S I PSI ■ 0 (P680) (P700) Figure 2 These photochemical reactions produce the oxidized re- action centers P680-*"h and P700 *4" and the reduced acceptor molecules Q- and X~. The energy for moving electrons ■j* from water to NADP against an electrochemical gradient comes from the energy that is obtained by transferring the electrons from the reaction centers to the primary acceptors (57). Q is believed to be a plastoquinone molecule in a specialized environment (62) which transfers an electron to a pool of plastoquinone molecules. Electrons are then transferred in a series of steps from the plastoquinone pool to cytochrome f, plastocyanin, and finally to P700+. * The identity of X is unclear, but it may be one of the iron-sulfur centers which are located very close to P700 in PSI particles (92). The electrons are transferred sequentially from these initial iron-sulfur centers to ferredoxin (Fd), ferredoxin-NADP+ reductase (Fd-Red), and finally the NADP . The reactions in which water electrons are transferred to P680+ are unknown (35). The coupling of the water oxidizing complex (35) with the reaction center of PSII is the most labile portion of the electron transport scheme presented in Figure 2. The complete series of electron transport steps from water to NADP not only provides reducing equivalents for biosynthesis, but also generates a proton gradient across the thylakoid 10 membrane which is the driving force for ATP synthesis (46) . Piqment-Protein Complexes Three types of chlorophyll-protein complexes exist in the chloroplast membrane. The evidence for these chlorophyll-protein complexes has come from studies in which chloroplast membranes were treated with anionic detergents (102). The detergent extracts were then sub jected to electrophoresis in SDS-containing polyacryl amide gels which separated the slower migrating chloro phyll-protein complexes from the fast migrating free pigment. After optimizing the solubilization and electro phoresis conditions, more than a half-dozen chlorophyll- protein bands can be resolved in the gels (2,3,50,111). A chlorophyll a containing protein with an apparent molecular weight of 70,000 has been assigned to the PSI reaction center (2,3,50,111) and is known to contain P700 (3). A second chlorophyll a containing poly peptide with a molecular weight of approximately 45,000 is thought to be the PSII reaction,center (2, 3, 50, 111). The third chlorophyll-protein complex has a mole cular weight of approximately 25,000 and contains equal amounts of chlorophyll a and chlorophyll b. This third complex has been assigned as the light-harvesting 11 chlorophyll a/b protein (LHC) (2,3,50,111). The remain ing chlorophyll-protein bands in the detergent extracts of chloroplasts are thought to be oligomers of PSI and LHC. Further evidence for the three types of chlorophyll- protein complexes is that purified preparations of each have been obtained by disrupting chloroplast membranes followed by purification on sucrose gradients or anion exchange columns of various types. Preparations of LHC have been developed in which chloroplast membranes are solubilized with SDS (64, 101), Triton X-100 (101), or digitonin (91, 110). Active preparations of PSII re action center complexes have been obtained using either digitonin (91, 110) or Triton X-100 (107). Photochemically-active chlorophyll a protein complexes of .the PSI reaction center have been isolated by detergent solubilization with Triton X-100 (68, 76, 82, 95), digitonin (91, 110), or a combination of both (11). The compositions of the different preparations vary somewhat. The chl/P700 ratios range from 30-110, but all of the preparations have chla/chlb ratios greater than seven which indicates the absence of LHC. Some preparations contain cyto chromes f and b6 (95) while others do not (11, 68, 76). The overall polypeptide compositions of the various pre parations are quite different (11, 76) with one exception. Many of the preparations contain a polypeptide with an apparent molecular weight of approximately 70,000 which is known to contain P700 (3). It has been sug gested that a 20,000 dalton polypeptide is involved in the binding of plastocyanin (11, 44). Another group has implicated two polypeptides of approximately 25,000 daltons as being involved with the light-harvesting chlorophyll a molecules of PSI particles. These are the first reports of attempts to determine the function of the polypeptides that are isolated with the P700 re action center. Preparations of PSI particles also contain ^-carotene and several non-heme iron-sulfur centers. The ^3-carotene is thought to be present not only for light-harvesting purposes, but also to prevent irreversible oxidation of excited chlorophyll molecules in the presence of oxygen (57, 81). The iron-sulfur centers are thought to be part of a network of electron acceptors for P700 (92). Plastocyanin Plastocyanin is a 10,000 dalton copper protein which acts as an electron carrier between PSII and PSI in chloroplasts (13, 58). The plastocyanin from many sources has been isolated and sequenced (13). The protein contains one atom of copper per molecule which undergoes 13 a one electron oxidation-reduction between the Cu (II) and Cu (I) forms. The redox potential of the isolated, protein is approximately +370mV (13) although the redox potential is thought to be approximately +340mV in the thylakoid membrane (71). The oxidized form of the pro tein has a characteristic blue color with three visible absorption bands (13) - a major peak at 597nm with two minor maxima at 460nm and 775nm. Early studies suggested that plastocyanin is loca ted on the inside of the thylakoid membrane. This con clusion was made because antibody against plasto cyanin was not able to inhibit electron transport asso ciated with PSI unless chloroplasts were sonicated in the presence of antibody (48). However, more recent work with hydrophillic covalent modifiers has suggested that a small portion of the plastocyanin molecule is exposed on the outer surface of the thylakoid membrane (94). Because acetone treatment, detergent treatment, or soni- cation is required to extract plastocyanin from thylakoid membranes, the plastocyanin molecule must be at least partially embedded in the membrane (13, 32, 58). Several lines of evidence suggest that plastocyanin is located between cytochrome f and P700 in the chloro- plast electron transport chain. Chloroplasts depleted of plastocyanin require the addition of purified plasto- cyanin for cytochrome f photo-oxidation (84). Chloro- plast fragments from a mutant of the algae C. reinhardti which lacks plastocyanin were found to require the addi tion of purified plastocyanin in order for cytochrome f photo-oxidation to be observed (36). Incubation of chloroplasts with HgC^/ which preferentially replaces the copper of plastocyanin with Hg +2 , completely in hibited cytochrome f photo-oxidation by inactivating plastocyanin (60). An elegant kinetic study has recently shown that a plastocyanin molecule complexed with P700 is the immediate electron donor to P700+ (45). Cation Effects on Chloroplast Membranes Evidence from many years of study implicate mono- and divalent cations in the regulation of a broad spectrum of photosynthetic processes. In the presence of light, 4. the transfer of electrons from water to NADP by the electron transport chain also results in the pumping of protons from outside the thylakoid membranes into the loculus (8). The protons deposited in the loculus are bound to negative charges on the membrane surface which are probably protein carboxyl groups (8). In intact chloro plasts, this results in a A pH across the thylakoid membrane of 2.5 units, with the loculus having a pH of 5.4 and the stroma a pH of 7.9 (8). The pumping of protons into the 15 +2 loculus is accompanied by an efflux of Mg ions from the loculus into the stroma to maintain electrical neutrality (8). These divalent cations are then free to affect the other photosynthetic processes. Cations regulate electron transport in the thylakoid membrane by regulating the turnover rates of the reaction centers in PSII and PSI. Cations regulate turnover rates by controlling the distribution of excitation energy be tween the two photosystems. By equalizing the amount of excitation energy that reaches each reaction center, the two reactions centers are able to turnover at comparable rates in order to achieve maximum flow of electrons "i" from water to NADP . There are two reasons for needing such a mechanism. First, a larger portion of the total chlorophyll (especially chlorophyll b associated with LHC) in the thylakoid membrane is associated with PSII instead of PSI (18, 57). Second, the type of light ab sorbed by each photosystem is different with PSII preferring red light and PSI far red light (57). Under conditions in which more light is absorbed by PSII, a portion of the light energy is transferred to PSI by the LHC. When con ditions allow more light to be absorbed by PSI, a portion of the energy is directed to PSII. This process is called 4-2 spillover. Low concentrations of Mg ions (2-5mM) or high concentrations of monovalent cations (lOOmM) inhibit 16 the spillover mechanism. This effect of salts on energy distribution has been correlated with the rate of turn over in the two reaction centers (6,7,38,41,56,73,77,78, 79, 104). Several groups have collected evidence that suggests that the site of divalent cation effects on chloroplast energy transfer is located in the LHC (4,17,39,85,86). The thylakoid membranes contain two binding sites for cations (39). Covalent modification of carboxyl groups with a water-soluble carbodiimide in the presence of a nucleophile prevented binding of cations to the membrane which inhibited membrane stacking and spillover (85). The majority of the radioactively labelled nucleophile was found to be incorporated into the LHC (86). Cations also produce dramatic structural changes in the thylakoid membranes which accompany the changes in energy distribution. Under low salt conditions, the thylakoids do not form grana. In the presence of cations, these membranes form grana stacks (1, 40, 55) which is accompanied by shrinkage of both the loculus volume and the thylakoid membrane thickness (8). This suggests that cation-induced rearrangement of the thylakoid membranes may cause a reorganization of the chlorophyll-protein com plexes to produce the observed changes in the regulation of energy distribution. The role of cations in regulating 17 membrane structure and energy distribution has recently been analyzed in terms of a model in which cations screen negative charges on the membrane surface (9, 80, 90). Cations reduce electrostatic repulsion (surface potential) which then allows membrane conformational changes. Cations have also been found to regulate the ability of artificial electron donors (54, 100) and plastocyanin (45, 100) to donate electrons to P700+ in broken spinach chloroplasts. After oxidation with light, the rate of 4. P700 reduction by negatively - charged artificial electron donors was accelerated by salts of mono-, di-, and trivalent cations (54, 100). As the charge on the cation increased, smaller concentrations of the cation •j- were required to obtain an equal effect. The rate of P700 reduction was not affected by the type of anion used. Cations have also been shown to stimulate the rate of P700+ reduction by plastocyanin added back to sonicated chloro plasts (100) or plastocyanin present in the membranes of broken chloroplasts (45). The cation stimulation of P700+ reduction was attributed to the screening of negative charges on the membrane by cations which decreased electro static repulsion between the donor and the membrane. This would support the model in which cations regulate photo synthetic activities at the thylakoid membrane surface by screening negative charges on the membrane. (90) 18 Cation Effects on Subchloroplast Particles Cation regulation of activity and structure has also been observed for the isolated chlorophyll-protein complexes. Divalent cations decrease the quantum yield for electron transport in TSF-II particles which consist of the reaction center of PSII in association with a large amount of LHC (27). Removal of the LHC produced a PSII core complex (TSF-IIa) in which cations stimulate electron transport (27) although this stimulation is dependent on the amount of Triton X-100 in the assay (66). Therefore LHC is the site of cation inhibition of energy transfer in TSF-II. The isolated LHC is known to have two binding sites for divalent cations (25, 26). The binding of divalent cations to isolated LHC causes the protein to aggregate (25, 26) which may be the source of cation inhibition of energy transfer in TSF-II particles and perhaps in the thylakoid membrane as well. Addition of cations to PSI subchloroplast particles results in a decrease in chlorophyll a fluoresence and an increase in the quantum yield for PSI electron trans- +2 port (87). Gross and Grenier I (42) found that Mg ions produced a 12% increase in the K -helical content of PSI i particles which was correlated with an increase in energy I transfer from light-harvesting chlorophyll a molecules | to P700. This increase suggests that cations regulate energy transfer in PSI particles by regulating protein structure. Cations also regulate the interaction of PSI particles with electron donors. Gross (43) found that cations stim ulate the rate of electron transfer from artificial elec- tron donors to P700 in PSI particles. Divalent cations have also been shown to stimulate P700+ reduction in PSI particles when purified plastocyanin was used as the electron donor (68). The purpose of this dissertation is to try to understand the mechanism by which cations regulate the activity of isolated PSI particles. Statement of Problem Isolated PSI particles have a negative charge at neutral pH values (91, 97) due to the fact that protein carboxyl groups are the predominant charged species present in the complex. Divalent cations are known to bind to PSI particles (86). The question arises as to how cations regulate PSI particle activity through their interaction with the negatively-charged protein complex. One possibility is that cations interact with specific portions of the PSI complex to alter the protein structure, which is reflected as a change in PSI activity. A second possibility comes from consideration of the surface poten tial model for cation regulation at the thylakoid membrane surface (90). According to this model, cations bind to 20 protein carboxyl groups by electrostatic attraction which results in a more positively-charged protein surface Therefore# the surface potential model suggests that PSI particle activity is affected by the net charge on the protein surface which is regulated by cations. If cations act by changing the net charge on the complex, then chang ing the net charge on PSI particles using chemical tech niques should produce the same effects on activity as those observed for cations. The chemical modification reaction used to alter the charge on the PSI particles involves reacting protein carboxyl groups with a water-soluble carbodiimide in the presence of ethylenediamine (74). The reaction takes place in four steps (65) which are schematically presented in Figure 3. The first step of the reaction is proton ation of the carbodiimide to form the cation. The car bodiimide cation is attacked by the carboxylic acid anion to form the O-acylisourea intermediate. This interme diate is protonated in step III and then reacts with one amino group of ethylenediamine to form the amide. The t N,N - disubstituted urea is formed as a byproduct. In this scheme, a negatively-charged carboxyl group is re placed by a positively-charged amino group. PSI particles will be chemically modified using Figure 3. Chemical Modification of Protein Carboxyl Groups with a Water-Soluble Carbodiimide and Ethylenedia mine. 21 22 NR Protein Protein }—C—O—C o - NHR +N, NHR Protein J— C—0 —C Protein V-C—0 —C NHR NHR NHR IV ( Protein V-C—0 —C + NH,(CH.,LNH,-> Protein }—CNH (CH ) NH NHR R.NHCNH r, Figure 3 23 the reaction described above and the modified PSI particles will then be characterized. The activity of modified PSI particles will be compared with PSI par- +2 tides to which Mg ions have been added. The activities which will be examined are energy transfer within the PSI particle and the interaction of PSI particles with charged electron donors. Because spinach plastocyanin is a negatively- charged protein (30), carboxyl groups on the surface of plastocyanin may be involved in the interaction of this electron donor with PSI particles. For this reason, plastocyanin carboxyl groups will also be modified as shown in Figure 3. After characterization of the modi fied plastocyanin, its interaction with PSI particles will be examined. These experiments should give insight as to whether cations govern PSI particle activity by specific inter actions or by regulating the net charge on the protein surface. Materials and Methods Isolation of PSI Particles PSI particles were isolated from fresh market spinach by a method adopted from Shiozawa et al. (95) as modified by Gross and Grenier (42). The first step involved the isolation of chloroplast lamellae from 2-3 pounds of washed and deveined spinach leaves. The leaves were homogenized for 25 seconds in a Waring blender in the presence of cold (4°C) isolation buffer which consisted of 0.5M sucrose, 0.1M NaCl, and 50mM Tris-HCl pH 8.2. The homogenate was filtered through eight layers of cheesecloth into a beaker surrounded by ice. The chloroplast lamellae were collected from the filtrate by centrifugation at 7000xg for ten minutes at 0°C in a Sorvall RC2-B centrifuge. The pelleted material was resuspended in 0.1 M NaCl, ImM EDTA, and 50mM Tris-HCl pH 8.2 and the lamellae collected by centrifugation at 12000xg for fifteen minutes at 0°C. The chloroplast lamellae were re suspended in 50 mM Tris-HCl pH 7.4 and the chlorophyll concentration of the suspension was measured. The chlorophyll concentration of the suspension was adjusted to 1 mg ch1/ml with 50mM Tris-HCl pH 7.4. 24 25 The resuspended membranes were solubilized with detergent by adding 2 ml of Triton X-100 for each 25 mg of chlorophyll. This suspension was stirred for fifteen minutes at 4°C followed by centrifugation at 12000 x g at 0°C to remove unsolubilized material. The supernatant from the final centrifugatibn step was applied to a 5cm x 5cm column of hydroxylapatite previously eguilibrated at 4°C with lOmM sodium phosphate pH 7.0. The column was washed with 10 mM sodium phosphate pH 7.0 until the eluate was free of chlorophyll. The column was then washed with 1.5-2.0 liters of 1% Triton X-100 (w/v) and 50 mM Tris-HCl pH 7.4 until the eluate was free of chlorophyll. The column was washed again with 10 mM sodium phosphate pH 7.0 (approximately 500 ml). A glass rod was then used to slurry the hydroxylapatite column in approximately 100-200 ml of 300 mM sodium phospahte pH 7.0 containing 0.05% (w/v) Triton X-100. This was done to assure that all portions of the hydro xylapatite were equally accessible to the final elution buffer so that the PSI particles eluted as a concentrated protein solution in a minimum of volume. This mani pulation was incorporated into the procedure after it was observed that regions of the hydroxylapatite column 26 became excessively compacted during previous column washings. After the hydroxylapatite settled once again, the column was washed with 300 mM sodium phos phate pH 7.0 containing 0.05% (w/v). Triton X t*100 to remove the PSI particles. Phosphate buffer was removed from the PSI preparation by dialysis using one of two procedures depending on the intended use for the PSI particles. The first procedure involved diluting the PSI particles 1:1 with double distilled deionized water followed by dialysis against several changes of water until the protein precipitated. The precipitated PSI particles were collected by centrifugation at 30,000 x g and resuspended in water using a hand homogenizer. The second procedure involved dialysis of the PSI particles against three changes of 0.05% (w/v) Triton X-100. Chlorophyll Determinations The chlorophyll content of either chloroplast lamellae or PSI particles was measured by the method of Arnon (5). A known volume of chlorophyll sample was* diluted with water to give a total aqueous volume of one milliliter. Four milliliters of 100% acetone were added to the aqueous sample and the solution 27 centrifuged at 2000 r.p.m. for five minutes to remove insoluble material. The absorbance of the supernatant was measured at 663 nm and 645 nm versus an 80£ acetone blank. The chlorophyll a and chlorophyll b concentration was calculated from the following empirical equations which are based on a 0.05 ml chlorophyll sample in 80% acetone. chi a (mg/ml) =1.27 (A663) - 0.269 (A645) chi b (mg/ml) - 2.29 (A645) - 0.468 (A663) The value obtained from these equations was corrected for chlorophyll samples larger than 0.05 ml (eg. the calculated value was divided by 10 for a chlorophyll sample of 0.5 ml). Occasionally the chlorophyll content of dilute solutions of PSI particles in Triton X-100 was determined by the following equation in which the absorbance of the solution at 435 nm is related to the chlorophyll concentration. . . , . A435 /*s = £ 7 5 5 Isolation of Spinach Plastocyanin Two tasks were performed the day before the isolation began. First, the leaves from 20 pounds of market spinach were washed, deveined, and stored at 4°C in 28 plastic bags. Second, eight liters of acetone were placed in a -20°C freezer. The chloroplast lamellae from 20 pounds of spinach were isolated according to the method of Davis and San Pietro (28) at 4-6°C in a cold room. The leaves were homogenized for 25 seconds at top speed in a four liter Waring blender using cold isolation media consisting of 50 mM Tris-HCl pH 7.8, 0.4M sucrose, and 10 mM NaCl. The homogenate was filtered through eight layers of cheesecloth and the lamellae collected from the filtrate by centrifugation at 10,000 x g for 20 minutes at 0°C. A Sorvall G/3 rotor with six 500 ml polyethylene centri fuge tubes was used for collecting the lamellae which helped to decrease the time required for chloroplast membrane isolation. A clean paint brush was used to resuspend the pelleted material in 1-2 liters of cold 50 mM Tris-HCl pH 8.0 containing 0.1 M NaCl. Plastocyanin was isolated from the resuspended membranes according to the method of Davis and San Pietro (29). Cold acetone (-20°C) was added to the resuspended membranes to give a final concentration of 33% acetone (v/v). This solution was stirred for 15 minutes at 6°C. The acetone treated membranes were collected by centrifugation at 10,000 x g for 10 minutes 29 at 0°c using the G/3 rotor and 500 ml polyethylene tubes. The 33£ acetone supernatant contained the■extracted plastocyanin. Cold acetone (-20°C) was added to the 23% acetone supernatant to give a final concentration of 80£ acetone (v/v). The precipitated protein was allowed to settle so that approximately 80£ of the light green supernatant could be removed with a siphon. The precipitated protein was collected by centrifugation at 10,000 x g for 10 minutes at 0°C. A glass homogenizer was used to resuspend the protein pellet in cold 5mM Tris-HCl pH 8.0. The resuspended protein was dialyzed overnight at 6°C against four liters of 5 mM Tris-HCl pH 8.0. Dialysis tubing with a 3500 molecular weight cutoff was used. The dialyzed protein was applied to a 2.5 x 20 cm column of DEAE-cellulose equilibrated in 5 mM Tris-HCl pH 8.0 at 6°C. After application of the sample, the column was washed with a solution containing 50 mM Tris- HCl pH 8.0 and 0.1M NaCl to remove ferrodoxin: NADP- reductase which eluted from the column as a brown solution. When the elution of the reductase began, the column was washed with 50 mM Tris-HCl pH 8.0 containing 0.2M NaCl. This increase in ionic strength removed a mixture of brown protein as well as the plastocyanin which was reduced 30 (colorless) at this stage of preparation. The fractions containing plastocyanin vere located by the addition of a few drops of 10 mM K^Pe(CN)g which oxidized the plastocyanin to its blue Cu(II) form. The fractions containing plastocyanin were combined and slowly stirred at 6°C while solid ammonium sulfate was added to 60% saturation. After the ammonium ■ sulfate was dissolved, the solution was allowed to incubate at 6°C for 15 minutes before centrifugation at 10,000 x g for 15 minutes to remove precipitated proteins. The supernatant, which contained plasto cyanin, was dialyzed against three changes of 50 mM Tris-HCl pH 8.0 (4 liters / change) for one hour, three hours, and overnight respectively to remove the ammonium sulfate. Because the volume of the supernatant doubled during dialysis, the dilute plastocyanin was concentrated by adsorption on a 2.5 x 5 cm column of DEAE-cellulose equilibrated in 50 mM Tris-HCl pH 8.0. The plastocyanin must be reduced before application to this column because the oxidized form of the protein does not completely bind to DEAE-cellulose under these conditions. When the supernatant was blue after dialysis, a few grains of ascorbic acid were added to reduce the protein. After 31 adsorption of the plastocyanin on DEAE-cellulose, a solution of 50 mM Tris-HCl pH .8,0 containing 0.2M NaCl was used to remove the protein. A few drops of 10 mM K^FetCNjg were used to locate fractions con taining plastocyanin. The fractions containing plastocyanin were con centrated by ultrafiltration using an Amicon cell with a UM2 membrane. The concentrated plastocyanin was chromatographed at 6°C on a column of Sephadex G-75-40 equilibrated with 50 mM Tris-HCl pH 8.0. The purest fractions of plastocyanin (A275/A597ox ^5.5) were combined, concentrated by ultrafiltration, and rechromatographed on the Sephadex G-75 column. The purified plastocyanin had an A275/A597ox ratio of 1.2-1.5. This isolation procedure required approximately 10 days to complete and yielded from 30 to 50mg of purified plastocyanin. Determination of Plastocyanin Concentration The concentration of plastocyanin was determined from the absorbance at 597 nm of the oxidized form of the protein using an extinction coefficient of 4.9 mM-’*' cm-*’ (29) . The absorbance (Aox) at 597 nm of the oxidized protein was measured after addition of a few crystals of potassium ferricyanide to an appropriately diluted sample. The absorbance at 597 nm due to light scattering ( Ared ) was measured after reducing the protein with a few crystals of ascorbic acid. The difference in these readings (Aox-Ared) corresponds to the amount of plastocyanin. Diphenvlcarbazone (DPCN) Disproportionation PSI electron transport was measured using DPCN disproportionation which was monitored as a decrease in -f absorbance at 485 nm (- 3nm) using an Aminco-Chance split beam/dual wavelength spectrophotometer. The assay mixture contained control or modified PSI part icles (10 jig chl/ml), 10 mM Tris-HCl pH 8.2, 0.15 mM DPCN (diluted into the assay from a stock solution prepared in methanol), and 0.05% (w/v) Triton X-100. A projector lamp with a variable voltage supply for varying the output from the lamp was used to illuminate the sample. Red actinic light (A> 650 nm) was isolated by placing 3 cm of water and a red long-pass cutoff filter (Corning CS-2-64) between the lamp and the sample compartment. Actinic light intensities were measured using a Kettering-Yellow Springs Instruments radiometer. A 620 nm short-pass cutoff filter (Bausch and Lomb No. 33 90-1-62-0) was used to prevent actinic light from reaching the photomultiplier. Rates were calculated using an extinction coefficient of 3.0 mM ^cnT'^ at 485 nm for DPCN (96). Determination of P700 Content in PSI Particles The P700 content was determined by measuring the light induced decrease in absorbance at 700 nm (3 nm slit width) using an Aminco DW-2a spectrophotometer operated in the double beam mode. The assay mixture contained a known amount of PSI particles in the range of 10-30 jtig chl/ml, 10 mM Tris-HCl pH 8.2, and from 2 to 7 mM sodium ascorbate. The amount of Triton X-100 in the assay mixture was variable depending on the experiment but never exceeded 0.05% (w/v). The sample was illuminated with blue actinic light (X^560 nm) from a projector lamp using a Corning 4-97 filter and a Bausch and Lomb 90-1-560 interference filter. This filter combination allowed steady state illumination 4 2 of the sample with a light intensity of 4.7x10 ergs/cm /sec which was sufficient to oxidize all of the P700. Two Schott 695 nm long-pass cutoff filters were used to protect the photomultiplier from actinic light. The P700 content was calculated using an extinction coefficient Kinetics of P700 Reduction The rate of P700+ reduction was measured with an Aminco DW-2a spectrophotometer in the double beam mode using the same wavelength settings, filter comb inations, and illumination conditions described for determination of P700 content. The assay mixture contained PSI particles (10 ^g chl/ml), 10 mM Tris-HCl pH 8.2, and residual Triton X-100 which was added to the assay with the PSI particles. The assay mixture was titrated with increasing concentrations of electron donor. Following the steady state of oxidation of P700, the initial rate of P700+ reduction was measured after each addition of electron donor. When DCIP, TMPD, or plastocyanin was used as an electron donor, sodium ascorbate was included in the assay mixture at a concentration of 2 mM for isolated PSI particles and 50 y*.'M for EDA-PSI or GEE-PSI in order to keep the electron donor reduced. The background rate of P700+ reduction by sodium ascorbate was subtracted from the measured rates. In all cases, oxygen served as the terminal electron acceptor (43). When a sample of PSI particles is subjected to continuous illumination in the presence of an electron donor, two processes occur simultaneously. P70Q is "f" being oxidized by light while P700 is being reduced by the electron donor. When a poor electron donor such as sodium ascorbate was used, the rate of oxidation was much faster than the rate of reduction which resulted in all of the P700 being oxidized during continuous illumina tion. However, when high concentrations of a good electron donor such as plastocyanin were used, the steady + state rate of P700 reduction was able to compete with the steady state rate of P700 oxidation. This resulted in a decrease in the magnitude of the absor bance change at 700 nm produced by oxidized P700. Such a decrease in signal results in an underestimation of the true rate of P700 reduction. The rate of P700 reduction (v) can be described as v = k (P700ox) where k is a rate constant and (P700 ox) is the con centration of oxidized P700. When the P700 is totally oxidized, v is proportional to k. If the P700 is not completely oxidized, the observed rate will be less than the true rate. Under conditions where a decrease in P700 signal was observed, the rate of P700 reduction was corrected using the equation 36 P700 totalJ (P700ox J where v' is the corrected rate, vobs is the observed rate, P700 total is the maximum P700‘signal measured at low donor concentrations, and P700ox is the observed P700 signal. Lineweaver-Burk analysis was used to analyze the corrected rate of P700 reduction in the presence of the electron donors listed above. A least squares error calculation was performed to determine the error in K and V to within one standard deviation, m max Kinetics of P700 Oxidation The initial rate of P700 oxidation was measured as a decrease in the absorbance of the reaction center at 430 nm (5nm slit width) upon continuous illumination. The assay mixture contained PSI particles (20 ytg chl/ml), 5 mM Tris-HCl pH 8.2, 2 mM sodium ascorbate, 0.33 mM DCIP, and a small amount (0.05% w/v) of Triton X-100 which was added with the particles. Either a Baird Atomic 650 nm interference filter (half band width = 12 nm) or a Baird Atomic 710 nm interference filter (half band width = 12 nm) was used to isolate red actinic light from a projector lamp which was connected to a variable voltage supply for varying light intensity. A Corning 4-96 filter and a Bausch and Lomb 90-1-620 interference filter were used to protect the photomulti 37 plier. The initial rate of P700 oxidation, was determined as a function of light intensity using an extinction -1 -1 coefficient for P700 of 45 mM cm at 430 nm (59). This measured rate may be a slight overestimation of the rate of P700 oxidation due to a possible contribution to the signal from the electron acceptor P430 present in PSI particles. The contribution of reduced P430 to the signal could be as much as 20% (59) depending on the rate of electron transfer from P430- to oxygen. The purpose of the assay is to measure the efficiency of light utilization by PSI particles under limiting light conditions which is reflected as the formation of both P700+ and P430”. The rate of change in the 430 nm signal as a function of light intensity is the important factor and not that P700 and P430 both contribute to the signal. Kinetics of Cytochrome f Photooxidation The photooxidation and reduction of the cytochrome f present in the PSI particle preparation was measured using an Aminco DW-2a spectrophotometer in the dual-wavelength mode of operation. The measuring and ref erence wavelengths were 554 nm and 540 nm, respectively. A 2 nm slit width was used. The assay 38 contained PSI particles (25-30 yug chl/ml), 10 mM Tris-HCl pH 8.2, and 50yiM sodium ascorbate. The s a m p l e was illuminated by a projector lamp using the filter combination described for DPCN disproportionation. Rates were calculated using an extinction coefficient —1 —1 of 29 mM cm for cytochrome f (109). Absorption Spectra Visible absorption spectra were recorded on the Aminco DW-2a spectrophotometer operated in the double beam mode (lnm slit width). The absorbance of samples containing control or modified PSI particles (10 jig chl/ml), 10 mM Tris-HCl pH 8.2, and 0.05% (w/v) Triton X-100 was recorded from 400 to 700 nm. Samples of oxidized plastocyanin in 50 mM Tris-HCl pH 8.2 were scanned from 400 to 825 nm. The plastocyanin was oxidized just before recording the spectra by the addition of potassium ferricyanide which was then removed from the protein by gel filtration using a Sephadex G-25 column equilibrated in 50 mM Tris-HCl pH 8.2. The reduced minus oxidized absorption spectra of cytochrome f and cytochrome b^ present in PSI particles were recorded by operating the spectrophotometer in the double-beam mode (lnm slit width). ■Both the sample and 39 reference cuvettes contained either control or modified PSI particles (25-30 jig chl/ml), .10 mM Tris-HCl pH 8.2, and Triton x-100 ( 0.05a; w/v) added with the protein. Samples of EDA-PSI also contained 0.5M NaCl (added as a solid) to prevent precipitation of the cationic modified protein in the presence of ferricyanide which is a polyanion. A few crystals of potassium ferricyanide were added to the stirred reference cuvette. A few crystals of ascorbic acid were added to the stirred sample cuvette and the spectrum of cytochrome f recorded from 500 to 600 nm. A few grains of sodium dithionite were then added to the stirred sample cuvette and the combined spectrum of cytochrome f and cytochrome b& recorded from 500 to 600 nm. The extinction coefficient of cytochrome f is £5 53.5 = 29 mM 1cm” 1 (109). The extinction coefficient of cytochrome bg is £5 63-600 = 21 mM"1cm"1 (109) . Discontinuous Polyacrylamide Gel Electrophoresis of Plastocyanin Discontinuous buffer SDS polyacrylamide gel electrophoresis was performed by a method adopted from Kirchanski and Park (61). The gel conditions are given in Table 1. Tube gels were composed of a 9.0 cm long 40 TABLE 1 The Protocol for Discontinuous. Buffer SDS Polyacrylamide Gels Final Resolving Gel Stock Solution '"Volume Concentration (ml) Acrylamide/Bis- 3035/0.8% (w/v) 9.8 7.5% aery1amide Tris-HCl pH 9.8 2M 26.4 1.31M SDS 20% 0.2 0 .1% Water 3.5 t e m e d 100% 0.016 0.04% Ammonium Per 10% 0.5 0.1253 sulfate Stacking Gel Acrylamide/Bis- 30%/0.8% (w/v) 1.66 5% acrylamide Tris-HCl pH 6.1 1M 1.0 0.1M SDS 20% 0. 05 0 .1% Water 7.3 TEMED 100% 0.016 0.19% Ammonium Per 10% 0.025 0.03% sulfate 41 resolving gel and a 1.0 cm long stacking gel. Electro phoretic samples were prepared by diluting plastocyanin with sample buffer to give a final concentration of 65 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 1% 2-mercaptoethanol, 200 mM sucrose, and 0.003% bromophenol blue. Samples were immediately immersed in a boiling water bath for five minutes. The running buffer consisted of 0.192 M glycine, 0.025 M Tris, and 0.1% (w/v) SDS. Samples containing 10 ^ag of protein in 50-200 ^al were applied to the gels and electrophoresis performed.at 1.0 milliampere per tube. Gels were removed from their tubes, fixed overnight in 50% methanol/10% acetic acid (v/v), and followed by incubation in several changes of 5% acetic acid. The gels were stained with 0.04% Coomassie Brilliant Blue G-250 in 3.5% perchloric acid for 90 minutes at room temperature and then destained in 5% acetic acid (53). The apparent molecular weight of control and modified plastocyanin was calculated from a standard curve constructed by plotting the relative mobility of standard proteins as a function of the log of their molecular weights. The standard proteins were bovine serum albumin (68,000), glutamate dehydrogenose (53,000), aldolase (40,000), chymotrypsinogen (25,700), RNase A 42 (13,700), and cytochrome c(ll,700), The relative mobility was measured as the distance traveled by each protein divided by the distance traveled by the bromophenol blue. Discontinuous buffer polyacrylamide gel electro phoresis was performed by adopting .the buffer system described by Kirchanski and Park (61) to the method of Brewer (15). The gel conditions are given in Table 2. Tube gels were composed of a 9.0 cm long resolving gel and a 1.0 cm long stacking gel. Control or modified plastocyanin was dialyzed against electrode buffer which consisted of 0.03 M glycine and 0.005M Tris. Solid sucrose was added to the dialyzed protein sample to a concentration of 10% (w/v). Approximately 10 yug of protein in 50-200 jil were applied to the gels and the electrophoresis performed at 12 volts per tube. The gels were removed from the tubes and immediately stained and destained as described above. The protein pattern was recorded by scanning the gels at 600 nm on a Guilford gel scanner. Chemical Modification of PSI Particles PSI particles were modified in the presence or absence of Triton X-100. Isolated PSI particles dialyzed 43 TABLE 2 The Protocol for Discontinuous Buffer Polyacrylamide Gels Final Resolving Gel Stock Solution Volume- Concentration (ml) Acrylamide/Bis- 30%/Q.8% (w/v) 7.4 7.5% acrylamide Tris-HCl pH 9.8 2M 5.6 0.375M Water 16.8 TEMED 100% 0.017 0.058% Ammonium Per 10% 0 . 21 0.07% sulfate Stacking Gel Acrylamide/Bis- 30%/0.8% (w/v) 0.82 2.5% acrylamide Tris-HCl pH 6.1 1M 0.6 0.06M Water 8.5 TEMED 100% 0.006 0.058% Ammonium Per 10% 0.03 0.03% sulfate 44 against 0.05% (w/v) Triton X-100 were used for reactions in which the protein was modified in the presence of this detergent. The PSI particles (1-2 mg chi) were added to a solution of nucleophile which had been previously adjusted to pH 6.0 with NaOH. This "I" mixture was adjusted to pH 6.0 - 0.05. EDC was added to this reaction mixture in the form of a concentrated stock solution prepared immediately before use. The final concentrations of the components in the reaction were PSI (10-80 p g chl/ml), 0.2M nucleophile, and 0.1M EDC. Triton X-100 was added to a final concentration of 0.05% (w/v) for modifications performed in the presence of this detergent. The reaction mixture was + stirred at room temperature for one hour at pH 6.0 - 0.1. The reaction was stopped by diluting the reaction mixture with an equal volume of 0.5M Tris-succinate buffer pH 6.0. The modified PSI particles were treated in one of two ways depending on the Triton X-100 content. When the reaction mixtures contained no Triton X-100, the modified PSI particles were collected by centrifugation at 30,000 x g at 4°C followed by two washings with distilled water. The washed PSI particles were resuspended 45 in a glass homogenizer in distilled water. For reaction mixtures containing Triton X„ioo, the modified PSI particles were dialyzed at 4°C against three changes of either distilled water or 0.05% (w/v) Triton X-100 depending on the intended use. The modified PSI particles were stored at 4°C. 14 For the labelling experiments, 5 yuCi of c- ethylenediamine were added to the reaction mixture containing unlabelled ethylenediamine before the addition of EDC. Radioactive ethylenediamine was also added to control reaction mixtures in order to follow the removal of free ethylenediamine during later dialysis. At some point during the one hour reaction, aliquots of the control and modified mixtures were removed and used to determine the specific activity (dpm^umole ethylenediamine) of the mixture. After removal of free ethylenediamine following the modification reaction, PSI particles (50-100 yug chi) and 0.6 ml of 30% H2®2 were comkinec^ in a liquid scintillation vial in a total aqueous volume of 1.0 ml. The vials were capped and heated in an oven at 65°C for 1.5 hours to oxidize the chlorophyll. After cooling the vials to room temperature, .10 ml of scintillation cocktail (83) were added to each vial. The cocktail was prepared by dissolving two volumes of a toluene solution with one volume of scintillation grade Triton X-100. The toluene solution consisted of 4.0g PPO and O.lg POPOP per liter of toluene. After mixing, the vials were placed in a Beckman LS—230 liquid scintilla tion counter and the amount of incorporated ethylenediamine determined. Gel Filtration Chromatography of PSI Particles Gel filtration of the PSI particles before and after chemical modification with ethylenediamine was performed using a 2.0 x 50 cm column of Sepharose CL-6B. The column was equilibrated with lOmM Tris- succinate pH 7.0, 0.05% (w/v) Triton X-100, and 1.0M NaCl at 4°C. Three milliliter samples containing PSI particles (90yig chl/ml), 0.05% Triton X-100, and 1.0M NaCl were applied to the column. Four milliliter fractions were collected at a flow rate of 10 ml/hr. After elution, the sodium chloride was removed by dialysis and the fractions analyzed for their chlorophyll and P700 content. Determination of the Isoelectric Profile for Control and Modified PSI Particles. Light scattering due to isoelectric precipitation of PSI particles was monitored as an increase in absorbance at 540 nm using a Model 139 Hitachi Perkin- Elmer spectrophotometer. Samples which contained control or modified PSI particles (10 jag chl/ml) and 0.1% Triton X-100 were placed in a stirred cuvette. The pH of this solution was adjusted in a stepwise fashion with 0.1 M HCl or 0.1M NaOH. The absorbance at 540 nm was measured as a function of pH. Chemical Modification of Plastocyanin The plastocyanin was dialyzed against 50mM sodium borate pH 8.0, 50 mM NaCl to remove Tris buffer present from the isolation. Ultrafiltration with an Amicon cell and UM2 membrane was used as a method to concentrate the plastocyanin to a concentra tion of 0.3 to 1.0 mM. Because the kinetic and redox properties of the modified plastocyanin did not depend on whether the protein was modified in the oxidized or reduced form, no attempt was made to standardize the redox state of plastocyanin before modification. The plastocyanin was then diluted with a known volume of a stock solution of ethylenediamine adjusted to pH 6.0 to give a final concentration of 0.2M ethylene diamine. The pH of this mixture was adjusted to 6.0^0. 48 with 0.1M NaOH or 0.1M HC1 as required. Solid EDC was added to the stirred solution to give a final concen tration of 50 mM and the reaction allowed to proceed for 30 minutes at room temperature. The reaction was stopped by passing the reaction mixture through a 1.5 x 45 cm column of Sephadex G-25 equilibrated in 50mM sodium borate pH 8.2 to remove unreacted ethylenediamine and carbodiimide from the modified plastocyanin. Control plastocyanin was treated identically except that the carbodiimide was not added. This procedure minimized exposure of plastocyanin to ethylenediamine. For the labelling experiments, 5-20 ^iCi of ^4 C- ethylenediamine were added to the reaction mixture con taining cold ethylenediamine before the addition of carbodiimide. At some point during the reaction, aliquots of the reaction mixture were removed and used to determine the specific activity (dpm/^umole ethylene diamine) of the mixture. After elution of the plasto cyanin from the Sephadex G-25 column, known amounts of plastocyanin were placed in liquid scintillation vials in a total aqueous volume of one milliliter; Ten milliliters of the Triton X-100/toluene scintillation cocktail (83) for aqueous samples were added to each vial. After mixing, the vials were placed in a Beckman LS-320 liquid scintillation counter to measure levels of radioactivity after which the number of moles of ethylenediamine incorporated per mole of plastocyanin was determined. Determination of the Redox Midpoint Potential for Control and Modified Plastocyanin The redox midpoint potential of control and modi fied plastocyanin was determined by the method of Davis and San Pietro (29). This procedure involved measuring the absorbance of oxidized plastocyanin at 597 nm as a function of the redox potential of the media. The redox potential of the assay media was established using the potassium salts of the ferricyanide/ferrocyanide -3 -4 (Fe(CN)g / Fe 5 ) couple. The Nernst equation for this couple is Q* where E is the electrical potential^ E = 0.420 volts is the standard electrical potential for this couple at pfr=7.0, T=25°C, all concentrations at 1.0M; n-1 for the one electron transfer process; and (K^Fe(CN)g) and (K^Fe(CN)g) are the concentrations of potassium 50 ferricyanide and potassium ferrocyanide, respectively. This equation can be rearranged to give (K3Fe(CN) 6) which was used to calculate the ratio of the reagents to be used to establish redox potentials in the range of 0.34 to 0.48 volts. A series of samples with different redox potentials were prepared which contained an equal amount of control or modified plastocyanin, 50mM Tris-HCl pH 8.2, and a total ferricyanide plus ferro cyanide concentration of 0.33 mM. The plastocyanin concentration in the sample was in the range of 20-25 jaM. The absorbance at 597 nm was determined for each sample and a linear plot of log ((PC ) / (PC .)) ox red versus E constructed. (PC ) and (PC ,) are the con- ox red centrations of oxidized and reduced plastocyanin, re spectively. The redox potential of plastocyanin is the value of E where log ((PC ) / (PC )) = 0. ox red Tryptic Digestion Of Modified Plastocyanin 14 Tryptic digestion of C-labelled modified plastocyanin in the presence of 2M urea was performed by a procedure adapted from the method of Smyth (98). A known amount of modified plastocyanin (4—6 mg protein, 30000-50000 cpm/mg) was dialyzed extensively against 0.01M NH^HCO^ and then lyophillized to a powder. The lyophillized modified plastocyanin was denatured over night in 8M urea. The denatured protein was then diluted with NH^HCO^ buffer (pH 8.0) to give final con centrations of 1% (w/v) plastocyanin, 50 mM NH^HCO^, and 2M urea. TPCK-trypsin was added in a small volume (0.02ml) of 0.001 M HC1 to the stirred solution and the hydrolysis allowed to proceed at room temperature for four hours. After four hours, a second aliquot of TPCK-trypsin was added which gave a total trypsin con centration of 0.01& (w/v). The hydrolysis was con tinued for 16 hours at room temperature. After hydrolysis, the urea was removed from the peptides by passing the hydrolysate through a 1.5 x 46 cm column of Sephadex G-10 equilibrated with 0.01M NH,HCO_. Two and one-half milliliter fractions were 4 3 collected at a flow rate of 15 ml/hr. and the elution of the peptides monitored by their absorbance at 225 nm. The fractions containing peptides were pooled and lyophillized to a powder. The powder was dissolved in water and lyophillized extensively once again to insure 52 the complete removal of the volatile NH^HCO^ buffer. The peptides were resuspended in a minimal volume {25-50 jil) of water. Peptide Mapping of Modified Plastocyanin Peptides Peptides produced by tryptic digestion of modified plastocyanin were separated using the two dimensional peptide mapping system described in detail by Bennett (12). Sheets of Whatman 3MM chromatography paper (47 x 56 cm) were prewashed by descending elution of the paper with freshly prepared solvent consisting of n-butanol, glacial acetic acid, pyridine, water (90: 18:60:72, v/v). After evaporation of solvent, 1-2 mg of plastocyanin peptides in water were applied to a spot near the upper right corner of the paper (12). The sample was applied in one or two drops at a time followed by evaporation of water by a hand held dryer. The application of the sample was restricted to a spot with a maximum diameter of 1.5 cm. Decending chromatography with the solvent described above (freshly prepared before each elution) was used to separate the peptides in the first dimension. After placing the paper in the chromatography tank, approxi mately 300 ml of solvent were placed in the bottom of 53 the tank in an open 2x9x13 inch glass container. After closure of the tank, the solvent in the glass container was allowed to saturate the atmosphere in the tank with solvent vapors for one hour. The reservoir was then filled with 140 ml of solvent through a port in the top of the tank and the elution allowed to proceed. After elution, the paper was re moved from the tank and the solvent allowed to evapor ate at room temperature. A better separation of peptides was observed using repetitive elutions with solvent followed by evaporation of solvent from the paper after each run. Three elutions of increasing duration (5 hrs., 8 hrs., 12 hrs.) were performed as described above. The chromatogram was then dried in an 80°C oven for 15 minutes to remove residual solvent from the paper. Excess paper above the sample origin and below the solvent front was trimmed from the chromato gram. High voltage electrophoresis was used to separate the peptides in the second dimension using a buffer (pH 3.6) containing 100 ml of glacial acetic acid and 10 ml of pyridine diluted to 3000 ml with water. The chromatogram was sprayed with this buffer until the paper was completely wet. Strips of Whatman 3MM paper 54 were used to blot up excess, buffer. Electrophoresis was carried out at a potential of 3200 volts for 1.5 hours at 10-15°C. The buffer and varsol in the electro phoresis tank had to be precooled for four hours in order to maintain this temperature. The peptides migrated away from the cathode (+) toward the anode {-) due to the fact that the peptides carried a posi tive change at pH 3.6. After electrophoresis, the chromatogram was allowed to dry at room temperature followed by heating in an 80°C oven for 15 minutes. Peptides were located by lightly spraying the chromato gram with a ninhydrin spray (0.025% ninhydrin in abso lute ethanol: 2N acetic acid, 75:25, v/v) and the spots developed by heating in an 80°C oven for five minutes. The paper containing each spot was cut out and placed in a scintillation vial with 10 ml of scintillation cock tail for nonaqueous samples. This cocktail consisted of 4.0g PPO and O.lg POPOP per liter of toluene. A Beckman LS-320 counter was used to measure levels of radioactivity in the spots. The paper was removed from the vial, rinsed in toluene, and the toluene allowed to evaporate at room temperature overnight. 55 Elution of Plastocyanin Peptides from Whatman 3MM Paper The portion of the chromatogram which contained a single spot was placed in a round teflon cup approxi mately 8mm in diameter and 25 mm deep. The cup contain ing the piece of paper was placed in a 15 ml conical centrifuge tube. A few drops of 0.001 M HC1 were used to moisten the paper in the cup. Centrifugation at 1600 r.p.m. for five minutes was used to force the dilute HC1 through the paper and out the holes in the bottom of the teflon cup. The HCl containing the eluted peptides was collected in the bottom of the centrifuge tube. For complete elution of the peptides, the process of moistening the paper followed by centri fugation was repeated many times until approximately 1 ml of HCl was collected in the bottom of the conical centrifuge tube. Identification of Plastocyanin Peptides bv Amino Acid Analysis Peptides eluted from the Whatman 3MM paper with 0.001 M HCl were placed in hydrolysis tubes and the dilute HCl removed using an aspirator which was aided by heating the solution with a hand held dryer. One milliliter of 6N constant boiling HCl and 2 mg of phenol were added to the hydrolysis tube. The tube was con nected to a vacuum pump and evacuated for 5 minutes to remove oxygen. The peptides were then hydrolyzed under vacuum at 110°C for 24 hours. The 6N HCl was removed using the aspirator described above. The hydrolyate was dissolved in 0.5 ml of 0.2M citrate buffer (pH 2.2) and then filtered. Identical volumes of hydrolysate (approximately 0.2 ml) were analyzed for amino acid content with a Beckman Model 119 CL analyzer 14 and for C-ethylenediamine content using the liquid scintillation counting system described previously for aqueous solutions. The peptides were identified by their amino acid composition. The amount of ethylene diamine incorporated per mole of labelled peptide was calculated from the amino acid composition of the peptide and the amount of glutamic acid/aspartic acid 14 and c-ethylenediamine in the peptide hydrolysate. Separation of Modified Plastocyanin Species bv DEAE-Sephadex Chromatography DEAE-Sephadex G-50 was swollen for 2 hours in a boiling water bath in the presence of 0.1M Tris-HCl pH 8.0. The buffer was decanted and the gel equilibrated against several changes of 50 mM Tris-HCl pH 8.0 at 6°C. A 2.5 x 22 cm column of DEAE-Sephadex equilibrated in 50 mM Tris-HCl pH 8.0 was prepared in the cold room (6°C). A small amount of Sephadex G-50 was layered on top of the column to prevent disruption of the ion exchange gel upon application of sample. Modified plastocyanin (10-20 mg protein) was concentrated to a volume of 5 ml by ultrafiltration using an Amicon cell with a UM2 membrane. A few crystals of potassium ferricyanide were added to the concentrated protein. The completely oxidized modified plastocyanin sample was then applied to the column. The column was washed with 125 ml of 50 mM Tris-HCl pH 8.0 at a flow rate of 60 ml/ hr. and 6.5 ml fractions were collected. The column was then eluted with a 300 ml linear gradient of sodium chloride (0 to lOOmM) in the presence of 50 mM Tris- HCl pH 8.0. Following the gradient, the column was washed with 0.1M NaCl, 50 mM Tris-HCl pH 8.0 until all of the protein had eluted. The elution of the modified plastocyanin from the column was followed by the protein absorbance at 275 nm. The separated modified plasto cyanin species were pooled and concentrated by ultra- filtration, 58 Proton NMR of Control and Modified Plastocyanin Control or modified plastocyanin was reduced with ascorbate. The ascorbate was then removed from the protein by gel filtration on Sephadex G-25 equilibrated in 50 mM sodium phosphate pH 8.0. The protein was concentrated by ultrafiltration using an Amicon cell with a UM2 membrane. This ultrafiltration step was very time consuming because phosphate buffer inhibits the flow rate of the UM series of membranes. After determining the plastocyanin concentration, a known amount of protein was lyophillized to a powder in a 10 ml conical centrifuge tube. The protein was then lyophilLized twice from 99.8% D^O to remove exchange able protons. The lyophillized sample was stored at 5°C. Just before NMR spectroscopy, the samples were dissolved in D2O so that the final sample contained plastocyanin (10 mg protein/ml) and approximately 0.5M phosphate buffer. The sample of control plastocyanin dissolved completely in I^O. However, the modified plastocyanin contained a significant amount of white precipitate which was probably denatured protein. This denatured protein was removed from the modified plasto cyanin sample by centrifugation at 2000 r.p.m. for 59 five minutes. The samples were placed in NMR tubes and a teflon plug placed over the sample which confined the sample to the bottom of the tube. A sample volume of approximately 0.4 ml was used. Proton NMR spectra were obtained with a Bruker WM-300 FT-NMR spectrometer set up for quadrapole de tection and phase correction. The 300 MHz spectra were the result of 1000 scans of a 5000 Hz band width. Each scan involved excitation of the sample with a 3 ^isec pulse followed by 1.63 seconds of data acquisition in which 16,300 time domain data points were collected. No time delay between scans was employed. Computer assisted line broadening of the spectra using a line width of 2.0 Hz was performed to improve the signal to noise ratio. Chemical shifts were obtained relative to the water peak which was used as a lock signal. A chemical shift of 4.5 parts per million ( £ (ppm)) relative to DSS (2,2-dimethyl silapentane 5-sulphonic acid) was assigned to the water peak. All spectra were obtained at ambient temperature. Materials TMPD was obtained from Aldrich Chemical Company as well as diphenylcarbazone and sulfanilic acid which were recrystallized before use. DEAE-cellulose and hydroxylapatite were obtained from Biorad Laboratories. Ascorbic acid and Spectrapor 3500 molecular weight cutoff dialysis tubing were obtained from Fisher Scientific. Fresh spinach was obtained from local 14 markets. C-ethylenediamine dihydrochloride was obtained from either New England Nuclear or Amersham. EDC, ninhydrin, and 6N constant boiling HCl were ob tained from Pierce Chemical Company. Triton X-100 was obtained from Research Products International. Sephadex G-75-40, Sephadex G-25, DEAE-Sephadex G-50, Sepharose CL-6B, DC1P, protein standards, ethylene diamine dihydrochloride, glycine ethyl ester hydro chloride, 2-aminoethanol, and taurine were obtained from Sigma Chemical Company. TPCK-trypsin was obtained from Worthington Enzymes. All other chemicals were reagent grade. Double distilled deionized water was used in all experiments. RESULTS AND DISCUSSION Isolation of PSI Particles The isolation procedure for PSI particles involved an initial extraction of spinach chloroplast membranes (200-300 mg chi) with 7.4% (w/v) Triton X-100. The solubilized membrane components were adsorbed on a hydroxylapatite (calcium phosphate) column and washed with 50mM Tris-HCl pH 7.4 containing 1% (w/v) Triton X-100. A more extensive washing with 1500-2000 ml of this buffer was performed instead of 300-500 ml as suggested by the original procedure (95). This de creased the chl/P700 ratio from 150-250 to a value of 80-150. This procedure yielded a PSI preparation which con tained 5-10% of the chlorophyll in the isolated chloro plast membranes. The preparation contained cytochrome f and cytochrome bg and had a high chi a/chl b ratio which indicated the presence of only traces of LHC (Table 3). The advantage of this PSI preparation is that a large amount of PSI particles can be obtained in one day. Other types of PSI preparations (11, 68, 76) have lower chl/P700 ratios and no cytochrome f 61 Table 3 Properties of the Isolated PSI Particles Chlorophyll / P700 80-150 Chlorophyll a / Chlorophyll b > 6 Cytochrome f / P700 0.9 i 0.1 Cytochrome bg / P700 3.3 - 0.7 63 or cytochrome bg. However, these PSI preparations of higher purity involve much longer isolation procedures that contain a sucrose gradient centrifugation step which limits the amount of material that can be purified. Chemical Modification of PSI Particles Using Ethvlenediamine or Glycine Ethyl Ester as a Nucleophile When control PSI particles, EDA-PSI, or GEE-PSI were illuminated with high intensity blue light 4 2 (1=4.7 x 10 ergs/cm /sec), there was a rapid oxidation of all the P700 (Figure 4). After the actinic light + was turned off, the reduction of P700 in control PSI particles was very slow when 6.7mM ascorbate was used as the electron donor. Addition of 5mM MgCl^ to control PSI particles caused an increase in the rate of P700 reduction. This divalent cation stimulation of P700+ reduction by ascorbate has been observed by others (43,68). The magnitude of the light induced absorbance change at 700 nm in control PSI, EDA-PSI, and GEE-PSI was found to be identical on a chlorophyll basis. This indicated that the modification had no effect on the total amount of P700. However, modifica tion of PSI particles with ethylenediamine or glycine ethyl ester resulted in a twenty fold increase in the Figure 4. Comparison of the light-induced P700 assay for control PSI particles, EDA-PSI, and GEE-PSI. The assay was performed as described in the Materials and Methods for measurement of P700+ reduction. Ascorbate (6.7 mM) was used as the electron donor. 64 65 OJ Control- Mg-H- Control + 5mM Mg-H- Modified- Mg-H- Modified + 5mM Mg-H- 20 sec Figure 4 66 rate of P700+ reduction (Figure 4). Identical results were obtained for PSI modified in the presence and absence of 0.05% (w/v) Triton X-100. Determination of the Optimal Conditions for the Chemical Modification of PSI Particles by Ethvlenediamine The optimal chemical modification conditions de scribed in the Materials and Methods were determined from a series of experiments which utilized the increase in P700+ reduction by ascorbate as the assay. When the ethylenediamine concentration was varied (Figure 5), the rate of P700 reduction increased at ethylenediamine concentrations above 0.05M and began to saturate at 0.4M. A concentration of 0.2M was chosen for further experiments because 80% of the observable increase in + P700 reduction occurred at this concentration of ethylenediamine. Notice that at those ethylenediamine concentrations (0 and 0.05M) where EDC was in excess, the rate of P700 reduction was equal to or less than the control rate. The PSI particles also appeared to aggregate when modified at these low concentrations of ethylenediamine. The effect of varying the EDC concentration is shown in Figure 6 . The optimal carbodiimide Figure 5. The concentration dependence of ethyl enediamine on the modification reaction. The reaction mixtures contained PSI particles (20 ;ug chl/ml), 0.1 M EDC, and varying amounts of ethylenediamine. The components were allowed to react one hour at pH 6.0 The reaction was stopped using 0.5M Tris-succinate buffer pH 6.0. The EDA-PSI was collected and washed with distilled water as described in the Materials and Methods. The rate of P700+ reduction by 6.7 mM ascorbate was measured as described in the Materials and Methods. EDA-PSI (□— □ ). Control PSI particles (---- — ). 67 00 00 O J\) -P> 69 od O ro The pH profile for the reaction is shown in Figure 7. The increase in P700 reduction was found to be maximal at both pH 5 and pH 6 . pH 6 was chosen for further modifications due to the fact that PSI particles have an isoelectric point near 5 (See Figure 11). The rate of P700+ reduction for modified PSI decreased at pH 4. Modification at pH 4 also resulted in PSI particle aggregation similar to that observed under conditions where EDC was in excess of ethylenediamine. The modification reaction was found to be inde pendent of the concentration of PSI particles in the reaction mixture. A series of reaction mixtures were prepared which contained 0.2M ethylenediamine, 0.1M EDC, and varying concentrations of PSI particles. The reaction was performed at pH 6.0 for one hour. The reaction was stopped by addition of 0.5M Tris-succinate pH 6.0. The EDA-PSI was washed and the rate of P700+ Figure 7. The pH profile for the modification of PSI particles. The reaction mixtures contained PSI particles (20 ^ag chl/ml), 0.2M ethylenediamine, and 0.1M EDC. The components were allowed to react for one hour at various pH values. The reaction was stopped by addition of 0.5M Tris-succinate pH 6.0. -The PSI particles were washed and the rate of P700+ reduction measured as described in the Materials and Methods using 6.7 mM ascorbate as an electron donor. EDA-PSI (□— a ) . Control PSI particles Co-o)t 72 oro4^ Figure 74 reduction by 6.7mM ascorbate measured as described in *4* the Materials and Methods. Identical rates of P700 reduction were observed when PSI particles were modified at concentrations between 10 and 80 jug chl/ml. Con centrations outside this range were not tested. The time course of the reaction is seen in Figure 8 . The rate of P700 reduction increased with time and reached a maximum at approximately 60 minutes. A re action time of 60 minutes was chosen for the chemical modification reaction. Storage of EDA-PSI Control PSI particles and EDA-PSI prepared under optimal conditions were stored at 4°C in unbuffered aqueous solutions. Figure 9 shows that the stimulated + rate of P700 reduction in EDA-PSI began to decrease after 14 days of storage. Because of this effect, all experiments were carried out with EDA-PSI which had been stored for less than two weeks. The Effect of 1-ethvl -3- (3-dimethvlaminopropvl) Carbodiimide on PSI Particles Several observations indicated that EDC inhibited PSI particles under certain conditions. When PSI Figure 8 . Time course of the modification of PSI particles. The reaction mixture contained PSI particles (20 )ig chl/ml), 0.2M ethylenediamine, and 0.1M EDC at pH 6.0. Aliquots of reaction mixture were removed at known times and the reaction stopped by addition of 0.5M Tris-succinate buffer pH 6 .^. The PSI particles were washed and the rate of P700 reduction by 6.7 mM ascorbate measured as described in the Materials and Methods. EDA-PSI (a □ ). Control PSI particles (0-0). 75 — o oro-&a>a>oi\)-& Initial Reduction Rate (^.moles P700+-mgchrl-hr"1) (^.moles Rate Initial Reduction a> CD ro o o o o o ro o o Time (Minutes) Figure 9. The effect of storage at 4 C on the activity of EDA-PSI. Control PSI pagticles (----- ) and EDA-PSI {□— □ ) were stored at 4 C in unbuffered solutions. The rate of P700+ reduction by 6.7 mM ascorbate was assayed as described in the Materials and Methods. 77 00 T ro T Initial Reduction Rate (/mmoles P700+-mgchr'-hr_l) (/mmoles Rate Initial Reduction ro o OJ O O Time (Days) C n ro H- iQ 79 particles were modified under conditions where the con centration of EDC exceeded that of ethylenediamine, the modified PSI particles showed an inhibition of P700 re duction by ascorbate (Figures 5 and 6 ). These modifica tion conditions also caused an aggregation of the particles. To examine this phenomenon, PSI particles were incubated with 0.1M EDC at pH 6.0. These condi tions resulted in the formation of large PSI particle aggregates. However, the chl/P700 ratio was the same before and after EDC treatment, which means that all of the reaction centers remained functional. The aggregated *|* particles showed a 75% decrease in the rate of P700 reduction, using 6.7mM ascorbate as the electron donor. This may be the result of ascorbate becoming less accessible to P700+ in the PSI aggregates. Measurements of P700 oxidation as a function of light intensity also revealed an inhibition of energy transfer from light- harvesting chlorophyll a molecules to the reaction center in PSI particles treated with EDC. During experiments to optimize chemical modifca tion conditions, it was also observed that PSI particles aggregated when the reaction was performed at pH 4.0 even though ethylenediamine (0.2M) was in excess of EDC (0.1M). One way to explain these results is that an 80 excess of EDC can be created in a functional sense by performing the modification at a pH where very little unprotonated amine is present to act as a nucleophile. This was examined by comparing ethylenediamine and glycine ethyl ester which have amine pK^s of 7.5 and 7.75, respectively, with 2-aminoethanol and taurine which have amine pK^s of 9.5 and 9.0 (see Table 4). The reaction of PSI particles with EDC was performed at pH 6.0 in the presence of these amines. Aggregation of PSI part icles occurred for the two compounds with amine pK^s of 9.0 or above. No aggregation occurred for ethyl enediamine or glycine ethyl ester which have pK^s much closer to the reaction pH. The conclusion is that aggregation of PSI particles occurs when EDC is in excess by either adding more carbodiimide than nucleophile or by using amines which are not good nucleophiles at the reaction pH. One example in Table 4 does not follow this general conclusion. Sulfanilic acid should be a good nucleophile at pH 6.0 and yet aggregation of PSI particles was observed. Perhaps electrostatic repulsion between the negatively-charged PSI particles and sulfanilic acid prevented this nucleophile from attacking the inter mediate formed between EDC and protein carboxyl groups. 81 Table 4 The Effect of Modification of PSI Particles by Various Nucleophiles Nucleophile Structure Amine pKa Observation ethylenedia Stimulation of mine n h 2 (ch2)2&h 3 7.5, 10.7 'P700+ 'reduction Glycine Stimulation of ethyl ester NH2CH2CO-CH2CH 3 7.75 P700 reduction 2-aminoetha- Aggregation of nol NH2 (CH2)2OH 9.5 PSI particles Aggregation of taurine NH2 (CH2)2SO“ 9.0 PSI particles sulfanilic Aggregation of acid ^2<2 > S°3 3*2 PSI particles The modification reactions were performed at pH 5.0 with 0.2M nucleophile and 0.1M EDC as described in the Materials and Methods. P700+ reduction by 6.7 mM ascorbate was performed as described in the Materials and Methods. 82 The nature of the EDC promoted aggregation of PSI particles is unknown. It was observed that 1& (w/v) SDS could not break up the aggregated protein- Comparison of the Visible Absorption Spectra of Isolated PSI Particles with EDA-PSI and GEE-PSI Figure 10 shows the visible absorption spectrum of isolated PSI particles superimposed on the spectra of EDA-PSI and GEE-PSI. The spectra have not been corrected for light scattering or absorption flattening. Except for small differences in concentration and light scattering properties of the samples, the absorption spectra for the three types of PSI particles are identical. The Effect of Chemical Modification on the Net Charge of PSI Particles To determine if the modification altered the net charge of the PSI complex, light scattering was measured as a function of pH (Figure 11). The light scattering of the control was maximal at pH 5 due to precipitation of the protein at its isoelectric point. A PSI particle isoelectric point near pH 5.0 agrees with the results of other workers (91,97). EDA-PSI had an isoelectric pH of 10.3 indicating a change in the net charge of PSI Figure 10. Visible absorption spectra of PSI particles before and after chemical modification. The spectra of isolated PSI particles ( ) , GEE-PSI (--- ), and EDA-PSI (..... ) were measured as described in the Materials and Methods. 83 ABSORBANCE 5 , 0 0 0 4 iue 10 Figure 0 0 5 WAVELENGTH (nm) 0 0 6 0 0 7 03 Figure 11. Determination of the isoelectric pH for PSI particles before and after chemical modification. The isoelectric pH for isolated PSI particles (a— a ), GEE-PSI (•-•), and EDA-PSI (0-0} was measured as described in the Materials and Methods. 85 ABSORBANCE AT 5 4 0 nm F- iQ C i-J CD CJl *U I 00 o oo CTi 87 from negative to positive as the result of replacing carboxyl groups with amino groups. Incorporation of a large number of ^C-ethylenediamine molecules into each PS I complex (.Table 5) also supported the idea of a generalized change in the charge of the complex rather than the specific incorporation of a few ethylenediamine molecules. Although the reaction of EDC with protein carboxyl groups is the only pathway that leads to ethylenediamine incorporation, there are two other amino acids which can react with an EDC molecule. These are cysteine (20) and tyrosine (19). Because PSI particles from higher plants contain only traces of cysteine (103), the reaction of EDC with this amino acid was not a significant reaction. If the EDC had reacted with tyrosine residues on the protein, the isourea derivatives produced by this reaction would have contained a diraethylaminopropyl group from the original carbodiimide molecule. This tertiary amine could also contribute positive charges to the EDA-PSI when protonated. GEE-PSI also exhibited a very high isoelectric pH of approximately 10.3 (Figure 11). A shift in isoelectric pH to high values compared to the control would be expected since carboxyl groups were replaced by the neutral glycine ethyl ester moiety. However, the cationic nature of GBE-PSI implies that the amino groups of lysine and arginine outnumber the remaining carboxyl groups after modification of the protein. This would require that a majority of the carboxyl groups be modified since the mole percentage of amino acids with carboxyl groups is much larger than the mole percentage of amino acids with amino groups in PSI particles (103). Evidence for Two Types of Monomeric Units in PSI Particles. The labelling experiments also give some insight into the organization of the isolated PSI complex. The average molecular weight of the monomeric unit of PSI particles is estimated to be 100,000 - 200,000 (42, 103). The combined mole percentage of aspartic and glutamic acid residues in PSI particles has been estimated to be in the range of 15% (103). Using these values, the calculated value for the number of carboxyl groups associated with each reaction center is approximately 300. This value is lower than the value for moles of ethylenediamine incorporated per reaction center (see Table 5). Three possibilities exist which would explain why the amount of ethylenediamine incorporated is larger than the potential number of reactive carboxyl groups on 89 Table 5 Determination of Ethylenediamine Incorporations into PSI Particles3 Amount of moles ethylenediamine moles ethylenediamine Triton X-100 mole chlorophyll mole P700 0 6.4 644 0 5.4 533 0.05SS 5.5 616 aSee the Materials and Methods for details of this determination. 90 PSI. First, perhaps a portion of the reaction centers are inactivated upon isolation. If this were true, more than 50% of the isolated reaction centers would be 14 inactivated to account for the C-ethylenediamine results. The chl/P700 ratio of the PSI particle pre paration was the same when measured by either the light- induced P700 assay or by the chemical difference spectrum. Therefore the preparation did not contain photochemically inactive reaction centers. A second possible reason is that our PSI preparation may contain a few chloroplast proteins that are labelled but have no function related to PSI activity. Impurities hlone can not account for the large discrepancy between carboxyl group content and the amount of ethylenediamine incorporated. A more feasible explanation was suggested by Thornber et al. (103). They described two types of PSI monomers which are of the same molecular weight but differ in that one contains the P700 reaction center while the other does not. The presence of a large amount of PSI complex which is not photoactive would explain the large number of barboxyl groups modified per reaction center. The Effect of Chemical Modification on the Molecular Weight of the PSI Complex. 91 In addition to a change in the net charge, it was possible that the chemical modification reaction caused cross-linking of polypeptides through amino and carboxyl groups on the protein. This cross-linking could have been intramolecular between the several polypeptides (11, 76) within the complex or intermolecular between several intact complexes. SDS gel electrophoresis would have been an excellent tool to examine EDA-PSI for intra molecular cross-linking. This study was not possible because the positively-charged EDA-PSI formed insoluble aggregates in the presence of SDS. It can be speculated that intramolecular cross-linking probably did not occur. The high concentration of ethylenediamine used in the reaction mixture should have prevented protein amino groups from competing with free ethylenediamine as the nucleophile in the modification reaction. Still, intra molecular cross-linking can not be completely ruled out. Several lines of evidence suggest that intermolecular cross-linking of PSI monomers did not occur. High concentrations' of ethylenediamine would prevent this for the reasons stated above. The definitive experiment which argues against intermolecular cross-linking was gel filtration using Sepharose CL-6B. Sodium chloride (1 .0M) had to be included in the column buffer to prevent an ionic interaction between cationic EDA-PSI and the column material. This must be the result of trace amounts of anionic groups on Sepharose CL-6B. Control PSI particles, EDA-PSI, and the stock solution of PSI particles from which the first two originated each eluted in the same fractions from the gel filtration column (Figure 12)_. This ruled out intermolecular cross- linking as a factor in the stimulation of P700 reduction. Catalase (247,000 daltons) eluted in the same fractions as the PSI particles. Therefore the complex had a molecular weight of approximately 2 50,000 daltons before and after chemical modification. EDA-PSI may contain a small amount of a higher molecular weight component which caused a broadening of the chlorophyll peak. Each protein sample contained a small amount of aggregated protein in the void volume of the Sepharose column. The void volume peak is much larger in control PSI particles possibly due to the incubation of the control at pH 6.0 where isoelectric precipitation of the particles has begun (Figure 11}. The P700 is not symmetrical with the chlorophyll peak but instead is shifted toward the high molecular weight shoulder. This indicates the presence of either a lower molecular weight chlorophyll protein which contains no P700 or Figure 12. Gel filtration of PSI particles before and after chemical modification. The gel filtration was performed as described in the Materials and Methods. 93 ABSORBANCE AT 435 nm 0.8 0.4 0.8 0.4 0.8 0.4 ———1 — — 1— 0 iue 12 Figure 0 0 0 40 30 20 10 ISOLATED PSI CONTROL PSI EDA-PSI FRACTION ------95 perhaps free chlorophyll in Triton X-100 micelles. The Light and Dark Processes of PSI Particles The activity of PSI particles can be divided into those processes that require light and those dark processes which are independent of light. The light processes include excitation of chlorophyll a molecules by absorption of photons, transfer of excitation energy from light harvesting chlorophyll a to the reaction center, and the primary photochemistry of P700. The dark processes involve the interaction of PSI with the electron donor as well as the dark electron transfer steps between the donor and oxygen which served as the terminal electron acceptor in all of the photochemical assays. + 2 The Effect of Mg and Chemical Modification on DPCN Disproportionation by PSI Particles. PSI electron transport was measured as a function of light intensity using DPCN disproportionation (96, 106}. The relationship between the rate of electron transport and light intensity can be expressed by the equation (70, 89) 1/v = 1/Kd + 1/(Kl . I) 96 where v is the rate of electron transport in ^unoles DPCN/mgchl/hr. and I is the light intensity in ergs/ 2 cm /sec. Kt is the rate constant for the light-processes. KD is the rate of electron transport at infinite light intensity. Therefore changes in KQ indicate changes in those processes of PSI that are independent of light (i.e. dark processes) - It was found that PSI particles with P700 inactivated by boiling for 5 minutes produced a measurable rate of DPCN disproportionation. This background rate increased linearly with light intensity from 0.5 to 1.5 ^omoles DPCN/mg chl/hr. under the conditions of our measurements. Subtraction of this background rate from active PSI rates was performed before the plots of 1/v versus 1/1 were constructed. The cause of this background rate may have been light induced DPCN disproportionation by detergent solubilized chlorophyll molecules as described by van Ginkel (105). This free chlorophyll catalyzed rate was 3-5% of the active PSI rates. and Kp were determined from the slope and y- intercept, respectively, of the 1/v versus 1/1 plots. The results are seen in Table 6. The addition of MgC^ to control PSI particles caused a 4l£ increase in K^. +2 Mg ions and chemical modification both produced an Table 6 4*2 The Effect of Mg and Chemical Modification on DPCN Disproportionation by PSI Particles3 MgCl2 Kl X104 % increase % increase in K_ in Kd L i k d Control PSI 0 5.8 t 0.8 - 27 - 3 Control PSI 5mM 8.2 - 1.5 41 40 - 6 45 4 * EDA-PSI 0 8.4 ± 0.7 45 100 - 16 270 3Assay conditions are described in the Materials and Methods, Units for are [jamoles DPCN/mg chl/hrlj . fergs/cm^/secj-^. Units for Kp are jumoles DPCN/mg chl/hr. 98 increase in K^. The increase in was larger for EDA-PSI +2 particles. Therefore the binding of Mg ions and chemical modification both affect the light and dark processes of PSI particles. The Effect of Chemical Modification on the Light Dependent Processes of PSI Particles. The light-induced oxidation of P700 was used as a second independent method to examine the effects of chemical modification on the light processes of PSI particles. Under light limiting conditions, the initial rate of P700 oxidation is proportional to the actinic light intensity. The relative quantum yield is propor tional to the slope of the linear portion of a graph in which the initial rate of P700 oxidation is plotted as a function of light intensity. Measurements of the initial rate were made using interference filters to isolate actinic light at 650nm or 710nm.* The 650nm light is absorbed by light-harvesting chlorophyll a molecules in PSI particles while 710nm light is absorbed by the reaction center chlorophyll molecules. By examining rates of P700 oxidation under the two illumination conditions, the reason for the stimulation of the light processes can be attributed to either an increase in energy transfer 99 from light-harvesting chlorophylls to the reaction center or an effect on the reaction center itself. Control PSI particles, EDA-PSI, and the stock solution of PSI particles from which the first two originated are compared in Figure 13. The stock PSI was included in order to determine if the incubation at pH 6.0 affected the control PSI particles. The slope of the curve for control PSI particles is less than that for stock PSI when using 650nm actinic light- Thus incubation at pH 6.0 inhibited the energy transfer within PSI particles. This may have been due to iso electric precipitation which begins at pH 6.0 (Figure 11). When 650nm light was used for illumination, the slope of the curve for EDA-PSI is greater than the slope for stock PSI. Using 710 nm light, the slope is approximately the same for all three types of PSI particles. These - results indicate that a primary effect of chemical modification is to increase energy transfer from light- harvesting chlorophyll a molecules to P700. Gross and Grenier (42) demonstrated that the + 2 addition of Mg ions to PSI particles resulted in an increase in energy transfer from light-harvesting chlorophyll a to P700. They used circular dichroism +2 to show that the Mg ions also produced a large conform- Figure 13. The effect of chemical modification on P700 oxidation in PSI particles. See the Materials and Methods for the details of the assay. Control PSI particles (0-0). Stock solution of PSI particles (4 4 ). EDA-PSI (□— a). 100 Initial Oxidation Rate (^tmoles P700 -m g c h f'-h r" 20 40 20 30 50 10 - 0 iue 13 Figure n o i t a n i m u l l I m n 0 1 7 650 nm Illumination 650 0.2 I0' (rscr - -1) c e 2-s (ergs-cnrf '4 0 I x i 0.4 0.6 0.8 1.0 101 1.2 102 ational change. This conformational change must orient the light harvesting chlorophyll molecules into a more efficient network for transferring the energy from absorbed photons to the reaction center. The negative charges on the surface of PSI part icles must be a factor in determining the conformation of this protein complex. Chemically changing the charge +2 of the PSI complex, has the same effect as Mg ions on +2 energy transfer. Therefore, the role of Mg in regulation of energy transfer within the PSI complex is to bind to protein carboxyl groups to make the surface of the complex more positively-charged. This positively- charged environment creates a PSI particle conformation capable of more efficient energy transfer. The Effect of Chemical Modification on the Dark Processes of PSI Particles on the Oxidizino Side of P700. + The initial rate of P700 reduction can be analyzed by Michaelis-Menton kinetics (43) for comparison of divalent cation and chemical modification effects on the dark processes of PSI particles. The apparent K is a measure of the binding of the electron donor to PSI particles. The Mnax reflects the environment of + the electron transfer from the donor to P700 . Four 103 different electron donors were used to analyze the system. Ascorbate and DCIP are negatively-charged artificial electron donors. TMPD is a positively-charged artificial electron donor. The fourth electron donor is spinach plastocyanin which is a negatively-charged copper protein + that serves as the in vivo electron donor to P700 (13, 58). The results for the artificial electron donors are complex. A majority of the data can be explained on the basis of the electrostatic interaction between the electron donor and the PSI particles. An electrostatic repulsion exists between the negatively-charged control PSI particles and negatively-charged electron donors. Removal of this electrostatic repulsion would be expected if MgCl^ was added to screen negative charges on the molecules. This result was observed as a decrease in the K m for ascorbate when M gC^ was added to control PSI particles (Tables 7 and 8). Decreasing the negative charge on PSI particles by chemical modification of carboxyl groups should also eliminate electrostatic repulsion. The low K m's for ascorbate donation to P700+ in EDA-PSI and GEE-PSI support this idea. These results are consistant with those of Itoh (54) who found that addition of cations to broken chloroplast membranes caused an increase in the rate of P700 Table 7 Electron Donation to P700+ in EDA-PSIa Electron Donor______Control PSI______EDA-PSI -MgCl2 +5roM MgCl2 -MgCl2 +5mM MgCl2 t Ascorbate K b 278-12 88^5 3.5^0.1 7.6-0.3 m V c 3lil8 18-3 22^1 20±4 max DCIP K 7 9-0 3 16.5-6 1.9±0.1 2.8^0.1 m . . V 17±3 111^52 90^10 max 67-13 TMPD 135±6 2ooie 123^3 201-10 V 120-33 193^77 110^18 179^77 max Plastocyanin K d 32.6^1.2 1.3-0.1 2.0^0.1 m V d 263^130 130^56 289^103 max 104 a - J - The rate of P700 reduction was measured as described in the Materials and Methods. ^Units for K are mM for ascorbate and iiM for all other electron donors. c + Units for are umoles P700 /mg chl/hr. ^ ITkIX ^ No reaction. Table 8 Electron Donation to P700+ in GEE-PSI3 Electron Donor Control PSI Particles GEE-PSI -MgCl2 +5mM MgCl, -MgCl2 +5mM MgCl2 Ascorbate K 81^5 7.4^1.0 0.019-0.002 0.014^0.002 m 16.5i4.3 9.7±0.9 12 0 0.4 9.9^0.5 max .^ Plastocyanin K d 18.7-1.2 3.9^0. 3 m 0 .8±0.1 V d 261^120 134^56 131^69 max The rate of P700 reduction was measured as described in the Materials and Methods. Units for Km are mM for ascorbate and jjM for plastocyanin. CUnits for Vmax are jimoles P700+/mg chl/hr. No reaction. 106 reduction by negatively-charged artificial electron donors. He found that the increase in the rate was the result of lowering the surface potential of the negatively-charged membranes which allowed the electron donor a greater accessibility to P700. In this study, the Km values reflect the surface potential of PSI particles. A lowering of the surface potential +2 (electrostatic repulsion) by Mg or chemica 1 modification produced a decrease in Km for ascorbate. It would be predicted from this model that M g C ^ would inhibit the electrostatic attraction between ascorbate and the positively-charged modified PSI particles. This result was observed and was expressed as an increase in the K for ascorbate donation to EDA-PSI. m The data for DCIP and TMPD do not completely support a simple electrostatic model. Such a model would predict that MgC^ would decrease the for DCIP donation to P700+ in control PSI particles by decreasing electrostatic repulsion. Instead, an increase was observed (Table 7) which contradicts the model. The predicted decrease in Km for DCIP was observed for EDA-PSI. The model would suggest that an electrostatic attraction should exist between positively-charged TMPD and negatively- charged control PSI particles. MgC^ would be predicted 107 to inhibit this interaction. This predicted inhibition +2 by Mg was observed as an increase in the for TMPD donation to P700 in control PSI particles. However, the expected increase in Km for TMPD donation to positively-charged EDA-PSI was not observed. Therefore other factors such as protein conformation play a role in the binding of artificial electron donors to PSI particles. Electron donation from artificial donors to PSI particles is regulated by the net charge on the protein through a combination of factors. One factor is simple attraction or repulsion between the donor and the PSI complex. The protein conformation at the site of electron donation must also be a factor. The role of Mg +2 in these processes is to bind to carboxyl groups on the PSI particles to alter the net charge of the complex. This results in charge screening or protein conformational changes that regulate electron donation. Addition of MgC ^ increased the Vmax for DCIP and TMPD (Table 7) although the statistical significance of the TMPD data is questionable. However, Gross(43) did observe a statistically significant increase in the Vmax for TMPD donation to isolated PSI particles. The Vmax for DCIP donation to EDA-PSI was also increased compared 108 to control PSI particles. This increase in V max implies that MgCl2 and chemical modification alter t the environment of PSI particles where the electron is transferred from a donor to P700+ . This alteration would require a change in protein conformation that must he the result of changing the electrostatic environment +2 of the protein by the binding of Mg or chemical modi- fication. This conclusion is supported by the MgCl^ induced conformational change observed for isolated PSI particles (42). Divalent cations also play an important role in regulating electron transfer from plastocyanin to P700+ in both chloroplast membranes and isolated PSI particles. Tamura et al. (100) and Haehnel et al. (45) have shown "I* that plastocyanin donation to P700 in chloroplast membranes was stimulated when cations were present. They found that this stimulation was due to a decrease in membrane surface potential which allowed plastocyanin more accessibility to P700. There is an absolute requirement of divalent cations for the interaction of plastocyanin with control PSI particles. Negatively-charged spinach plastocyanin was not able to donate electrons to P700 the absence of MgCl2 (Figure 14). The addition of Mg+2 Figure 14. The effect of MgCl„ on electron donation by plastocyanin to P700 in PSI particles. The initial rate of P700+ reduction was measured as described in the Materials and Methods. 109 RATE (/xmoles P700+-m gchr'-hr"1 50 70 90 30 2 6 1 1 1 1 1 2 22 20 18 16 14 12 10 8 6 4 2 0 iue 14 Figure PLASTOCYANIN Mg+5mM PLASTOCYANIN-Mg Cl C\z 110 Ill allowed high rates of P700+ reduction- Cation stiraula- + tion of P700 reduction by plastocyanin has also been observed by others (68) who study isolated PSI particles. Chemical modification of PSI particles completely +2 abolished this Mg requirement. The stimulation + of P700 reduction by MgC^ or chemical modification of PSI particles was the result of lowering the for plastocyanin binding and not an effect on Vmax (Tables 7 and 8). The work of others (30) also supports the conclusion that MgC^ affects the binding of plasto cyanin. Compared to control PSI particles in the presence +2 of Mg3 ions, EDA-PSI and GEE-PSI have much lower K m 's for plastocyanin. This suggests that chemically re placing negative charges on PSI particles at the site of the PSI/plastocyanin interaction is more effective than charge shielding by divalent cations. However, the most dramatic effect is that either addition of MgC^ or making the PSI particles positively-charged by chemical techniques both facilitate the binding of +2 plastocyanin to PSI particles. The role of Mg ions must be to decrease the electrostatic repulsion between plastocyanin and PSI particles which are both acidic proteins. 112 The Effect of Chemical Modification on Cytochrome f Content and Photooxidation in’ PSI Particles In the chloroplast membrane, electrons are trans ferred in a series of steps from cytochrome f to plastocyanin which in turn donates the electrons to P700+ (36, 45, 84). When the membranes are solubilized by Triton x-100, cytochrome f is isolated as part of the PSI complex by our procedure (Table 3). When PSI particles were chemically modified, the amount of cytochrome f decreased by 7525 in both EDA-PSI and GEE- PSI as determined by reduced minus oxidized absorption spectra (Table 9). Two possibilities exist to explain this observation. First, the modification reaction may have released cytochrome f from the PSI complex. However, the cytochrome f should still be observed because the dialysis tubing used to remove ethylenediamine from the protein after chemical modification should have retained cytochrome f (molecular weight = 32500 (109)) in the sample. The second possibility is that the modi fication reaction had modified cytochrome f so that it was no longer capable of under going chemical oxidation and reduction. The cytochrome f present in PSI particles is capable of donating electrons to P700+ after the reaction 113 Table 9 The Effect of Chemical Modification on the Cytochrome Content of PSX Particles3 Isolated Ratio PSI Particles EDA-PSI GEE-PSI chlorophyll/P700 114 114 119 cytochrome f/P700 0.80 0.20, 0.15 cytochrome bg/P700 2.3 1.5 1.4 aSee the Materials and Methods for details of measuring the chlorophyll, P700, and cytochrome content of a sample. 114 center is oxidized by light. This is observed as an absorption change in the «><.-band of cytochrome f at 554nm {see Trace A in Figure 21). Ascorbate present in the assay serves as a source of electrons for oxidized cytochrome f. Because cytochrome f is not capable of donating electrons directly to P700 in the chloroplast membrane (36, 60, 84), the isolation procedure for PSI particles must alter the orientation of cytochrome f with respect to P700 so that electron transfer can occur. After chemical modification of PSI particles, only approximately 25% of the original cytochrome f in the complex remains. This remaining cytochrome f can no longer donate electrons to P700 . This also supports the statement that the chemical modification reaction has an inhibitory effect on cytochrome f. Isolation of Spinach Plastocvanin The plastocyanin isolation procedure described in the Materials and Methods consistantly yielded purified plastocyanin with an A275/A597ox ratio of 1.2 to 1.5. The purified protein eluted as a single peak by both Sephadex G-75 gel filtration and DEAE-cellulose ion exchange chromatography using linear NaCl gradients. The purified plastocyanin consisted of a single polypeptide 115 of 11,000 molecular weight as determined by SDS polyacrylamide gel electrophoresis. This molecular weight agrees with the molecular weight previously determined for plastocyanin (13). During the first isolation, the chloroplast membranes were isolated on consecutive days and then frozen until the treatment of the membranes with acetone was performed. The plastocyanin from this preparation had a A275/A597ox ratio of 1.8 which could not be lowered by any technique. This was probably due to the freeze- thaw process which has been shown to activate a poly- phenolase enzyme that destroys plastocyanin (32). For this reason, it is important that the plastocyanin isolation procedure be completed up to the point of re- suspending the acetone precipitated protein during the first day of the isolation. After this point, the remaining steps of the procedure can be completed at the convenience of the experimentalist. The isolated plastocyanin is stable for months when frozen at -20°C. Repeated freezing and thawing will denature the protein so that it is wise to freeze purified plastocyanin in aliquots that can be used in a short period of time. When in use, purified plastocyanin can be stored at 4°C for several weeks before the solution 116 becomes cloudy due to denatured protein. Chemical Modification of Plastocyanin Spinach plastocyanin was chemically modified using EDC to form an amide bond between protein carboxyl groups and one amino group of ethylenediamine. The chemical modification procedure described in the Materials and Methods was designed to minimize the exposure of plastocyanin to ethylenediamine. The ethylenediamine is a chelating agent which removes the copper ion from the protein. This chelating effect was first observed during initial experiments in which the ethylenediamine was dialyzed into the plastocyanin sample. After over night dialysis against 0.2M ethylenediamine pH 6.0, 60-80% of the copper was removed from the protein as judged from the A275/A597ox before and after dialysis. No increase in the A275/A59 7ox ratio was observed when ethylenediamine was added to plastocyanin immediately before chemical modification and then quickly removed by gel filtration after the reaction. Whether modified in the oxidized or reduced state, the properties of the modified plastocyanin were the same. Therefore no attempt was made to standardize the redox state of the protein before modification. 117 The protein concentration in the modification reaction mixture did no't affect the characteristics of the modified plastocyanin. Plastocyanin was modified at protein concentration from 100 ^iM to 500 jiM during this study. Since the concentration of ethylenediamine (0.2M) and EDC (0.05M) used in the modification reaction con- sistantly produced modified plastocyanin with the required properties, no attempt was made to examine the concentra tion dependence of these reagents. A pH of 6.0 - 0.1 was determined to be ideal for the modification reaction. A pH below this value was not considered to be feasible because the two histidine ligands to the copper have pK^'s near pH 5.0 (27). A pH significantly below 6.0 might disturb the copper environment due to partial protonation of the histidine residues. Plastocyanin modified at pH 6.5 still required +2 + Mg for electron donation to P700 in a manner similar to isolated plastocyanin. For these reasons, pH 6.0 was chosen as the pH for chemical modification of plasto cyanin . Absorption Spectra of Oxidized Plastocyanin Before and After Chemical Modification The Cu (II) ion in oxidized plastocyanin is coordinated 118 to the side chains of four amino acid residues in the protein. These ligands are the thiolate group of cysteine 84, the thioether group of methionine 92, and the imidazole nitrogens of histidines 37 and 87 (21) . The electronic transitions of this complex produce a plasto cyanin absorption spectrum characterized by a major absorption band with a maximum at 597 nm and two minor bands with maxima at 460 nm and 775 nm (99). Except for a small difference in sample concentration, the room temperature absorption spectra of control and modified plastocyanin are identical (Figure 15). Incorporation of Ethylenediamine Molecules into Plasto cyanin by Chemical Modification The incorporation of ethylenediamine into plasto cyanin by the modification of protein carboxyl groups 14 was examined by using C-ethylenediamine. The average incorporation was found to be 4.5 - 0.6 moles of ethyl enediamine per mole of plastocyanin. Including the carboxyl terminus of the protein, spinach plastocyanin contains 16 carboxyl groups. The modification of an average of four or five carboxyl groups resulted in the large alterations in electron donation capability and redox midpoint potential of plastocyanin which are Figure 15. Absorption spectrum of oxidized plasto cyanin. Absorption spectra were recorded as described in the Materials and Methods. Control plastocyanin (-----■ ). Modified plastocyanin (---). 119 ABSORBANCE 4 . 0 0.2 0 0 6 0 0 4 Figure 15 Figure 0 0 5 WAVELENGTH (nm) 0 0 7 0 0 8 120 121 discussed in the following pages. The molecular weight of control and modified plastocyanin was 11,000 which was determined by SDS polyacrylamide gel electro phoresis. This indicates that the alteration of plasto cyanin activity by the chemical modification process is the result of replacing negatively-charged carboxyl groups with positively-charged amino groups and not the result of protein cross-linking. Ethylenediamine can be incorporated into plastocyanin only by the EDC catalyzed modification of protein carboxyl groups. The EDC molecule is also known to react with cysteine (20) and tyrosine (19) to form stable S-acylisoureas and O-arylisoureas, respectively. Spinach plastocyanin contains only one cysteine which is one of the four copper ligands. Because the absorption spectrum of plastocyanin is not altered by chemical modification (Figure 15), it is concluded that EDC has not reacted with the cysteine residue. Only indirect evidence has been obtained to show that EDC has not reacted with the three tyrosine residues of spinach plastocyanin. EDC forms an O-arylisourea derivative with tyrosine which is moderately stable to acid hydrolysis (19) . In experiments to be discussed later, modified plastocyanin was hydrolyzed with trypsin and the peptide fragments separated and 122 analyzed for amino acid composition. The tyrosine residues were found in the appropriate peptides and in the expected relative concentrations. This suggests that the reaction of EDC with tyrosine is not a major factor in the modification of plastocyanin. However, the presence of a small number of modified tyrosine residues cannot be ruled out. Separation of Modified Plastocyanin Species The chemically modified plastocyanin was found to be a heterogeneous mixture of plastocyanin molecules modified to different extents. This was determined by using polyacrylamide gel electrophoresis which separated the plastocyanin molecules on the basis of charge. In this gel system, the most negatively-charged proteins will have the largest relative mobility. Native plasto cyanin has a large relative mobility of 0.83 (see trace F in Figure 16). The modified plastocyanin contained four major bands with smaller relative mobilities than native plastocyanin (see trace A in Figure 16). Two conclusions can be made from these results. First, all of the plastocyanin was chemically modified to some extent because the modified plastocyanin contained no protein which migrated with native plastocyanin. Figure 16. Polyacrylamide gel electrophoresis of chemically modified and native spinach plastocyanin. The electrophoresis was performed as described in the Materials and Methods. The gels were scanned at 600 nm after staining with Coomassie blue. Key: A. Modified plasticyanin mixture B. Modified plastocyanin fraction I (see figure 17) C. Modified plastocyanin fraction II (see figure 17) D. Modified plastocyanin fraction III (see figure 17) E. Modified plastocyanin fraction IV (see figure 17) F. Native plastocyanin 123 124 in O ii o o 10 < Figure 16 Second, the modified plastocyanin was a mixture of four major populations of plastocyanin which have different charges due to different extents of modification. The smaller relative mobilities of the modified plastocyanin species indicated that modified plasto cyanin was less negatively-charged than the control plastocyanin, which is an extremely acidic protein {pi = 3.0, ref. 30). This would be expected since the chemical modification reaction replaces negatively- charged barboxyl groups with positively-charged amino groups. Two lines of evidence indicated that the modified plastocyanin proteins carried a slightly negative or neutral charge. (1) The replacement of an average of five carboxyl groups on plastocyanin with amino groups results in a protein with twelve amino groups and eleven carboxyl groups. This modified protein would have an approximately neutral charge at pH 7. (2) The modified plastocyanin did not bind to CM-Sephadex which is a cation exchange resin that binds the positively-charged plastocyanin from Anabaena Variabilis (69) . The mixture of modified plastocyanin molecules can be separated by ion exchange chromatography on DEAE- Sephadex into four protein entities (Figure 17). The Figure 17- Separation of modified plastocyanin species by ion exchange chromotography on DEAE-Sephadex. The conditions for separation are described in the Materials and Methods. Each fraction contained 6.5 milliliters. 126 ABSORBANCE AT 2 7 5 nm 0.04 0.06 0.02 0.08 0 iue 17 Figure 20 FRACTION 40 8060 100 50 127 i o o s I 128 fractions were combined as shown in Figure 17 and then concentrated by ultrafiltration. Polyacrylamide gel electrophoresis was performed on each fraction and the results are shown in Figure 16. Fractions I, II, III, and IV correspond to-gel scans B, C, D, and E,respectively. The proteins in each fraction were correlated with the four proteins found on polyacrylamide gels. The proteins eluted from the column in an order that proceeded from most positively-charged to most negatively-charged as indicated by the relative mobility of each protein fraction in the polyacrylamide gel. .This would be expected since DEAE-Sephadex is an anion exfchange resin which will have a higher affinity for the more negatively-charged molecules. The incorporation of ethylenediamine into each fraction also followed this pattern in which the amount of label decreased in order from fraction I to fraction IV (see Table 10). All of these data agree with the conclusion that the chemical modification reaction produced a series of plastocyanin derivatives with different charges due to different amounts of ethylenediamine incorporation. The conditions described in the Materials and Methods for separation of the modified plastocyanin species were chosen for several reasons. A similar elution pattern was 129 Table 10 Incorporation of Ethylenediamine in the Modified Plastocyanin Species moles ethylenediamine Fraction3, mole plastocyanin Control 0 Mixture of fractions I, II, III, and IV 5.1 I 6.3 II 4.1 III 3.2 IV 2.1 aThe symbols I, II, III, and IV refer to the modified plastocyanin fractions shown in Figure 17. See the Materials and Methods for the details of this determination. observed if the modified plastocyanin was reduced with ascorbate before application to DEAE-Sephadex. In fact, a better separation of fractions I and II occurred when the reduced protein was used. However, the reduced protein was less stable during the separation which was observed as an increase in the A275/A597ox ratio of the separated protein fractions. For this reason, modified plastocyanin was oxidized before separation. Notice that the separation of fractions I and II occurs due to differences in the affinity of these protein species for DEAE-Sephadex at the same ionic strength (i.e. 50 mM Tris-HCl pH 8.0). If the concentration of initial equilibration buffer was increased from 50 mM to 100 mM, fractions I and II eluted in a single peak. Presumably the separation of fractions I and II would be more complete if the initial ionic strength of the column were decreased below 50 mM Tris-HCl pH 8.0. Fractions III and IV required an increase in ionic strength by addition of NaCl to the buffer in order to elute from the column. Under all conditions tested, the modified plastocyanin species eluted from the column in a large volume of dilute protein. The Effect of Modification of Plastocyanin on the . Kinetics of F700+ Reduction. 131 H* The reduction of P700 in PSI particles by isolated spinach plastocyanin required the presence of divalent cations as has already been discussed (Figure 14). Cation stimulation of P700+ reduction by plastocyanin has been previously observed for isolated PSI particles (68) and broken chloroplast membranes (45,100). This demonstrates the existance of electrostatic regulation + of the interaction between plastocyanin and P700 which has been discussed earlier in this dissertation. Initially, the ability of the mixture of modified “f* plastocyanin species to donate electrons to P700 was examined. The mixture of chemically modified plastocyanin +2 t no longer required Mg for electron donation to P700 . Chemical modification of plastocyanin effected the binding (K^) of plastocyanin to PSI particles and not the rate of electron transfer (vma:jC) from reduced plastocyanin to Am P700 (Table 11). Modified plastocyanin had a much lower than control plastocyanin, which indicated that replacing negative charges on the protein with positive charges was more effective in allowing plasto cyanin to interact with PSI particles than charge screening +2 +2 by Mg ions (Table 11, Figure 18). Adding Mg to modified plastocyanin caused an increase in the Km which indicated an inhibition of binding (Table 11, Figure 19). 132 Table 11 The Effect of MgCl2 and Chemical Modification on Plastocyanin Donation to P700 Control Plastocyanin Modified Plastocyanin Mixture -MgCl. +5mM MgCl, -MgCl2 +5mM MgCl, K b 2.1-0.08 m 36.5-2.0 5.7^0.3 V b 176^65 max 153^38 100^52 a These parameters were determined from the double reciprocal plots seen in Figures 18 and 19. ^No reaction. cUnits for K are 11M. m Units for Vmax are /imoles P700 /mg chl/hr. Figure 18. Comparison of MgCl^ .and chemical modi fication on electron donation from plastocyanin to P700 . See the Materials and Methods for details on the measurement of P700 reduction. 133 RATE'1 (/i.moles PTOO^-mgchr'-hr'1 0.2 0.15 5 0 . 0 0 0 iue 18 Fiqure 2 C O N T R O L P L A S T O C Y A N IN + 5 m M M g Cl g M M m 5 + IN N A Y C O T S A L P L O R T N O C 4 6 [PC]"' 8 FIED PLASTOCYANI - Cl2 C g M - IN N A Y C O T S A L P D E I IF D O M 10 12 14 16 134 Figure 19. The effect of MgC^ on the ability of modified plastocyanin to donate electrons to P700+ . See the Materials and Methods for details on the measure ment of P700+ reduction. 135 0.20 t _c J MODIFIED PLASTOCYANIN+5 mM Mg Cl. I 0.16 JE 0 CP E 1 0.12 + O O N- CL 0.08 if) Ui 0 0 3 4 5 8 £ 136 (T Ip c ]-' Pi 137 +2 However, this Mg effect on modified plastocyanin was variable in that some preparations showed little or no inhibition. +2 The variable effect of Mg on modified plasto cyanin is probably a reflection of the fact that modified plastocyanin is really a mixture of four plastocyanin derivatives. The K of the mixture of modified plasto- m cyanin species is some complicated function of the of each species. The overall trend in the values of the separated species (see Table 12) is consistant in all preparations of modified plastocyanin. However, the actual values of Kjn vary from one preparation to the next which will produce differences in both the +2 value of the mixture and the Mg effect on this The net charge on the plastocyanin molecule is an important factor in determining the ability of plastocyanin to donate electrons to P700+ . In the absence of cations, the ability of plastocyanin to donate electrons increased as the protein charge approached neutrality (Table 12). Control plastocyanin and modified plastocyanin fraction IV* were not capable of donating electrons to P700+ in +2 the absence of Mg . Fraction III was a very poor electron donor. Only fractions I and II, where the net protein charge is positive or near neutrality, can plastocyanin Table 12 P700+ Reduction by the Separated Modified Plastocyanin Species3 Fraction Net protein0 P700 Reduction charge at -MgCl +5mM MgCl pH 7.0 V K ^V K d max m max m Control -9 f 9.4^0.3 157^31 IV -5 f 2.3^0.1 74±12 III -3 5.1±0.2 12^2 2.3^0.1 89±24 II -1 3.6±0.4 154±62 1.8^0.2 134^63 I +3 0.4-0.03 220^97 1.4^0.06 174^59 aSee the Materials and Methods for details on the measurement of P70Q+ reduction. i . The symbols I, II, III/ and IV refer to the modified plastocyanin fractions shown in Figure 17. CEstimated from the known amino acid sequence of spinach platocyanin (Figure 22) and the amount of ethylenediamine incorporated into each species of modified plastocyanin (Table 10). Units for Km are jiM. 138 Units for Vmax are jjmoles P700 /mg chl/hr. ^No reaction. 139 + donate electrons to P700 effectively. Fraction I had the lowest which is consistant with the overall trend that the binding of plastocyanin increased as the net protein charge becomes more positive. In the presence of MgC^* this trend is continued. Although MgC^ allowed all of the plastocyanin molecules to donate electrons at high rates, the values still reflect the protein charge. In the presence of MgCl2 the decreased as the protein charge became more positive. Except for control plastocyanin and fractions III and IV which are very poor donors in the absence of MgC^ (Table 12), the values for Vmax were the same in all cases within experimental error. The absolute values for Vmax varY substantially. However, the large error + associated with the P700 assay at high rates make these differences in Vmax have little or no significance. Interpretation of small differences would provide more frustration than useful information. Other investigators have studied the plastocyanin/ PSI particle interaction by examining plastocyanin and plastocyanin analogs from several sources. Many algae have replaced plastocyanin with a small C-type cytochrome (C-553) which has an isoelectric point that can vary from acidic to basic depending on the source. In addition, the 140 algae Anabaena variabilis produces a very basic form of plastocyanin. Davis et al. (30) used plastocyanin and cytochrome C-553 from various sources as electron donors to P700 in the PSI particles from spinach. They concluded that the donor was able to interact with P700 more effectively as the charge on the donor became more positive. This agrees with the results presented in Table 12. Davis et al. (30) also observed that MgC^ increased the binding of donors with isoelectric points below 5.0 while inhibiting the binding of donors with isoelectric points above 5.0. This agrees with the results shown in Table 12. MgCl„^2 induced a decrease in K m for control plastocyanin and the modified plastocyanin species II, III, and IV which have net negative charges. The was increased for the positively-charged modified plastocyanin in fraction I (Table 12). All of the above experiments suggest an electrostatic regulation of the plastocyanin/PSI particle interaction. Neutral or basic donors bind effectively to the negatively- charged PSI complex. Acidic donors such as control spinach plastocyanin require cations to screen negative charges on the two molecules in order to facilitate their interaction. 141 One final observation should be made at this point. The mixture of modified plastocyanin molecules was capable of high rates of electron donation to EDA- FSI with low apparent values. However, the kinetics of the mixture are really a combination of the kinetics of the individual modified plastocyanin species. Further experiments are required to examine the interaction of the individual modified plastocyanin species with EDA- PSI. It might be predicted that EDA-PSI will not interact with modified plastocyanin fraction I because they are both positively-charged proteins. The Effect of Chemical Modification on the Oxidation- Reduction Potential of Plastocyanin The oxidation-reduction midpoint potential of plastocyanin was found to be altered by the modification process (Figure 20). A midpoint potential of approximately +367mV was found for control plastocyanin which was close to the value of 370 mV previously reported for spinach plastocyanin (13). Modified plastocyanin had a midpoint potential of +399 mV which is an increase of +33mV. One possible explanation for this change involves differences in the local concentrations of ferrocyanide and ferricyanide at the protein surface compared to the Figure 20. The effect of chemical modification on the redox midpoint potential of plastocyanin. See the Materials and Methods for details of the measure ment. Control plastocyanin (&-- &.) . Modified plasto cyanin mixture (0-0). 142 o CP PC ox. CL O o * Q> 04 L— -0.4 0.8 0.4 0.6 0.2 0.2 0.32 iue 20 Figure (volts)E 0.40 0.440.36 143 bulk solution. Regions of the modified plastocyanin molecule may be positively-charged as the result of replacing carboxyl groups with positively-charged amino groups. Ferrocyanide would be attracted more strongly to the positive protein surface because it carries more negative charge than ferricyanide. An increase in the local ferrocyanide concentration would artifactually increase the redox potential of modified plastocyanin. If this phenomenon were occurring, the redox potential of modified plastocyanin should be dependent on the total ferrocyanide plus ferricyanide concentration in the bulk solution (31). However, the redox potential of modified plastocyanin was identical when the measure ment was performed under conditions where the ferricyanide plus ferrocyanide concentration was held constant at either 0.33mM or 2.64mM. Therefore the positive increase in the redox potential indicates a stablization of the reduced Cu (I) state of plastocyanin compared to the oxidized Cu (II) state. Brill et al. (16) suggested that the steric constraints of a protein were not likely to provide a very flexible geometry of ligands for binding copper. This steric constraint would tend to stabilize the Cu (I) ion because it has less rigid stereochemical demands 145 (16). Cu(II) requires a four coordinate complex while Cu(l) can exist in a complex with only two ligands. The ability of thiol reagents to react with the cysteine ligand in the reduced form of plastocyanin and not the oxidized form (58) may indicate that the Cu(I) has a lower coordination number than the Cu(II) ion. The Cu(I) form of plastocyanin is stabilized because the constraints of the copper site in the protein make two coordinate geometry much easier to achieve than a four coordinate system. The coordination geometry of Cu(II) in poplar plastocyanin (21) and bean plastocyanin (99) has been found to be a flattened tetrahedral unit. The effect of this distortion of the copper geometry is to stabilize Cu(I) which gives plastocyanin a very positive redox potential of +370 mV. A high redox potential is required by plastocyanin because it is part of a photo synthetic electron-transfer sequence in which its redox partners cytochrome f and P700 have high redox potentials of +365 mV (24) and +450 mV (35), respectively. The further stabilization of Cu (I) in modified spinach plastocyanin may be due to conformational altera tions brought about by incorporation of ethylenediamine 146 molecules near the copper site. The incorporated ethylenediaraine molecules not only change the local ionic environment but would also create a modified amino acid residue with a more bulky side chain. These changes could produce additional steric strain in the copper environment which would stabilize the Cu (I). A portion of this strain must be the result of some type of ionic phenomenon because reduction of ionic inter actions by adding NaCl lowered the redox potential of modified plastocyanin in the direction of control plastocyanin by about 22 mV (Table 13). The difference in the redox potential of the control plastocyanin in Figure 20 and Table 13 may be the result of using different buffers in the assay. After separation, each of the modified plastocyanin species was found to have an increase in redox potential by at least +38mV compared to the control (Table 14). In the case of fraction IV, a large increase in redox potential is observed after the incorporation of only two molecules of ethylenediamine. Then the redox potential continued to increase gradually as additional ethylene diamine molecules were incorporated into fractions III, II, and I. These results are in contrast to the results ■f for P700 reduction in which the incorporation of two 147 Table 13 The Effect of Sodium Chloride on the Oxidation- Reduction Potential of Modified Plastocyanin3 Midpoint potential (mV) —NaCl +100mM NaCl control plastocyanin 383 380 modified plastocyanin 423 401 aThe redox midpoint potential was measured as described in the Materials and Methods except that 50mM sodium borate pH 8.0 was used as a buffer instead of 50mM Tris - HCl pH 8.0. 148 Table 14 The Redox Midpoint Potential of the Separated Modified Plastocyanin Species moles ethvlenedi amine Midpointc Fraction mole plastocyanin Potential CONTROL 0 367 I3 6.3 416 I i a 4.1 410 II i a 3.2 405 iva 2.1 405 aThese symbols refer to the modified plastocyanin fractions shown in Figure 17. These values are taken from Table 10. cSee the Materials and Methods for details on the measurement of plastocyanin midpoint potential. 149 ethylenediaraine molecules had only a small effect on the - - * | i ability of plastocyanin to donate electrons to P700 (Table 12). Therefore the modification of the first two carboxyl groups on plastocyanin has a profound effect on the copper environment while the donation of electrons from plastocyanin to P700+ requires that several carboxyl groups be modified. This may imply that the effect on the copper site is due to the modific&tion of a specific region of the protein while electron donation is regulated by the overall net charge on the protein. The Effect of Chemically Modified Plastocyanin on Cytochrome f Photooxidation in PSI Particles The PSI particles used in this study contained approximately one mole of cytochrome f for each mole of the P700 reaction center (Table 3). The cytochrome f present in PSI particles is capable of donating electrons to P700+ after the oxidation of the reaction center by light. This electron transfer process is observed as a decrease in absorbance at 554nm upon oxidation of cytochrome f (trace A in Figure 21). The light minus dark absorption spectrum showed a symmetrical peak around 554nm which indicated that the signal came from oxidized cytochrome f. Under the conditions employed, Figure 21. Cytochrome f photooxidation in PSI particles. Cytochrome f photooxidation was performed as described in the Materials and Methods. K e y : Trace Conditions A. Cytochrome f photooxidation performed in the presence of 50 yuM ascorbate B. Trace A plus 1 p. M modified plastocyanin fraction I C. Trace A plus 1 p. M modified plastocyanin fraction II D. Trace A plus 1 p. M modified plastocyanin fraction III E. Trace A plus 1 /i M modified plastocyanin fraction IV F. Trace A plus 1 jj. M control plastocyanin G. Traces A through F plus 20 mM MgCl^ 150 151 LIGHT ON LIGHT OFF 20 seconds Figure 21 152 approximately 50% of the cytochrome f present in the sample was oxidized by steady-state illumination. The oxidized cytochrome f is reduced by ascorbate present in the assay. The rate of ascorbate reduction of cytochrome f is very slow which is shown as the slow recovery of the 554 absorbance change when the actinic light is removed (trace A in Figure 21). In the presence +2 of Mg , the initial rates of both cytochrome f oxidation and reduction are increased (trace G in Figure 21). The effect of control plastocyanin and each of the separated modified plastocyanin species on cytochrome f photooxidation is also seen in Figure 21. In the presence of control plastocyanin (trace F) or modified plastocyanin fractions III (trace D) and IV (trace E), the initial rates of cytochrome f oxidation and reduction are the same as for ascorbate alone (trace A). Therefore control plastocyanin and fractions III and IV have no effect on cytochrome f photooxidation. The initial rate of cytochrome f oxidation is increased in the presence of modified plastocyanin fractions I (trace B) and II (trace C). The modified plastocyanin in fractions + I and II can donate electrons to P700 in the absence of MgC^ which must result in the reconstitution of the 4* native pathway for cytochrome f electrons to reach P700 . 153 This produced a stimulation of cytochrome f photo oxidation. Fractions I and II have no effect on cytochrome f reduction as would be expected since it, is ascorbate that reduced, cytochrome f (109) . When the assays shown in traces A through F (Figure 21) are repeated in the presence of 20mM MgC^/ the result is shown as trace G which is characterized as having fast initial rates of both cytochrome f oxidation and +2 reduction. This suggests that Mg increases the inter action of cytochrome f with P700 since both ascorbate and the various species of plastocyanin produce the same cytochrome f photooxidation. This is unfortunate since studies on the interaction of control plastocyanin with cytochrome f and P700 in PSI particles requires the presence of■e MgK/r "^2 The results shown in Figure 21 as well as other results discussed in this dissertation produce a compli cated picture of how electrons from ascorbate are trans ferred to P700+ in PSI particles. The data suggest that the four pathways shown below may operate alone or in some combination depending on the assay conditions. Ascorbate--- *P700+ (1) Ascorbate--- > cyt f -- > P700 (2) Ascorbate--- >cyt f -- > plastocyanin---- >P700 (3) Ascorbate---> plastocyanin-----^P700 (4) 154 Pathway (1) must exist for two reasons. Ascorbate alone produced fast rates of P700+ reduction in EDA- PSI where the cytochrome f content has been cut by 1 5 % as the result of chemical modification of PSI particles. Ascorbate can also reduce P70 0 in other preparations of PSI particles which contain no cytochrome f (68). Pathway (2) must exist because cytochrome f photooxidation is observed when only ascorbate is present (trace A in Figure 21). Because cytochrome f cannot donate electrons directly to P700+ in chloroplast membranes (36, 45, 60, 84), the isolation procedure for these PSI particles must alter the orientation of these components so that this pathway can exist. Other cytochrome f depleted PSI particles- (29) require the presence of both plastocyanin and MgClj before electrons from purified cytochrome f can be donated to P700+ . Control plastocyanin In the presence of MgCl^ and modified plastocyanin produce very fast rates of P700 reduction (Table 12). This supports the idea that electrons from ascorbate must pass through plastocyanin J. to get to P700 in PSI particles. Evidence for pathway (3) comes from the fact that modified plastocyanin fractions I and II stimulated the initial rate of cytochrome f photooxidation. Several lines of evidence 155 support the existence of pathway (4). When PSI part icles are illuminated, electrons are continuously being transferred from P700+ to oxygen. The P700+ is reduced by electrons at a rate which is equivalent to 4* the initial rate of P700 reduction. In the presence of 1 p. M modified plastocyanin fractions I and II, this + rate is approximately 150 and 40 ^jinoles P700 /mg chl/hr. , respectively, in the absence of MgCl^- Upon illumination, some of the electrons must initially come from cytochrome + f to P700 because cytochrome f oxidation is observed. However, the rate of ascorbate reduction of oxidized cytochrome f is too slow (approximately 2 ^pmoles cyt f/ mgchl/hr, see traces B and C in Figure 21) for a majority of the electrons to pass from cytochrome f to plasto- + + cyanin and then to P700 . The fast rates of P700 reduction suggest that a majority of the electrons are transferred directly from ascorbate to plastocyanin. The ascorbate reduction of plastocyanin is known to be a very fast process (58) which is not the rate limiting step of P700 ■ reduction in PSI particles (68). Pathway (4) is also supported by the fact that plastocyanin + produces fast rates of P700 reduction in EDA-PSI where the cytochrome f is no longer present. Therefore pathway + (4) is the major source of electrons for P700 reduction in the presence of plastocyanin while pathways (1) (2) , and (3) may- make only small contributions to the observed rate. In order to completely understand the role of cytochrome f in our PSI particles, much more extensive study is required which is beyond the scope of the work presented here. Identification of Modified Plastocyanin Peptides It was of interest to identify the portions of the plastocyanin molecule that are modified in order to examine structure-function relationships of the mole- 14 cule. This involved labeling the plastocyanin with C- ethylenediamine followed by specific hydrolysis with a protease and identification of the labeled peptides. Spinach plastocyanin contains six lysine residues and no arginine residues. Because trypsin hydrolyzes peptide bonds on the carboxyl side of lysine and arginine this proteolytic enzyme would be expected to cleave plastocyanin into the seven peptides shown in Figure 22. Native plastocyanin was found to be relatively stable towards extensive hydrolysis by trypsin. For this reason, plastocyanin was denatured in 8M urea before hydrolysis and the hydrolysis performed in 2M urea where trypsin retains its activity (9 8). The mixture of the Figure 22. The primary structure of spinach plastocyanin (reprinted from reference 93). The pep tides produced by trypsin cleavage of plastocyanin are shown. 157 h«------PI ------H2N - Val -Glu - Val -Leu - Leu - Gly -Gly - Gly - Asp-Gly - Ser - Leu - Ala -Phe-Leu - 1 5 10 15 ------Pro - Gly -Asp-Phe-Ser - Val -Ala - Ser-Gly - Glu - Glu - lie - Val -Phe-Lys- 20 25 30 P2 -Asn -Asn-Ala -Gly -Phe-Pro-His -Asn-Val - Val -Phe-Asp-Glu - Asp-Glu - 3 5 4 0 4 5 ------He - Pro- Ser - Gly - Val - Asp- Ala - Ala -Lys - lie - Ser - Met - Ser - Glu - Glu - 5 0 5 5 6 0 P 3 ------* + « ------P4 Asp-Leu -Leu-Asn-Ala - Pro -Gly -Glu -Thr -Tyr -Lys -Val -Thr -Leu-Thr - 6 5 7 0 7 5 P5 - Glu - Lys - Gly - Thr - Tyr - Lys - Phe - Tyr - Cys - Ser - Pro - His - Gin - Gly - Ala - 8 0 8 5 9 0 P 6------P7- - Gly -Met - Val - Gly - Lys-Val - Thr - Val -Asn-COOH 9 5 Figure 22 159 four species of modified plastocyanin was subjected to extensive hydrolysis by trypsin- After hydrolysis, the urea was removed from the peptides by gel filtration (Figure 23). The peptides that eluted in the void 14 volume (peak I) contained all of the C-ethylenediamine. Initially, peptide mapping of the void volume (peak I) and the lower molecular weight material (peak II) were performed separately. The main component of the lower molecular weight material was found to be plastocyanin peptide P5 (see Figure 22). All of the peptides were combined (I & II) for later peptide maps. The peptide map of the void volume material from Figure 23 is shown in the scaled schematic representa tion seen in Figure 24. The spots were located using the ninbydrin spray described in the Materials and Methods. Spot 1 was the sample origin. Spots 2-11 were spots which contained enough material for amino acid analysis after elution of the peptide from the paper and hydrolysis in HCl. The other spots contained too little material and were barely detectable with the ninbydrin spray. The overall pattern seen in Eigure 24 was very consistant from one peptide map to the next. However, the maps usually did not resolve spot 4 from both spot 2 and spot 5 which made it very difficult to identify the Figure 23. Removal of urea from modified plasto cyanin peptides after trypsin hydrolysis. The removal of urea from plastocyanin peptides was performed as described in the Materials and Methods. The void volume (blue dextran) was fraction 12. The included volume of the column (DNP-glycine) was fraction 70. 160 ABSORBANCE AT 225 nm 0.5 2.0 .0 0 iue 23 Figure 0 0 0 0 0 80 70 60 50 40 30 FRACTION 161 Figure 24. Schematic representation of the peptide map of modified plastocyanin. The peptides were separated and located as described in the Materials and Methods. A picture of the peptide map was then drawn to scale. 162 2 cm 2 cm 163 Fiqure 24 peptide in spot 4. The results of the amino acid analysis of the spots on the peptide map are seen in Table 15. Pep tide P2 was identified in spots 2 and 3 which contained 14 C-ethylenediamine. Peptide P3 was found in spots 6 and 7 which contained label. Peptide P4 was found in both a labelled (spot 8) and unlabelled (spot 10) form. This separation of identical peptides into more than one spot was the result of modified plastocyanin being a mixture of four plastocyanin molecules modi fied to different extents. Spot 5 contained a large amount of material and was heavily labelled. However, the amino acid content of spot 5 did not correlate with one of the plastocyanin peptides. This spot must consist of a mixture of components. One component must be peptide Pi because this was the only plastocyanin peptide not found in any of the spots on the peptide map. Other unknown components in spot 5 might be segments of plastocyanin that were not completely di gested by trypsin and therefore contained a combination of two or more of the peptides shown in Figure 22. The Physiological Role of the Modified Plastocyanin Amino Acids 165 Table 15 Identification of Peptides from Modified Plastocyanin i. c a c tiiyicu Spota Peptide*3 mole pe 1 sample origin contained 2 P2 1.3 3 P2 2.1 4 P6d d 5 not resolved contained 6 P3 1.9 CM T - * 7 P3 1 8 P4 0.8 9 P5 0 10 P4 0 11 P7 0 a Spots refer to the peptide map in Figure 24. k The naming system for plastocyanin peptides is in Figure 22. c See the Materials and Methods for details of this determination. j Due to an apparent overlap with spot 2, spot 4 was not positively identified as peptide P6. However, this spot contained the characteristic amino acids for peptide P6. Spot 4 contained carbon-14 due to overlap with spot 2. Peptide P6 should not be labelled since it does not contain aspartic or glutamic acid. A high degree of similarity exists between the sequences of plastocyanin isolated from a variety of sources (13). High resolution NMR studies indicate that the tertiary structures of many plastocyanins, including those from spinach and A. variabilis are very similar (33,34,72). The x-ray diffraction structure has been determined for poplar plastocyanin (21). Plastocyanin from spinach and poplar leaves have the same number of amino acids and a great deal of sequence homology (13,21). Davis et al. (30) pointed out that if the amino acid sequence of spinach plasto cyanin is projected onto the structure of poplar plasto cyanin, there is a ring of negatively-charged residues around the middle of the molecule (Figure 25). They also pointed out that if the sequence of A. variabilis plastocyanin is projected onto the structure of poplar plastocyanin, the negatively-charged amino acids in this ring are replaced by neutral or positively-charged amino acids. Because they observed a 100 fold decrease in the Km for A. variabilis plastocyanin compared to spinach plastocyanin, they suggested that the differences in the interaction of these donors with PSI particles were due to changes in the net charge of this ring. Amino acids 42-45, 59-61, and 68 (see Figure 22) of spinach Figure 25. The amino acid sequence of spinach plastocyanin projected onto the three dimensional structure of poplar plastocyanin. The three dimensional structure of poplar plastocyanin was reproduced from Colman et al. (21). The circles represent the JL. - carbons of each amino acid. The dark circles represent the charged amino acids from spinach plastocyanin. The letters N and C denote the amino terminal and carboxyl terminal residuestrespectively. 167 168 90 87 92 37 30 84 40 70 80 20 50 Figure 25 169 plastocyanin are acidic residues located in this ring which are highly conserved in higher plants (13). These sequences are lbcated within tryptic peptides 14 P2 and P3 which were labelled with C-ethylenediamine in our preparation of modified plastocyanin (Table 15). Using chemical modification to replace a portion of the negative carboxyl groups in this ring with positively- charged amino groups allowed modified plastocyanin to donate electrons to P700+ in the absence of cations. +2 Therefore the role of Mg ions is to screen the negative charges in this ring from the negative charges on PSI particles in order to allow the plastocyanin molecule to bind to PSI particles. The identification of the modified peptide which is involved in the shift in the plastocyanin redox potential proved to be difficult. Modified plasto cyanin fraction III (Figure 17) contained an average of three modified carboxyl groups (Table 10) which shifted +2 the redox potential (Table 14) while the Mg require ment for electron donation (Table 12) was retained. Because these modified carboxyl groups had a large effect on only the redox potential, the peptide map of 14 C-ethylenediamine labelled fraction III was examined in the hope that the peptide responsible for this effect 170 could be identified. The peptide map for fraction III was the same as for the mixture of modified plasto cyanin species seen in Figure 24. The same spots were labelled in both peptide maps as well. This meant that the three labelled carboxyl groups of fraction III were not specifically located in one peptide. Although the label in fraction III was spread s over the protein, peptide P2 in fraction III contained more label relative to the other peptides when compared with the peptides generated from the mixture of four plastocyanin species (Table 16). Peptide P2 contains residues 42-45 (Figure 22) which are highly conserved negatively-charged amino acids. The increase in the relative amount of label in peptide P2 of fraction III might imply that the redox potential of plastocyanin is affected by modification of residues 42-45. However, this cannot be stated as a fact since significant labelling of other peptides also occurred. If the modification of residues 42-45 in peptide P2 is responsible for the redox potential change, it is interesting to speculate as to how these residues effect the copper environment when they are not near the copper (Figure 25). The negatively-charged residues 42-45 are located near the positively-charged lysines at positions 171 Table 16 Comparison of Radioactivity Distribution in Modified Plastocyanin Peptides Spot ^ Peptide Id Relative Amount of Radioactivity c mixture fraction III 2 P2 2.4 3.1 3 P2 2.1 4.6 4 P6 0.8 1.6 5 not resolved 1.8 2.2 6 P3 1.7 1.7 7 P3 1.9 2.4 8 P4 1.0 1.0 9 P5 0 0 10 P4 0 0 11 P7 0 0 Spots are numbered as in Figure 24. Peptides are identified as in Figure 22. The peptide maps were prepared and the spots developed as described in the Materials and Methods. The individual spots were cut out and placed in scintillation vials with 10 ml of scintillation cocktail for nonaqueous samples. The amount of radioactivity (cpm) in each spot is reported as a value relative to the value for spot 8. 81 and 95. The polypeptide chain between residues 81 and 95 contain three of the copper ligands - cysteine 84, histidine 87, and methionine 92. The ionic interaction of lysines 81 and 95 with the aspartic and glutamic acid residues at positions 42-45 maybe important in determining the orientation of three ligands around the copper. Perhaps the incorporation of ethylenediamine at residues 42-45 creates electrostatic repulsion be tween the positively-charged ethylenediamine molecules and positively-charged lysines 81 and 95. This might alter the orientation of the three ligands so that the Cu (I) form of the protein is stabilized because the new orientation makes the formation of the four coordinate Cu (II) complex more difficult. Decreasing electro static interactions by adding NaCl helped to shift the redox potential back towards the control value (Table 13) which is consistant with this argument. NMR of Control and Modified Plastocyanin High resolution proton NMR studies have been used to study the tertiary structure (33, 34, 72) and electron transfer (22,74) in plastocyanin. The proton NMR spectrum of reduced control plastocyanin was com pared with the spectrum of the mixture of reduced modified 173 plastocyanin species to see if some change in tertiary structure can be correlated with the differences in redox potential. Figure 26 shows the aromatic region of the NMR spectrum for control and modified plastocyanin. The two peaks at 7.25 ppm and 7.30 ppm have been previously assigned to the two histidine protons (72). Since these histidines are two of the copper ligands, large changes in the copper environment upon chemical modification should be reflected in changes in these two resonances. However, the chemical shift for these two peaks was identical in control and modified plastocyanin. There fore the copper environments in the reduced forms of control and modified plastocyanin appear to be the same. Because the increase in redox potential indicates a stabilization of Cu (I), significant changes in the copper environment of control and modified plastocyanin may only occur when the oxidized protein must form the four coordinate Cu (II) complex. Resonances of protons in the copper environment cannot be observed when the protein is oxidized because Cu (II) is paramagnetic which increases the rate of proton relaxation (63,72). The increased rate of relaxation broadens the lines from resonances near the Cu (II), which includes the histidine protons, so that they can no longer be Figure 26. The aromatic region of the proton NMR spectra for reduced control and modified plasto cyanin. Samples of reduced protein were prepared and the NMR spectra taken as described in the Materials and Methods. The top spectrum is from control plasto cyanin. The bottom spectrum is from the mixture of modified plastocyanin molecules. 174 1.0 9 8 7 6 5 4 CHEMICAL SHIFT 8 {p p m ) Figure 26 176 observed. Therefore, the copper environment of the oxidized protein must be examined by other techniques such as visible circular dichroism or electron paramagnetic resonance to look for changes in tertiary structure near the copper site. The proton NMR spectrum for control plastocyanin (Figures 26 and 27) agrees very well with the previously published spectrum for spinach plastocyanin (72). The large HDO peak is seen at 4.50 ppm while the sharp peak at -0.15ppm is an artifact created by the electronics of the spectrometer. There are differences between control and modified plastocyanin which can be attributed to conformational differences and to the methylene protons of the incorporated ethylenediamine molecules. Resonances between 7.5 ppm and 9.0 ppm are from peptide bond protons which are buried inside the protein and cannot exchange with the D^O solvent (14). The differences in this region between control and modified plastocyanin (Figure 26) reflect differences in protein conformation. Differences in the 5-7 ppm region after modification such as the decrease in the relative intensity of peaks at 6.20 ppm and 6.55 ppm and the disappearance of peaks at 5.2 ppm and 5.7 ppm are also attributed to con formational differences (Figure 26). The modified Figure 27. The proton NMR spectra of the reduced forms of control and modified plastocyanin. Samples of reduced protein were prepared and the spectra recorded as described in the Materials and Methods. The top spectrum is from control plastocyanin. The bottom spectrum is from the mixture of modified plastocyanin molecules. 177 178 10 9 8 7 6 5 4 3 2 0 CHEMICAL SHIFT 8 (ppm) Figure 27 179 plastocyanin showed a large increase in peaks at 2.50, 2.80, and 3.20 ppm (Figure 27). These peaks appear where the methylene protons of ethylenediamine would be expected assuming these protons are similar to the methylene protons of lysine (14). Specific interpreta tion of these observed differences await the assignment of peaks in the spectrum with specific amino acid residues. Conclusions Protein carboxyl groups of PSI particles have been modified with a water-soluble carbodiimide in the presence of a primary amine which served as a nucleophile. Ethylenediamine was used as the nucleophile during this work although glycine ethyl ester was used in a few ex periments for comparison with ethylenediamine. The modifi cation of the particles was performed at pH 6.0 in the presence of O.lM EDC and 0.2M ethylenediamine. An excess of nucleophile was required in order to prevent the formation of insoluble aggregates of the particles by EDC. The EDC promoted aggregation of PSI particles inhibited both the light-dependent and independent activities of PSI par ticles. For a majority of the modified PSI particle prepa rations, 0.05% (w/v) Triton X-100 was present during the modification reaction although the activity of the modified PSI particles was the same whether Triton X-100 was present or absent. The chemical modification of negatively-charged PSI particles with either ethylenediamine or glycine ethyl ester produced a modified PSI complex which was positively- charged. Both EDA-PSI and GEE-PSI had an isoelectric pH of 180 181 10.3 compared to an isoelectric pH of 5.0 for isolated PSI particles. Such a large shift in isoelectric pH would require the modification of a large number of carboxyl groups. The incorporation of approximately 600 moles of ethylenediamine per mole of P700 reaction center supports the idea of a generalized change in the net charge of the complex rather than the specific incorporation of a few ethylenediamine molecules. Using a molecular weight of 200,000 and a com- ■ bined mole percentage of 15% (103) for the amount of aspartic and glutamic acid in PSI particles, a value of approximately 300 carboxyl groups per P700 was estimated. A value of 600 moles of ethylenediamine were incorporated in EDA-PSI. These results indicated that there are two types of PSI monomers. Both have the same molecular weight but one contains P700 while the other does not. This would ex plain the large number of carboxyl groups modified per P700. Both EDA-PSI and GEE-PSI had the same chl/P700 ratio as the starting material which indicated that the chemical modification reaction did not inactivate any of the re action centers. Gel filtration was used to determine that the PSI particles had a molecular weight of 250,000 before and after chemical modification. This means that no intermolecular cross-linking through protein carboxyl and 182 amino groups had occurred between separate complexes. The excess of nucleophile used in the modification reaction should have prevented intramolecular cross-linking between polypeptides within a single complex. Intramolecular cross- linking can not be completely ruled out since the poly peptide composition of modified PSI could not be determined by SDS gel electrophoresis because the modified protein formed insoluble aggregates with SDS. The chemical modifica tion reaction did not affect the visible absorption spec trum of PSI particles. Therefore the major effect of chemical modification was to change the net charge on PSI particles from negative to positive. This provided the op portunity to compare the activities of modified PSI par- +2 t i d e s with isolated PSI particles in the presence of Mg +2 to examine the possibility that Mg regulates PSI particle activity by altering the charge on the complex. Both chemical modification and the addition of Mg+^ ions affect the light dependent processes of PSI activity by increasing energy transfer from light-harvesting chloro phyll a molecules to P700. This would require a conforma tional change in the complex to orient the antenna chloro phyll molecules into a more efficient network for trans ferring excitation energy. Because chemical modification and Mg+^ have the same effect, the role of Mg+^ must be to 183 bind to protein carboxyl groups on PSI particles which gives the complex a positively-charged surface. This change in surface charge allows a more favorable protein conforma- *4“ 2 tion for energy transfer. A previously reported Mg in duced conformational change in PSI particles (42) supports this idea. The incubation of control PSI particles at pH 6.0 inhibited energy transfer presumably due to the fact that PSI particles begin to aggregate at this pH due to iso electric precipitation. Mg+^ ions regulate the dark processes of PSI particles on the oxidizing side of P700+ by regulating the inter action of electron donors with the complex. Chemical modifi cation of protein carboxyl groups, which makes the PSI par ticles positively-charged, had the same effect as Mg+^ on these interactions. A majority of the data can be explained on the basis of electrostatic interactions between the elec tron donor and the PSI particles. The most significant example of this electrostatic regulation is seen with the natural electron donor plastocyanin. The binding of plasto cyanin to PSI particles has an absolute requirement for Mg+^ cations. The chemical modification of the PSI complex completely substituted for MgC^* The chemical modification of carboxyl groups on plastocyanin, which is summarized in +2 more detail below, also substituted for Mg ions. 184 Therefore the role of Mg+^ ions must be to decrease the electrostatic repulsion that exists between plastocyanin and PSI particles, both of which are negatively-charged proteins. The modification of carboxyl groups on either com ponent in the plastocyanin/PSI particle interaction pro- duced the same result. This indicates that Mg performs its task by regulating the net charge on the surface of these two molecules and not by binding in a specific manner to either of these components. Spinach plastocyanin carboxyl groups were modified at pH 6.0 in the presence of 0.2M ethylenediamine and 0.05M EDC. The visible absorption spectrum and the molecular weight of the modified plastocyanin was identical to the starting material. The modified plastocyanin consisted of a mixture of four populations of plastocyanin molecules which were modified to different extents. These four modified plastocyanins can be separated by anion exchange chroma tography and have been labelled as fractions I, II, III, and IV respectively according to their elution from DEAE- Sephadex. Fractions I, II, III, and IV contain 6.3, 4.1, 3.2, and 2.1 moles of ethylenediamine per mole of plasto cyanin, respectively. This gives the plastocyanin molecules in fractions I, II, III, and IV a net charge at pH 7.0 of 185 approximately +3, -1, -3, and -5, respectively, compared to -9 for control plastocyanin. The chemical modification of plastocyanin affected the binding (Km ) of plastocyanin to PSI particles and not the rate of electron transfer (V ) from plastocyanin to max P700+. In the absence of Mg+^, the ability of the various modified plastocyanin molecules to bind to PSI particles increased as the charge on the plastocyanin molecule pro- 4* 2 gressed from negative to positive. The presence of Mg stimulated the binding of control plastocyanin and the modified plastocyanin in fractions II, III, and IV which have a net negative charge. Mg+^ inhibited the binding of modified plastocyanin in fraction I which has a net positive charge. These results support the conclusion that 4* 2 Mg regulates plastocyanin binding to PSI particles by reducing electrostatic repulsion between the molecules. 14 When modified plastocyanin labelled with C-ethylenedi amine was subjected to tryptic hydrolysis and two dimen sional peptide mapping, the plastocyanin peptides contain ing the acidic amino acid residues 42-45, 59-61, and 68 were found to be heavily labelled. These negatively-charged residues must be involved in the plastocyanin /PSI particle interaction. 186 Spinach plastocyanin has a redox potential of +367mV. All four modified plastocyanin species have a redox poten tial that is at least +38 mV higher than control plasto cyanin. The incorporation of ethylendiamine molecules near the copper site may produce steric strain in the orienta tion of the amino acid side chains that serve as ligands for the copper. This would favor the Cu(I) form of the pro tein because the two-coordinate geometry of the cuprous ion has less rigid stereochemical demands than the four co ordinate geometry of the cupric ion. A portion of this strain may be the result of some type of ionic phenomenon because the reduction of ionic interactions by the addition of lOOmM NaCl lowered the redox potential of modified plas tocyanin in the direction of control plastocyanin. The location of the modified plastocyanin carboxyl groups that are responsible for the change in redox po tential remains unclear at present. These groups are probably not in direct contact with the copper ligands be cause there were no observable changes in either the visible absorption spectrum of oxidized plastocyanin or the proton NMR resonances of the two C2-H protons from the histidine ligands. Therefore the cause of the redox poten tial change must be a more subtle change in conformation of the plastocyanin molecule. Modified plastocyanin fraction 187 III contained an average of only three modified carboxyl groups which caused a shift in the redox potential while A retaining the Mg+ requirement for electron donation. When fraction III was subjected to tryptic hydrolysis and pep tide mapping, the plastocyanin peptide which contained the acidic residues 42-45 was found to be enriched in its ethylenediamine content compared to the other peptides. However, more experimental work must be completed before it can be stated conclusively that the change in the redox po tential of plastocyanin is the result of incorporation of ethylenediamine into a specific portion of the protein. The PSI particles isolated by our procedure contain ap proximately one equivalent of cytochrome f for each P700 re action center. Although cytochrome f can not donate elec- trons directly to P700 in the thylakoid membrane, some as pect of the PSI particle isolation must alter the orienta tion of these two components so that electrons can be do nated directly from cytochrome f to P700+ in the isolated +2 complex. Mg stimulates the rate of cytochrome f oxidation which indicates that divalent cations increase the in teraction of cytochrome f and P700+. 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