Protein-Protein Interactions and Electron Transfer Associated with Cytochrome F and Plastocyanin from the Cyanobacterium Prochlorothrix Hollandica

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PROTEIN-PROTEIN INTERACTIONS AND ELECTRON TRANSFER ASSOCIATED WITH CYTOCHROME F AND PLASTOCYANIN FROM THE CYANOBACTERIUM PROCHLOROTHRIX HOLLANDICA.

Maria V. Baranova

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

August 2007

Committee:

George S. Bullerjahn, Advisor

J. Devin McAuley Graduate Faculty Representative

Michael Y. Ogawa

Neocles Leontis ii

ABSTRACT George S. Bullerjahn, Advisor

This dissertational work describes the minimal structural requirements of

interaction surfaces between two proteins involved in photosynthetic electron transfer cyt

f and PC from cyanobacterium Prochlorothrix hollandica analyzed by stopped-flow

absorption spectroscopy and HSQC NMR.

Two mutant P.hollandica cyt f, Y102G and Y102G/F100S, yielding a modified

surface-exposed loop region, were expressed and characterized to analyze the structurally

unique Prochlorothrix cyt f ‘pocket-like’ region involved in the PC-cyt f complex

formation and electron transfer. Stopped-flow studies showed that altering these residues

slows down ket more than one order of magnitude. We propose that Tyr102 and Phe100

are actively involved in complex formation between cyt f and PC and serve to minimize

distance between electron donor and acceptor. Thus, by removing these residues, the Cu-

Fe distance in cyt f -PC complex increases, slowing electron transfer rates.

In previous NMR studies(21) it was shown that PC from cyanobacteria interacts with cyt f differently than the comparable proteins in higher plants and algae. The PC-cyt f complex in the cyanobacterium Phormidium laminosum(21) involves a ‘head on’ contact between the hydrophobic (‘northern’) patch of PC with a hydrophobic surface

surrounding the cyt f heme, with an average Cu-Fe separation of 15Å. Moreover,

Prochlorothrix hollandica PC has a structurally distinct docking surface among other

cyanobacteria(23) that likely makes these interactions somewhat different with additional

interaction of PC with a flexible loop of cyt f forming a ‘pocket-like’ region in the

iii

vicinity of cyt f residues 99-104. There are two aromatic amino acids Tyr and Phe among them that face toward the PC and are possibly involved in protein-protein contacts.

A parallel study was initiated to study the interactions of mutated cyanobacterial PC with Photosystem I (PSI) and some non-physiological electron-transfer partners (Lysine

2+ peptide and tris (2,2’-bipyridine)ruthenium (II) Ru(bpy)3 ). Additionally, negatively

charged P.hollandica PC mutants, mimicking the higher plant protein, should explain

better the necessity of electrostatic interactions in the PC/cyt f complex in chloroplast

systems.

ACKNOWLEDGMENTS

I would like to thank my husband, Radiy Islangulov who came with this crazy idea to do a PhD so far away from home and family. There is no regret now and I am happy that we made this decision five years ago. Thank you for your enthusiasm, patience and strength. Also, thanks to my parents for letting me to do it, for their unlimited support and love during these long years. I will not make it without you!

I am deeply indebted to my advisor Dr George S. Bullerjahn, who encourages my independence and gave freedom in work. Thank you George for your advice, help and comfortable atmosphere in the lab. I would like to acknowledge my committee members:

Dr. Michael Y. Ogawa, Dr Neocles Leontis and Dr J. Devin McAuley for you suggestion, corrections and advices. I thank Dr Haoming Zang and Lucy Waskell (University of

Michigan, Ann Arbor) for collaboration and stopped-flow equipment, Dr Venkatesha

Basrur for MALDI-TOF analysis, Dr Marcellus Ubbink for HSQC NMR studies, Dr

Eugene Danilov for help and advice and Dr Vintonenko for initial introduction to this scientific area. I thank Center for Photochemical Sciences for giving me an opportunity to study in Bowling Green State University and Department of Biological Sciences for financial support during last years.

I wish to thank my host family – Diane and Scott Regan for being my American parents, for their care, love and support.

Special thanks to my friends Natalia, Sergey Rybas and Irina, Armen Ilikchan for keeping me sane.

Dedicated to my father, Vladimir Konstantinovich Baranov, who always teach me to do my best or not to do it at all. I think I did my best!

And to my mother Olga Vladimirovna Baranova who use to tell me that the problem must be stated in order to be solved. Thank you mom!

TABLE OF CONTENTS Page

CHAPTER I. INTRODUCTION...... 1

CHAPTER II. CYTOCHROME F FROM THE CYANOBACTERIUM

PROCHLOROTHRIX HOLLANDICA. EXPRESSION IN ESCHERICHIA COLI AND

PHYSICAL CHARACTERIZATION...... 13

Introduction ...... 13

Materials and Methods ...... 20

Results and Discussion ...... 25

CHAPTER III. PROTEIN-PROTEIN INTERACTIONS AND ELECTRON

TRANSFER BETWEEN PROCHLOROTHRIX CYTOCHROME F AND

PLASTOCYANIN – MUTAGENESIS OF A CYT F LOOP REGION...... 38

Introduction ...... 38

Materials and Methods ...... 47

Results and Discussion ...... 52

Concluding Remarks...... 66

CHAPTER IV. STUDIES OF MUTANT P. HOLLANDICA PLASTOCYANIN

INTERACTIONS WITH PHYSIOLOGICAL AND NON-PHYSIOLOGICAL

PARTNERS. (TRIALS AND FUTURE PERSPECTIVES)...... 67

Introduction ...... 67

Materials and Methods ...... 73

Results and Discussion ...... 78

FUTURE PERSPECTIVES ...... 86

REFERENCES ...... 87 vii

LIST OF FIGURES

Figure Page

1. Photosynthetic electron transport chain ...... 2

2. Schematic representation of transient complex formation, illustrating the

three-step mechanism, where long-range electrostatic interactions

preoriented partners to optimal configuration by surface diffusion. Once

the specific complex is formed, electron transfer occurs and the products

dissociate ...... 7

3. Structure of the C-type heme ...... 14

4. Hexacoordination of the cyt f heme...... 14

5. Ribbon diagram of the soluble domain turnip cyt f ……...... 15

6. Model of the topography of cytochrome f in the thylakoid membrane ...... 15

7. Expression vectors ...... 21

8. Amino acids sequences alignment of mature cytochrome f. The sequences were

all taken from the NCBI protein data base. N-terminal peptide leader and C-

terminal polypeptides were truncated. Conservative amino acids highlighted in

red. Yellow patch represent the heme binding site and green patch shows the loop

region involved in protein interactions. Higher plants are in blue: Br- Brassica

rapa; Sa-Spinacia aleracea; Pa- Poplar alba. Cyanobacteria are in green: Syn –

Synechocystis PCC 6803; Nos – Nostoc PCC 7906; Pl – Phormidium laminosum;

Ph – Prochlorothrix hollandica. Green alga Chlamydomonas reinhardtii – Cr is

black ……………………………………………………………………………...... 26 viii

9. SDS-PAGE of purified cytochrome f ……..…...... 30

10. Mass Spectrum indicating molecular mass of cytochrome f as 27.3 kDa…………. 31

11. Visible absorption spectrum of reduced cyt f in 10mM phosphate buffer, 1mM

sodium ascorbate, pH 6.0; Black line, cytochrome f from P.hollandica; Red line,

Cytochrome f from P.laminosum; Green line, cytochrome f from turnip. B,

Enlargement of absorption spectrum of α- and β-peaks...... 32

12. Absorption spectra of oxidized (dashed line) and reduced (solid line) P.hollandica

cytochrome f from 610nm to 350nm. Eleven micromolar cytochrome f in 10mM

sodium phosphate buffer pH 7.5 with 1mM sodium ascorbate (reduced) and 5

mM potassium ferricyanide (oxidized)...... 32

13. Reduced minus oxidized absorption spectrum (green line) of the pyridine

hemochrome produced from treatment of P.hollandica cytochrome f. Oxidized

cytochrome f spectrum (black line), Reduced cytochrome f spectrum (red line) ...... 33

14. CD spectra of P.hollandica cytochrome f. CD spectra of 40uM solutions of

cytochrome f at 25°C. (A) CD spectra of wild-type and mutants of turnip

cytochrome f from Gong et al.(36). (B) CD spectra of P.hollandica cytochrome f.

The secondary structure content was estimated using the program k2d...... 34

15. Spectrophotometric titration of cytochrome f from P.hollandica...... 35

16. Redox titration of P.hollandica cytochrome f. A 6 µM solution of cytochrome f in

10 mM phosphate buffer pH 6.0 with 90mM NaCl containing 2mM potassium

ferricyanide was titrated with 400 mM potassium ferrocyanide at 22 °C. After

each addition the absorption spectrum was recorded…………...... 36 ix

17. A stick diagram revealing the details of the complex interface...... 39

18. The structure of the plastocyanin/cytochrome f complex from (A) chloroplast and

(B) cyanobacteria...... 44

19. Sequence alignment of cyanobacterial cytochrome f from Phormidium laminosum

and Prochlorotrix hollandica...... 52

20. 3D structure of cytochrome f from P. laminosum (A) and P. hollandica (B).

Enlarged “pocket-like” region of P.holladica cyt f (C)……………………………. 53

21. Ribbon diagram of P.laminosum (A) and P.hollandica (B) cyt f . ‘Pocket-

like’ region indicated with the red arrow. Amino acid residues that were

mutated highlighted in red ...... 54

22. Partial MALDI-TOF data of WT and mutants of P.hollandica cyt f...... 56

23. MALDI-TOF data of enlarged region around 27 kDa for the WT and mutants of

P.hollandica cyt f...... 57

24. Redox titration of P.hollandica cytochrome f WT(black line), Y102G mutant (red

line ) and Y102G/F100S mutant (green line). A 6µM solution of cytochrome f in

10mM phosphate buffer, pH 6.0 with 90 mM NaCl, containing 2 mM potassium

ferricyanide was titrated with 400 mM potassium ferrocyanide at 22 °C. After

each addition, the absorption spectrum was recorded ...... 58

25. CD spectra of P.hollandica cytochrome f wild type and mutants. CD spectra of

40µM solutions of cytochrome f in 10mM phosphate buffer pH 6.0 at 25°C...... 59

26. Rates of electron transfer from cytochrome f to wild-type and the

Y12G/P14L(Dbl) mutant of plastocyanin both from cyanobacteria Prochlorotrix

hollandica. Electron transfer from reduced cytochrome f (0.15 uM) to oxidized x

plastocyanin was monitored at 421 nm. The reactions were carried out at 5°C in a

buffer consisting of 10 mM potassium phosphate pH 6.0 and 90 mM NaCl. (A)

Oxidation of WT cyt f by WT PC (black ) and Dbl mutant PC (blue). (B)

Oxidation of Y102G cyt f mutant by WT PC (red) and by Dbl mutant PC (cyan).

PC (magenta)………………………………………………………………… ...... 61

27. Rates of electron transfer from cytochrome f WT and mutants to PC wild-type

(A); and from cyt f WT and mutants to PC Y12G/P14L mutant (B). Line colors

are coded as on Fig. 26………………………………………………………….… . 62

28. Binding curve for the interaction of (A) WT plastocyanin with WT and mutants of

cyt f. (B) Dbl mutant PC with WT and mutants of cyt f. The increase in

absorbamce ar 410nm was measured when plastocyanin was present at

concentrations between 0 and 60µM. Protein were dissolve on 10mM phosphate

buffer, 90 mM NaCl, pH 6.0 at 25ºC. Colors code same as for the Fig. 16 and 17) 64

29. X-ray structure of the Cu center from poplar plastocyanin ...... 68

30. Cu-cysteine bonding interactions. (A) ‘Normal’ bonding with weak π and strong

σ transfer transition; (B) Strong π and weak σ charge transfer spectra...... 69

31. Structure of poplar plastocyanin. Copper highlighted with the blue arrow...... 70

32. Scheme of interaction between poplar Pc and Lys peptide ...... 71

33. Expression plasmid pVAPC10 ...... 73

34. Protein expression vectors. A, pVAPC plasmid with 4 mutations of petE gene

fused together with P.laminosum PC peptide leader. B, pVAPC vector with the

fused whole natural petE gene together their own promoter and PC leader peptide. 76 xi

35. P.hollandica plastocyanin structure. A. Wild-type plastocyanin B. Mutant

plastocyanin with the negative amino acids on the surface. Blue indicates original

amino acids, Red – new involved negative amino acids. Green – initially mutated

arginine to glutamine ...... 79

36. SDS-PAGE of PC WT and mutants. Coomassie staining ...... 81

37. CD spectra of WT and mutants of plastocyanin. All samples were in 50 mM

Tris/HCl buffer………………………………………………………………...... 82

38. SDS-PAGE Western blotting of E.coli BL21(DE3)pLysS strain periplamic

fraction. IPTG was added to the final concentration 0.5 mM. Total protein

concentration 1 ug/ul...... 83

2+ 39. Dependence of kobs on PC concentration in electron transfer from *[Ru(bpy)3]

2+ to PC in 100 mM Tris buffer with [Ru(bpy)3] = 50 µM ...... 85

xii

LIST OF TABLES

Table Page

1. PCR primers ...... 21

2. Leader sequences. Positively charged amino acids highlighted in red ...... 28

3. Physicochemical parameters of cytochrome f...... 36

4. Molecular masses of wild type and mutant cyt f holoproteins...... 55

5. Midpoint redox potentials and extinction coefficients for the WT and mutants of

P.hollandica cyt f...... 59

6. The second order rate constant for electron transfer to WT and

Y12G/P14L mutant of plastocyanin ...... 63

7. Binding constant, KA for the interaction of cytochrome f and plastocyanin

from the cyanobacteria P.hollandica ...... 65

8. Amino acids sequence alignment of plastocyanin from P.hollandica and spinach... 78

9. P.hollandica amino acid sequences of the negative patch for wild-type and

mutant PCs…………...... 79

CHAPTER I

INTRODUCTION

Photosynthesis had been studied scientifically for at least 300 years, going back to Joseph

Priestley, the discover of oxygen, who also investigated the the influence of green plants on “air restoration” (26) i.e. replacement of O2 in air breathed by animals. Ten Nobel Prizes have been awarded for photosynthesis research in a last century (61). There are plenty of books and other publications written about different aspects of photosynthesis (38), but people are still studying this process and continue to discover photosynthetic organisms and new aspects of photosynthetic processes and their mechanisms. Scientists understand this process for new ideas to create artificial nanoscale devices and semibiological hybrids that carry out many of the functions of the natural photosynthetic process (7, 44), for application in more efficiently harvesting solar energy in a renewable fashion.

Photosynthesis can be thought of simply as a process of carbon dioxide fixation performed by plants. The overall reaction is the reduction of CO2 by electrons from water yielding glucose for cell metabolism, and molecular oxygen production released to the atmosphere as a waste product. However, in a more precise context, photosynthesis means the consequence of light-induced electron transfer processes that are controlled by redox reactions powered by primary charge separation within specific photochemical reaction centers with chlorophyll as an energy converter. Water provides the source of electrons flowing from membrane-bound donor and acceptor proteins(8).

Photosynthetic light reactions – electron transport

Most of the photosynthetic proteins found in chloroplasts and cyanobacteria are directly or indirectly involved in long-range electron transfer processes. The photosynthetic apparatus

2 consist of membrane-bound static protein complexes as well as mobile protein partners, which forms transient complexes with the static complexes to shuttle electrons between them.

Photosystem II (PSII), cytochrome bf, photosystem I (PSI) and ATP-synthase located in the thylakoid membrane (Figure 1). PSII is the enzyme that accumulates four high potential electrons from four single-photon events that extract four electrons from two bound water molecules, liberating dioxygen. PSI sequentially accepts these electrons via the cyt b6f complex to yield a light-driven 2-electron reduction of NADP to yield cellular reducing power. All of these static complexes are composed of many subunits. In order to integrate the system into a chain of electron donors and acceptors, there are mobile components that operate between membrane-bound complexes. These include plastocyanin or cytochrome c6, which shuttle electrons between cytochrome b6f complex and PSI; lipid soluble plastoquinone, which transports electrons from PSII to cyt bf; and ferredoxin/flavodoxin, that catalyzes cyclic electron transfer from PSI back to cyt bf via plastoquinone. The resulting proton gradient is used by the

ATP synthase (ATPase) to form ATP. Reduced ferredoxin and flavodoxin is also an electron donor to NADP, and this reducing power is consumed during the reactive light-independent reactions that reduce CO2 to glucose.

Figure 1. Photosynthetic electron transport chain

The photosynthesis process will be finally understood only when the mechanism of electron transfer and the associated energy transduction in this process can be explained in greater detail. Thus, understanding of photosynthesis will require determining precise kinetic and thermodynamic parameters for electron carriers involved in electron transfer process and the mechanism of their interactions(8).

Mobile electron carriers

Hydrophobic quinines like plastoquinone are immersed in and move freely within the phospholipid bilayer of the thylakoid membrane, but the metalloproteins plastocyanin and cytochrome c6 are soluble redox carriers located inside the thylakoid lumen. The oxidized

2+ 3+ proteins – both plastocyanin (Cu ), or cytochrome c6 (Fe ) in some organisms – each accept one electron from cytochrome f in the cytochrome b6f complex to become reduced. The reduced copper or heme protein moves freely inside the lumen to dock to the PSI complex, specifically donating the electron to the photooxidized chlorophyll molecule P700+ of PSI, then dissociating from the photosystem to repeat the cycle. It is interesting that PC is the only electron carrier in higher plants, whereas in cyanobacteria and some algae cyt c6 together with PC can be synthesized, depending on the copper availability in the media (85). The protein-protein interactions between cyt f and plastocyanin are the focus of this dissertation.

Principles of electron transfer reactions

Thermodynamic parameters, including midpoint redox potential, have been determined experimentally for numerous electron carriers, through potentiometric titration(41) or cyclic voltammetry (45, 123) methods appluing the Nernst equation (Eq 1) that defines the tendency of the oxidation-reduction couple to donate or accept electrons relative to a standard oxidation- reduction cell.

E=E0 + (RT/nF) ln([oxidized]/[reduced]) (Eq 1)

In Eq 1, E is the oxidation-reduction potential relative to a standard hydrogen half –cell, E0 is the half-reduction potential for the component under ‘standard conditions’, R-gas constant, T- the absolute temperature, F-Faraday constant, n-number of electrons transferred during the reaction; [oxidized] and [reduced] are the concentrations of oxidized and reduced species.

The kinetic parameters of a electron transfer component, including an oxidation rate of the electron carriers, have been studied through the rapid flow techniques(17) or laser-flash absorption spectroscopy (73, 95, 128).

Thermodynamics and kinetics of the outer-sphere processes, such as protein-protein interactions of the electron transfer reactions, can be described by the Marcus equation (Eq 2) that in general terms correlates the Gibbs energy of activation (∆G**) with the driving force

(∆G0) of the reaction.

∆G** = (λ/4)(1 + ∆G0/λ)2 (Eq 2), where λ is the reorganization energy, ∆G0 is the standard free energy of the reaction corrected required to bring the reactants together or driving force; λ/4 is the intrinsic barrier of the reaction for the outer-sphere electron transfer.

The rate of reaction can be calculated from the activation energy according to the Eyring equation (Eq 3), then by combination Eq 2 and Eq 3 will give other equation for the rate of the reaction (Eq 4).

** k = k0 exp (-∆G /RT) (Eq 3)

0 2 k = k0 exp [-(∆G + λ) /4λRT] (Eq 4)

To take into account specifics of electron transfer processes in biological systems, such as inter or intra protein long-range interactions, that occur between redox-active prosthetic groups buried inside the proteins, the rate constant of electron transfer (kET) will be proportional to the

2 2 electronic coupling factor (HAB = H0 (exp (-βd)) that correlates overlapping of donor and acceptor molecular orbitals. Those corrections will give an equation for the rate constant of electron transfer (Eq 5).

0 2 kET = k0 exp (-βd) exp [-(∆G + λ ) /4λRT] (Eq 5) where d is the distance between donor and acceptor and the β-distance factor dependent on the medium between donor and acceptor. Thus, according to this equation we can see that kET will exponentially decrease with the distance. k0 is characterized as the maximum rate of electron transfer when donor and acceptor are in van der Waals contact and λ = - ∆G0 and is usually 1013 s-1. Reorganization energy, λ, is the energy needed to deform the nuclear configuration from the reactant to the product state. For exothermic reactions, the rate will increase with increasing driving force where - ∆G0 < λ (normal region), whereas for the endothermic reaction, when -

∆G0 > λ the rate will start to decrease by increasing driving force (inverted region). Electron transfer reactions usually have low driving forces, sometimes ∆G0 > 0, and often, span distances greater than 10 Å. Thus, most bimolecular reactions occur in the normal region, whereas some systems with inverted region kinetics are known(117, 129).

To sum, the rate of electron transfer reactions are dependent on driving force, reorganization energy, distance and the nature of the medium in which electron must traverse. In other words, electron transfer depends on nature of the complex formation between two or more proteins.

Protein-protein interactions

Static complexes for example, are characterized by high binding affinity (107-109M-1), and long lifetimes (dissociation rate constant of s-1 to min-1). Examples of such complexes include antibody-antigen interactions, enzyme-inhibitor complexes and signal transduction protein complexes. In contrast to that, transient complexes exist that have much weaker binding affinity, in the 103 M-1-106 M-1 range and a shorter lifetime (dissociation rate constant 102-104 s-1). Good examples for these type of interactions occur in the electron transfer chain, which requires readily reversible protein interactions to ensure continuous electron flow between redox partners(10). Thus, transient complex formation requires a fine balance of specificity and stability. Specificity is necessary to facilitate fast productive binding while the affinity must be low in order to maintain the transient in reversible nature of the complex. Electrostatic interactions, due to charged patches on the partner proteins, can produce pre-orientation of diffusing reactants and increase affinity between the proteins, where upon subsequent contact, rearrangements are required to achieve the complex optimal for electron transfer. Specific complex formation and stability is determined by other non-covalent hydrophobic interactions that can occur without additional rearrangements(20). The three step transient complex formation described by Hervas et al(51) is shown in Fig. 2.

Long-range interprotein electron transfer involves donor and acceptor proteins with redox centers that are separated by relatively long distances, and the overall redox process may require several reaction steps including specific binding of proteins, protein rearrangement that optimizes the coupling between redox centers and chemical transformations such as proton transfer, and the actual electron transfer step.

Figure 2. Schematic representation of transient complex formation, illustrating the three-step mechanism, where long-range electrostatic interactions preorient partners to optimal configuration by surface diffusion. Once the specific complex is formed, electron transfer occurs and the reaction partners dissociate.

Hervas et al proposed three mechanisms of interprotein electron transfer based on the reactivity of plastocyanin and cytochrome c6 towards PSI(51). According to that, PSI reduction by the donor proteins can follow through different mechanisms: collisional mechanism (type I), a mechanism required complex formation (type II) or complex formation with rearrangement of the interface (type III).

PSIox+ Donorred PSIred+Donorox (Type I)

PSIox+ Donorred [PSIox/Donorred] PSIred+Donorox (Type II)

* PSIox+ Donorred [PSIox/Donorred] [PSIox/Donorred] PSIred+Donorox (Type III)

The type I model is found in some cyanobacteria in which donor proteins do not form a kinetically detectable transient complex (21). This type is observed for the weak interactions and moreover, might be controlled by electrostatic repulsion between partners(9, 99). For example, there is no complex formation between plastocyanin and PSI from thermophilic cyanobacteria

Synechoccocus elongatus(9) and Phormidium laminosum(99). Type I kinetics are characterized

+ by monophasic decay of the absorbance of photo-oxidized P 700 at 820 nm upon reduction by plastocyanin, and linear dependence of the observed pseudo-first-order rate constant kobs on the plastocyanin concentration(99). The type II model is found in most other cyanobacteria and algae, in which the transient complex can be either electrostatic or hydrophobic(51, 78). It also exhibits monophasic kinetics; however kobs represents a saturating value at infinite plastocyanin concentration, which provides strong evidence for the complex formation followed by intramolecular electron transfer. The Type III model is observed mostly in eukaryotic organisms, in which the intermediate complex is first formed by electrostatic attractions and the further reorientation mainly involves hydrophobic interactions(27, 49, 51). Type III shows biphasic kinetics, which provides evidence for the formation of an additional reaction complex. Thus, rearrangement must occur before intracomplex electron transfer (41). As was stated before, the kinetic mechanisms of each protein across whole range of photosynthetic organisms could suggest type I is most ancient and Type III having evolved most recently as chloroplasts evolved from cyanobacteria (31). Similar comparisons can be made between interactions seen between plastocyanin and its electron donor, cytochrome f. Indeed, in cyanobacteria, plastocyanin interacts with cyt f largely through hydrophobic interactions; whereas in chloroplast systems, plastocyanin binds cyt f through extensive electrostatics in addition to hydrophobic interactions

(21)

Analysis of protein-protein interactions

X-ray crystallography and NMR are the two main tools for determination of obtain structure of proteins and their complexes. Both techniques have their advantage and disadvantages. The main problem with X-ray crystallography is the preparation of the suitable crystals, especially for a

9 membrane-bound protein complex. At the same time, approximately 85 % of protein structures in Protein Data Base (PDB) have been determined by X-ray diffraction (14). The major advantage of NMR structures is the ability of the method to provide representation of the proteins behavior in near native conditions and to study weak protein-protein interactions, which may co-crystallize. Chemical shift perturbation mapping is the fastest NMR method for obtaining information about protein interactions. In this approach one of the proteins is labeled with 15N and changes in its heteronuclear spectra (typically the 1H-15N HSQC) are monitored during a titration with the unlabeled protein partner (3, 21, 23, 130). Changes in the chemical shifts and line width of the resonances contain information about the complex interface. Namely, complex formation gives rise to changes in the chemical environment of nuclei at the interface; such that the chemical shift (δ) of these nuclei differ between bound (δbound) and free (δfree) forms. Mapping there chemical shift changes (∆δBind) to specific amino acid side chains will identify the surface of labeled protein mediating the interaction with the partner. At the same time, a binding curve can be obtained by plotting the ∆δBind for single resonance as a function of the molar ratio of interacting proteins (22).

Other methods to obtain information about the structure of the complex are covalent cross- linking techniques (82, 111), chemical modification of amino acid residues(1) and mutagenesis

(35, 67, 107). Chemical cross-linking techniques involve the use of reagents to stabilize interactions by distance-dependent covalent binding between two proteins (usually amide or sulfite derivatives) that yield adducts of complexed proteins. Then, peptide mapping and protein sequencing studies can reveal linkage sites between two proteins. For example, Morand et al in their chemical cross-linking experiment demonstrated that Asp44 of plastocyanin can be linked to Lys 187 of cyt f, suggesting that in the complex formation these residues are close to each

10 other (82). Another study showed that intracomplex electron transfer is might be inhibited by chemical cross linking, which indicates that the initial complex may not be optimal for the efficient electron transfer and that rearrangement within the complex is nessesary (94).

Chemical modification techniques yield specific adducts to the amino acid residues functional groups. For example, modification of carboxyl groups on plastocyanin with ethylendiamine to produce basic groups resulted in decreased cytochrome f oxidation depending on the number of carboxyl groups modified (110). Another experiment involving modification of amino groups on cytochrome f with 4-chloro-3,5-dinitrobenzoic acid to produce acidic groups yielded decreased rates of cytochrome f oxidation by plastocyanin and ferricyanide dependent on the number of amino groups modified (112, 113).

Site-directed mutagenesis is one of the most powerful techniques for studding protein structure- functional relationships and specificity of protein-protein interactions in vitro that allows pretty quickly change protein structure or surface by changing one or several amino acids. Namely, this method provides information on the role of specific residues involved in interactions. For example, studies with site-directed mutants of pea plastocyanin suggested that the surface exposed tyrosine residue (Tyr83) of plastocyanin is involved in both binding and electron transfer reaction with cytochrome f(48). Replacement of Tyr83 by Phe83 resulted in an 8-fold decrease in the overall rate of electron transfer and this could be accounted for the decreased association constant with no change in the intrinsic rate of electron transfer in the binary complex. The decreased binding suggests that the hydroxyl group of Tyr83 plays a role in binding to cyt f (48). Also, site-directed mutagenesis technique was used for the study of the role of charged residues on the surface of plastocyanin. Schlarb-Ridley et al neutralized the charges on the eastern negatively charged surface of plastocyanin to check dependence on ionic strength

11 and established that negative charges accelerate the electron transfer reaction from cytochrome f and increase that dependence. Conversely, removal of positive charges slowed this reaction.

Research aims

This research project is focused on interactions between the mobile protein PC and the protein complexes of cytochrome b6f and PSI. Namely, this project seeks to determine the minimal structural requirements for the homospecific complex between a cyanobacterial PC-cyt f, and the electron transfer rate for the reaction. Actually, cyanobacteria have been widely used as a source of photosynthetic proteins (PC, cyt c6, cyt f, PSI and others) for in vitro and in vivo studies of photosynthetic electron and energy transfer. The major advantage for studying cyanobacteria is their relatively fast growth and (for some proteins), the easier extraction of proteins from the cell in compared to the higher plant chloroplasts. Additionally, analyzing subtle differences in reactivity between cyanobacterial and chloroplast photosynthetic complexes

(such as the Type I/II vs III mechanism described above), may help reveal common rules for biological electron transfer.

The cyanobacterium Prochlorothrix hollandica from which the proteins we are studying were obtained, is a very interesting organism due to the fact it contains both chlorophylls a and b, whereas most other cyanobacteria have only chlorophyll a and phycobilins as light harvesting pigment. The major difference of Prochlorothrix hollandica from other cyanobacteria is the structure of photosynthetic apparatus that looks more like that seen in plant chloroplasts (76).

Thus, in these studies was our interest to compare kinetics and binding interactions of PC- cyt f in this organism to those observed in higher plants, algae and other cyanobacteria.

Furthermore, we have prior structural information showing that the surfaces involved in protein-protein interactions in Prochlorothrix plastocyanin are structurally different than most.

Analysis of how this altered surface interacts with other reaction partners can identify novel co- evolved regions necessary for specific protein binding. Previously, there have been a lot of homo and heterospecific complexes studied among the spinach (69), turnip(36), pea (48),

Chlamydomonas reinhardtti (107), Phormidium laminosum(21) cyts f and PCs, focusing on their thermodynamic and kinetic properties. Some studies were also done for the P.hollandica PC interaction with PSI and P.laminosum cyt f but there has been no homospecific complex to examine the natural process of electron transfer and interaction for this unusual organism. In order to provide this information, I developed an expression system that increases the production of P.hollandica cyt f for in vitro interaction studies. Due to better understanding of the thermodynamic and kinetic properties of electron transfer and binding affinity between these two proteins, we can obtain a clearer picture of the minimum structural requirements for protein- protein interactions leading to productive electron transfer. This study will involve kinetic studies of wild type and mutant PC and cyt f, and will also involve HSQC NMR studies to map the interacting protein surfaces.

CHAPTER II CYTOCHROME F FROM THE CYANOBACTERIUM PROCHLOROTHRIX HOLLANDICA. EXPRESSION IN ESCHERICHIA COLI AND PHYSICAL CHARACTERIZATION

Introduction

Hemoproteins are metalloprtoteins containing a heme prosthetic group, either covalently or noncovalently bound to the protein. Among them are hemoglobin, myoglobin, peroxidase, catalase, nitrate and sulfite reductase, and cytochromes (59).

The term “heme” is usually understood as any tetrapyrrolic chelate of iron. These proteins are involves in oxidation-reduction processes when iron usually transformed from +2 to the +3 oxidation state. A hemochrome is defined as a low spin compound of heme in which fifth and sixth coordination sites are occupied by strong field ligands regardless of the oxidation state of the iron (59).

Cytochromes are generally membrane-associated proteins and found in the mitochondrial inner membrane, endoplasmic reticulum and also in the chloroplast of photosynthetic organisms. They characterized by an intense adsorption band between 510nm and 615 nm (92). All cytochromes are assigned to the groups a, b, c and d according to the nature and the binding of the heme prosthetic group. Cytochrome f of the photosynthetic electron transfer chain belongs to the c-type cytochromes characterized by a heme attachment sequence motif, Cys-X-X-Cys-His, located near the N-terminus. The cytochrome f heme site is covalently attached to the polypeptide through thioether bonds to Cys21 and Cys24 (for all mature cyts f). The fifth ligand to the heme iron is His25 and the sixth to the iron is the α-amino group of Tyr1(75) (Figures 3 and 4).

Figure 3. Structure of the C-type heme(58).

Figure 4. Hexacoordination of the cyt f heme(58).

Tyrosine is a very unusual ligand for the c-type cytochromes, where commonly methionine stabilizes the iron center. Initially, several decades ago, it was thought that lysine was the 6th ligand for the cytf heme, according to EPR analysis, magnetic circular dichroism studies(101,

103), Raman(56) and NMR spectroscopy(96). Possibly, because of this unusual axial ligation, cyts f have the highest midpoint potentials known for c-type cytochromes (+320 to +365 mV)(66).

Cytochrome f is an intrinsic membrane component and the largest subunit of the cytochrome bf complex of the photosynthetic electron transfer chain, transferring an electron from the Rieske

FeS protein to plastocyanin in the thylakoid lumen. Thus, cyt f plays a central position in the photosynthetic electron transfer between PSI and PSII(66).

The crystal structures of soluble (lumen-side) domain of cytochrome f reveal two structural domains: a small β sheet-domain located above a larger domain, consisting of an antiparallel β- sandwich structure and short heme-binding peptide forming a three-layer structure (Figure 5).

This larger domain is attached to the thylakoid membrane by single membrane-spanning helix located near the C-terminal of the polypeptide (residues 251-270 of the 285-residue cytochrome) and anchored by positively charged Arg or Lys residues (Figure 6).

Figure 5. Ribbon diagram of the soluble domain turnip cyt f(21).

Figure 6. Model of the topography of cytochrome f in the thylakoid membrane(40)

This ‘anchor’ is commonly proteolytically cleaved during purification or extraction of cyt f chloroplast thylakoid membranes and the soluble 252 residue hemo-protein exhibits an identical oxidation-reduction midpoint potential and optical spectra compared to the native protein(40) .

The endogenous proteolysis present in the thylakoid membranes and its action on cyt f is likely initiated by organic solvent treatment of the membrane during cyt f extraction(114). The proteolytic cleavage site is highly conserved in cyt f from all eukaryotes but the physiological function of this proteolysis system is not clear(40).

Cytochrome f is one of the most widely studied electron transfer proteins. However, extraction directly from the tissues (mainly leaves) has many steps, low yield, and is hard to perform due to poor solubilization in different organic solvents. Additionally, aggregation and the presence of other pigments and chlorophylls may co-extract with cyt f and shield its absorption spectrum(24). For example, preparation of cyt f from the cyanobacterium Spirulina maxima was characterized by a low yield of 0.47µmol from 500 g of dry membranes(69); in another study, 0.15 µmol of cyt f from 1800 g of tobacco leaves was obtained(39). In addition, only 36.6 mg of cyt f was obtained from 60 kg of Japanese radish leaves(114). Also, together with chlorophyll pigments, S. maxima has an abundance of phycobiliproteins that have broad absorption spectra in the visible region and cause great difficulties in locating during purification cytochrome f by its absorption spectrum. (69). None of the known procedures for quantitative cyt f extraction from the thylakoid membranes were entirely satisfactory, until cyt f yield from plants or cyanobacteria was increased by heterologous gene expression in the periplasm of

Esherichia coli(39).

Protein expression

Today, recombinant protein expression is a fairly common procedure. A key element in the development of an effective expression system is the construction of a vector plasmid (circular

DNA) that includes the appropriate promoter (the DNA sequence which directs RNA polymerase binding and transcription initiation), together with the gene of interest. The requirements for the suitable expression system are first of all highly efficient expression plasmids, the proper host strain, and the use of inexpensive media components. A single-subunit protein lacking a prosthetic group can easily be produced in bacterial hosts, whereas proteins that require a typical eukaryotic modification such glycosylation, necessitate a eukaryote as host, for example,

Saccharomyces cerevisiae. Optimizing the suitable host system depends on series of parameters, such as culture growth time, and temperature. The gram-negative bacterium Escherichia coli was the first organism to be employed for recombinant protein production due to its known physiology and genetics. However, the lack of glycosylation and other key eukaryotic functions required for expression of eukaryotic secreted proteins impose restriction on its use. Thus, other prokaryotic organisms such as Pseudomonas fluorescens, Staphylococcus carnosus, as well as eukaryotic S. cerevisiae, Arxula adeninivorans and Yaroowia lipolytica together with mammalian and plant cell hosts have been utilized for high level protein expression. It is not always clear which expression system to choose due to the advantages and disadvantages inherent in each. The choice is wide and complicated, thus selecting the right system has become an art, and expression of a new recombinant protein is akin to artistry (33). There are plenty of expression systems available, but there is always an element of unpredictability in the behavior of any given protein in a system.

For example, the expression of spinach plastocyanin by the Lundberg research group was examined in several different E.coli strains with different vector systems under the control of different E.coli promoters: trc, tac, trp and lac. Only the plasmid with the lac promoter gave a satisfactory protein yield(87). Lac promoter, as well as bacteriophage T7 promoter are widely used for protein overexpression(36, 65, 77, 98, 119). The E.coli host strain is also very important for the protein expression due to the design if the regulatory system. For example,

E.coli BL21 stain and its derivatives are common expression hosts: BL21(DE3),

BL21(DE3)pLysS or BL21(DE3) pLysE bearing the T7 RNA polymerase gene (λDE3 lysogen) produce T7 RNA polymerase for the expression of the target protein whose gene is under control by a T7 promoter (109). For more stringent control, some hosts carry either pLysS or pLysE plasmids which encode the production of T7 lysozyme to help regulate induced (controllable) overexpression of target protein(88). Finally, the plasmid vector for the target gene plays a significant role and choosing the proper one is ofter complicated. Many are commercially available(88), but the most popular are pET and pUC based plasmid systems and their derivatives. In summary, the expression of optimal protein level depends on host cell genetic background, culture conditions (temperature, growth phase) and vector configuration.

For the production of recombinant cytochrome f several expression systems are known(36,

98). Expression of cytochromes is complicated by the requirement that heme c be correctly ligated to the nascent synthesized apoprotein. This to improve the expression of c-type cytochromes in E.coli an extra plasmid, pEC86, is co-transformed into the host cells. This plasmid expresses the Rhizobium sp. cytochrome c maturation genes ccmABCDEFGH that catalyze heme attachment in the periplasm(4). Nonetheless, incorporation of the heme group is complicated, as the heme ligation occurs outside the cell in the periplasm, thus the apoprotein

19 must be efficiently secreted to interact with the heme ligation machinery. Secretion depends on the presence of a peptide leader sequence fused to cyt f to allow transport to the periplasm(98).

By analogy with the high-plant chloroplasts, cyanobacterial cytochrome f is expected to have a functional location in the thylakoid lumen. This would require translocation of cytochrome f across the cyanobacterial thylakoid membrane from its presumed site of synthesis in the cytoplasm. It is known that the protein is synthesized with a N-terminal presequence which is cleaved to from the mature polypeptide by peptidase upon translocation(57). Such sequences are somewhat similar to those mediating translocation of polypeptides across the bacterial plasma membrane, and it has been reported that the specificities of the plant thylakoid and E.coli processing peptidases are identical(46). The leader sequence consist of a positively charged N- terminal region, a hydrophobic region and a cleavage site for both of higher-plant chloroplast thylakoid transfer domain and for the bacterial transfer domain(122).

Thus, in this chapter I discuss the development of a recombinant cyt f expression system and the detailed characterization of the product. Expressed wild type and mutant cyts f and plastocyanin from Prochlorothrix will be analysed for reactivity as described in Chapter 3.

Materials and Methods

Expression plasmid pBAR

The expression plasmid pBAR was made from the previously characterized pVAPC plasmid(6) developed in our lab for the expression of plastocyanin from P.hollandica. The petA gene encoding cytochrome f from P.hollandica was amplified by PCR from the pUC19li5PCX(124) plasmid and cloned into a deletion derivative of the pVAPC plasmid. Specifically, a truncated gene, petE, encoding PC from P.hollandica was excised from the pVAPC plasmid (Figure 7A) by double digestion with Nhe I and Bam HI restriction enzymes, and the plasmid was dephosphorylated with bacterial Calf Intestinal Alkaline Phosphatase (CIP) to prevent the self- ligation of the plasmid. At the same time, the P.hollandica petA gene, bearing an in frame fusion to the P.laminosum plastocyanin (PC) 5’ leader peptide coding sequence was amplified by PCR from the plasmid pUC19li5PCX (Figure 7B) developed by Nadejda Vintonenko in our lab(124).

As mentioned before, the leader peptide coding sequence is required for transit to the E.coli periplasm. To eliminate the C-terminal intrinsic membrane domain of cyt f, the petA gene was truncated after the codon encoding Arg-250. This allows cyt f to be isolated as a soluble protein.

Pst I restriction sites were located on the both sides of this fragment. In order to generate an in- frame construct having compatible terminal restriction enzyme sites in pVAPC plasmid, PCR primers were designed to contain 5’NheI site and 3’ BamHI instead of PstI sites allowing ligation to the Nhe I and Bam HI site positions, respectively in the pVAPC plasmid. The PCR primers NheI F and BamHI R are presented in Table 1 below.

The resulting PCR product was inserted into the 2.1-TOPO vector (Invitrogen, Carlsbad, CA), digested by Nhe I and Bam HI and purified by agarose gel electrophoresis. The petA gene hybrid with the P.laminosum PC leader peptide was ligated into the Nhe I/Bam HI-digested pVAPC

A B C NheI - 175 NheI - 177

T7 pr BamHI - 462 omoto T7 pro r PC mo lea ter der lac i i p Z r r e pr o om PstI - 568 o t o E te r P C p le a d e e r t A

p m pBAR pVAPC A pUC19PCX

A 3441 bp 3560 bp t A 4072 bp BamHI - 1093 e m p p

Am p

Figure 7. Expression vectors

(A) pVAPC plasmid – P.hollandica PC expression vector used as a template for the pBAR

vector

(B) pUC19li5PCX plasmid – trial vector for the cyt f expression used as a template for the petA

gene hybrid amplification.

(C) pBAR plasmid – cyt f expression vector plasmid, yielding plasmid pBAR (Figure 7C), in which expression of petA is under the control of bacteriophage T7 lacking petE under the control of T7 RNA polymerase. Restriction enzymes

Nhe I and Bam HI, Alkaline Phosphatase and T4 DNA ligase were purchased from New England

Biolabs Inc., Beverly, MA. Plasmid pBAR was sequenced in the University of Chicago Cancer

Research Center DNA sequencing facilities to confirm the orientation of the insert and the lack of PCR-induced mutations in the coding region.

Primer Sequence NheI F 5’ –CTTTAAGAAGGAGAGCTAGCATGAAGTTGATTGCTC- 3’ BamHI R 5’ –GTGGAATTGTGAGCGGATCCCAATTTCACACAGGA- 3’

Table 1. PCR primers

Cytochrome f expression in E.coli and purification.

Plasmid pBAR was co-transformed into E.coli DH 5α (Invitrogen,Carlbad, CA) together with the pEC86 plasmid, encoding genes for the Rhizobium sp cytochrome c maturation cassette responsible for the heme ligation functions(100). Due to variation in petA expression among different transformants, initially several single colonies were picked and checked for expression efficiency. A glycerol stock of the best expressing colony was used for inoculation of 10 ml LB media supplemented with ampicillin (100 mg/l) and chloramphenicol (34 mg/L) and allowed to grow overnight (8 – 12 hr) at 37 °C. One L of LB media in 2L Erlenmeyer flasks were inoculated with 10ml of the initial inoculate and growth for 18 – 24 hr at 37°C with shaking (250 rpm).

Following this period, the temperature was decreased by 28 – 30 °C and culture incubated for another 20 – 24 hr. The lower temperature is required because the heme ligation enzymes encoded by pEC86 have an optimal temperature of 30 °C. Cells were harvested by centrifugation at 3000 × g at 4°C.

The E. coli periplasmic fraction containing cytochrome f was extracted by using cold osmotic shock as described by Karlsson et al.(68) The crude protein extract was fractionated by ion exchange chromatography by using a self-poured column (3.1 cm x 24 cm) containing Whatman pre-swollen DE52 resin (Whatman, Kent, UK) and equilibrated by 50 mM Tris buffer (pH 7.5).

The column was washed with 15 – 20 column volumes of 50 mM Tris-HCl buffer, 200 mM

NaCl and cyt f was eluted with 50 mM Tris-HCl (pH 7.5), 300 mM NaCl. The eluted protein was dialyzed twice against Milli-Q water containing 0.5mM phenylmethylsulphonylfluoride (PMSF) and 1 mM benzamidine twice followed by dialysis against pure Milli-Q water to remove salt from the solution. PMSF and benzamidine were used in order to inhibit residual protease activity to avoid degradation of the protein. Desalted pure samples (125 – 150 ml) were frozen at -80°C

23 and lyophilized in Labconco concentrator (Labconco Corporation, Kansas City, MO). Dry protein was redissolved in 10 mM phosphate buffer (pH 7.0 - 7.5), containing 1mM sodium ascorbate with final yield 2 – 3 mg of pure protein from 1 L of E.coli culture. The purity level and molecular weight of cyt f was monitored by SDS-PAGE. Cytochrome f samples were boiled for 3 – 5 min and then applied to a 16% polyacrylamide gel. Proteins were stained by Coomassie blue and heme staining of cytf was visualized by N,N,N’N’-tetramethylbenzidine and H2O2 by modified method of Thomas et al.(37, 115). Cytochrome f concentration was calculated from the

-1 -1 absorbance coefficient of reduced cytochrome f (ε554= 26.0 mM cm ) and absorption spectra were obtained on a HP8453 UV/Vis spectrophotometer (Hewlett-Packard Company, Palo Alto,

CA). The molecular mass of purified cyt f was confirmed by MALDI-TOF on a 2000 Brucker

Daltonics Mass Spectrometer Omniflex model (Bricker, Milan, Italy) by Dr Venkatesha Basrur at MUO, Toledo, OH.

Determination of the absorption coefficient of recombinant cytochrome f.

For the accurate determination of cytochrome f concentration, the absorption coefficient of newly expressed recombinant protein was determined by quantification of the heme concentration by using the pyridine hemochrome assay as described by Metzger et al.(79) .

Specifically, the oxidized pyridine hemochrome from approximately 1.5 – 2.25 µM cytochrome f was produced by 320µM potassium ferricyanide in a mixture with 0.1 N NaOH and 30 % (v/v) pyridine. After a steady absorbance of oxidized pyridine hemochrome at 550nm spectrum was recorded, a few grains of solid sodium dithionite were added to reduce the cyt f. Additional sodium dithionite was added until a stable absorbance reading was reached to ensure complete reduction and a spectrum was recorded from 520 to 580 nm. The reduced-oxidized spectrum was

24 obtained by subtraction of the oxidized from the reduced spectra and extinction coefficient was

-1 -1 taken ∆ε550 = 24 mM cm according to Berry and Trumpower(15) to calculate the concentration of the cytochrome f heme. The absorption spectrum of unmodified reduced cytochrome f solution of known heme concentration was recorded and the absorbance coefficient at 554 nm calculated.

CD spectroscopy of cytochrome f

CD spectra of cytochrome f were recorded on an Aviv 62DS CD spectrometer. Cytochrome f

(39.5 µM in 10 mM sodium phosphate buffer pH 6.0) was scanned from 193 nm to 260 nm for

10 scans at 25°C. The integration time was 0.5 s. Spectra were expressed as molar ellipticity

(degrees cm2 dmol-1) and the secondary structure content was estimated using the program k2d(2, 60).

Determination of redox potential of recombinant cytochrome f

Redox potentials were determined as described previously(5, 24, 42). Approximately 6 µM cytochrome f in 10mM phosphate buffer pH 7.5, 90mM NaCl containing 2mM potassium ferricyanide was titrated with 400mM potassium ferrocyanide at 22°C. The absorbance change at

554 nm after each addition was monitored with HP8453 UV/Vis spectrophotometer (Hewlett-

Packard Company, Palo Alto, CA). A value of + 430 mV (Eº) for the midpoint potential of the ferricyanide/ferrocyanide couple and Nernst equation (Eq 6) was used to calculate the midpoint redox potential of cytochrome f.

Em = Eº - 59,2/n log [ferricyanide] / [ferrocyanide] (Eq6)

Results and dicsussions

Primary structure of cyt f.

The amino acid sequence of the mature cytochrome f from P.hollandica was aligned with cyt f from higher, plants, algae and other cyanobacteria (Figure 8) and together the proteins show 33

% identity. The functionally most important and longest conserved region is located near the N- terminus (residues 17 – 27) and includes the sequence Cys-Ala-Asn-Cys-His, which acts as the heme-binding site. These two cysteine residues (Cys21 and Cys24) are the only conserved cysteine residues in the polypeptide and they provide the thio-ether links to the heme. His25 is the only conserved histidine and acts as fourth ligand to the heme iron. The sixth ligand for all these organisms is usually Tyr1, but in one case the coordinating amino acid is Phe1 in

Marchantia polymorpha.

In higher plants this polypeptide has very high degree of conservation (85 – 93 %). However, cyanobacteria, by comparison show only 40 – 55 % identity among mature cyt f polypeptides.

For example, P.hollandica cyt f shares 55 % of sequence identity with the higher plant Brassica rapa. Cyanobacterial cyts f are more variable and the mature proteins exhibit maximum 75 % identity as a group. Moreover, cyt f leader peptides are variable in length and have only 36 % residues shared (40). Comparison of P.hollandica mature cyt f sequence with other cyanobacteria gives 62 – 67 % identity. Many of the unique residues of P.hollandica cyt f are in regions of the protein that may be important in protein/protein interactions. One such region may be a loop expending from amino acids 99 to 104 of the mature protein. An objective of this dissertation is to determine whether this loop is functionally important in interactions with plastocyanin.

26 Br YPIFAQQNY- ENPREATGRIVCANCHLASK PVDIEVPQAV LPDTVFEAVV KIPYDMQLKQ Sa YPIFAQQGY- ENPREATGRIVCANCHLANK PVDIEVPQAV LPDTVFEAVV RIPYDMQLKQ Pa YPIFAQQGY- ENPREATGRIVCANCHLANK PVGIEVPQAV LPDTVFEAVV RIPYDMQLKQ Cr YPVFAQQNY- ANPREANGRIVCANCHLAQK AVESEVPQAV LPDTVFEAVI ELPYDKQVNS Syn YPFWAQETAP LTPREATGRIVCANCHLAQK AAEVEIPQAV LPDTVFEAVV KIPYDLDSQQ Nos YPFWAQQTYP ETPREPTGRIVCANCHLAAK PTEVEVPQSV LPDTVFKAVV KIPYDTSAQQ Pl YPFWAQQNY– ANPREATGRIVCANCHLAAK PAEIEVPQAV LPDSVFKAVV KIPYDHSVQQ Ph YPFYAQYNY- DSPREATGKIVCANCHLAKK TVEIEVPQAV LPDTVFKAVV KVPYDLDIQQ

Br VLANGKKGA LNVGAVLILP EGFELAPPDR ISPEMKEKIG NLSFQNYRPN KKNILVIGPV- Sa VLANGKKGG LNVGAVLILP EGFELAPPDR ISPEMKEKMG NLSFQSYRPN KQNILVIGPV- Pa VLANGKKGA LNVGAVLILP EGFELAPPDR ISPEMKEKIG NLSFQSYRPA KKNILVIGPV- Cr -LANGKKGD LNVGMVLILP EGFELAPPDR VPAEIKEKVG NLFYQPYSPE QKNILVVGPV- Syn VLGDGSKGG LNVGAVM-LP EGFKIAPPDR LSEGLKEKVG GTYFQPYRED MENVVIVGPL- Nos VGADGSKVG LNVGAVLMLP EGFKIAPEDR ISEELQEEIG DTYFQPYSED KENIVIVGPL- Pl VQADGSKGP LNVGAVLMLP EGFTIAPEDR IPEEMKEEVG PSYLFQPYAD DKQNIVLVGP- Ph VQADGSPSG LNVGAVLMLP EGFKLAPPER VDEELMEEVG DFYYLVTPYS ETDENILLAGP

Br -PGQKYSEITFPILAPDPATNKDVHFLKYPIYVGGNRGRGQIYPDGSKSNNTVYNATAGG--IISKILRK Sa -PGQKYSEITFPILAPDPATKKDVHFLKYPIYVGGNRGRGQIYPDGSKSNNTVYNSTATG--IVKKIVRK Pa -PGQKYSEITFPILSPDPAAKKDTHFLKYPIYVGGNRGRGQIYPDGSKSNNTVYNATAAG--IVSKIIRK Cr -PGKKYSEMVVPILSPDPAKNKNVSYLKYPIYFGGNRGRGQVYPDGKKSNNTIYNASAAG-KIVAITALS Syn -PGEQYQEIVFPVLSPDPAKDKSINYGKFVHLGA-NRGRGQIYPTGLLSNNNAFKAPNAG--ISEVNAL- Nos -PGEQYQEIVFPVLSPNPATDKNIHFGKYSVHVGGNRGRGQVYPTGEKSNNNLYNASATGTIAKIAKEED Pl LPGDQYEEIVFPVLSPNPATNKSVAFGKYSIHLGANRGRGQIYPTGEKSNNAVYNASAAG-VITAIAKAD Ph LPGEDYQEMIFPILSPNPATDAGVYFGKYSIHLGGNRGRGQVYPTGELSNNNAFSASIAGTIAAI---ED

Br EKGGYEITIVD-ASNERQVIDIIPRGPELLVSEGESIKLDQPLTSNPNVGGFGQGDAEIV-QDPLR Sa EKGGYEINIAD-ASDGREVVDIIPRGPELLVSEGESIKLDQPLTSNPNVGGFGQGDAEVVLQDPLR Pa EKGGYEITITD—APEGRQVIDSIPPGPELLVSEGESIKLDQPLTSNPNVGGFGQGDAEIVLQDPLR Cr EKKGGFEVSIE-KANGEVVVDKIPAGPDLIVKEGQTVQADQPLTNNPNVGGFGQAETEIVLQNPAR Syn EAGGYQLILT--TADGTETVD—IPAGPELIVSAGQTVEAGEFLTNNPNVGGFGQKDTEVVLQNPTR Nos EDGNVKYQVNIQPESGDVVVDTVPAGPELIVSEGQAVKAGDALTNNPNVGGFGQRDAEIVLQDAGR Pl DGSAEVKI-R--TEDGTTIVDKIPAGPELIVSEGEEVAAGAALTNNPNVGGFGQKDTEIVLQSPNR Ph NGFGFDVTIQ--PEDGDAVVTSILPGPELIVAVGDTVEAGQLLTTNPNVGGFGQMDSEIVLQSSSR

Figure 8. Amino acids sequences alignment of mature cytochrome f . The sequences were all taken from the NCBI protein database(62). N-terminal peptide leader and C-terminal polypeptides were truncated. Conserved amino acids highlighted in red. The yellow patch represent the heme binding site and green patch shows the loop region studied in this dissertation involved in protein interactions. Higher plants are in blue: Br- Brassica rapa; Sa-Spinacia aleracea; Pa- Poplar alba. Cyanobacteria are in green: Syn – Synechocystis PCC 6803; Nos –

Nostoc PCC 7906; Pl – Phormidium laminosum; Ph – Prochlorothrix hollandica. Green alga

Chlamydomonas reinhardtii – Cr is black.

A Novel P.hollandica cyt f expression system was developed allowing high yield of the protein. Expression vector pBAR (Figure 6C) was designed based on the pVAPC plasmid

(Figure 6A) that was previously used for the production of P.hollandica PC. The petE gene encoding plastocyanin was removed from the pVAPC plasmid by double digestion of NheI and

BamHI restriction enzymes and was replaced by P.hollandica petA gene (encoding cyt f) truncated after 750bp (Arg-250) with the Phormidium laminosum PC leader peptide sequence fused to the 5’ end of this gene. The P.laminosum PC leader peptide was necessary to direct the cyt f protein to the Escherichia coli periplasmic space, where heme ligation occurs in gram- negative bacteria. The plastocyanin leader peptide was chosen due to prior reports describing successful high yield production of cyt f in cyanobacteria P. laminosum(98). Thus the transportation of overexpressed cyt f into periplasm is more efficient by a PC leader peptide than by the endogenous cyt f leader. By comparing of different leader sequences (Table 2), we can conclude that P.hollandica and P.laminosum cyt f leaders are 32 % identical at the level of amino acid sequence, and are of similar length, which is much longer then the rest of the known leader peptides. These peptides are also more positively charged due to these similarities and the lack of cyt f expression when the cyt f leader peptide sequence is fused to the cyt f gene in E.coli, I decided to generate a fusion of the P.hollandica cyt f gene to the Phormidium PC leader peptide sequence. Thus, P.laminosum PC leader sequence was fused by PCR to the P.hollandica petA gene and ligated into pUCli5PCX plasmid under control of lacZ promoter (Figure 6B). This promoter did not give significant yield for the cyt f expression(124). Thus, the petA gene hybrid was amplified by PCR with NheI F and BamHI R primers (Table 1) from the pUC19li5PCX plasmid and ligated into the TOPO vector. Next, flanking regions were modified by the

28 introduction of 5’-Nhe I and 3’- Bam HI restriction sites and ligated into pVAPC plasmid under the control of the T7 RNA polymerase promoter.

Leader sequence N-terminal domain Hydrophobic stretch Cleavage site References P. hollandica cytf MLMKMHSSMAHLQSQFRSLSQ VVLVAIAALTLWVGLDTLV PQSAAA This work

P. laminosum cytf M NF KVCSFP S RRQS IAAFVR VLMVILLTLGALVSSDVLL PQPAAA (98)

P. laminosum PC MKLIAQISR SLSLALFALVLMVGSFVAVM SPAAA (98)

Pa. versutus cytc-550 MK ISIYATLAALSLAL PAVA (98)

E.coli cyt c MR FLLGVLMLMIS GSALA (98)

Anabaena MKK IFSLVLLGIALFTFAFS SPALA (81) sp.PCC7119 cyt c6

Table 2. Leader sequences. Positively charged amino acids highlighted in red.

The use of a chimeric gene encoding the transit peptide from one organism and the coding sequence from a mature protein from another is a common technique for the expression of thylakoidal proteins in E.coli and other hosts(80, 99). Some cyanobacterial proteins such as cyt c6 have been expressed successfully in E.coli with transport into periplasm by using their own peptide leader(28, 81). In our case the pBAR plasmid was co-transformed together with the pEC86 plasmid into E.coli strain DH5α. This strain was not designed for high-level controlled protein expression and it was a big surprise to get a high yield of protein. On average 2 – 3 mg of pure cyt f was obtained per liter of initial culture. The maximum obtained yield was 4.6 mg/l, which is comparable with the P.laminosum cytf expression system(98). Co-transformation in other E.coli strains, such as BL21(DE3), BL21(DE3)pLysS and MV1190 with our expression

29 plasmid system, was not successful and we were not able to get cells bearing both plasmids to yield significantly amounts of cyt f .

Purification of recombinant cyt f. Total periplasmic protein was extracted from E.coli by cold osmotic shock(68), and applied to a column with the anion exchange resin diethylaminoethyl cellulose (DE52). Cyt f from

P.hollandica is highly negatively charged, -19 by comparison to a net charge of -12 seen in other cyanobacteria, eg. P.laminosum cyt f(47, 98). This negatively charged protein bound tightly with the positively charged resin, yielding a straightforward purification. The column (3.1 x 24/ 230 ml) was washed with 4L of 50mM Tris buffer pH 7.5, and 200mM NaCl and cytochrome f was eluted by 300mM ionic strength of the same buffer and dialyzed against double distilled water.

In order to protect cyt f from degradation, proteases inhibitors were added during dialysis. Cyt f was next concentrated by lyophylization. Dry material was dissolved in 10mM phosphate buffer, pH 7.5.with 1mM sodium ascorbate. This reducing agent is necessary to obtain the complete reduction of cyt f that was oxidized during dialysis in the aerobic environment. Protein purity and size were checked by parallel heme and Coomassie stainings of SDS-PAGE (Figure 9).

As shown below, gel filtration did not yield effective purification of the crude extract. However, ion exchange chromatography yielded a near pure preparation. P.hollandica cyt f is more negatively charged than the closely related P.laminosum cyt f thus, the cationic resin is an effective sorbent for selective purification. Thus, we generally limited purification process to one ion exchange column.

30 A B

M1 1 2 3 4 M2 5 6 7 M 1 1 2 3 4 M2 5 6 7

75 75 51 51 50 50

36 37 36 37 28 28 25 25 20 20 15 15 6 6

Figure 9. SDS-PAGE of purified cytochrome f.

(A) Heme-stain using tetramethylbenzidine and H2O2;

(B) Coomassie stain;

M1, Precision Plus protein standard; M2, Broad range protein standard

Lane 1, Crude extract P.hollandica cytochrome f; Lane 2, P.hollandica cyt f run through gel filtration (Sephadex G-75); Lane 3, P.hollandica cyt f purified by gel filtration (Sephadex G75) and ion exchange chromatography (DE52);

Lane 4, P.hollandica cyt f purified only by ion exchange (DE52); Lane 5, Turnip cyt f (from Dr.

W.Cramer)-positive control; Lanes 6 and 7, P.hollandica cyt f purified only by ion exchange chromatography (DE52), and to be analyzed by NMR.

We were concerned about an additional minor band around 54kDa. As far as size exclusion chromatography by Sephadex G-75 as well as G-25 prior to the ion exchange column did not improve the purity level, and MALDI-TOF MS (Figure 10) did not show any additional species larger than 27.3kDa, it was concluded that this extra band might be a dimer, which formed solution under nondenaturing conditions(16, 64).

Figure 10. Mass Spectrum indicating molecular mass of cytochrome f as 27.3 kDa.

Protein characterization.

The reduced spectrum of cytochrome f from P.hollandica is identical to cyt f from higher plants and other cyanobacteria as seen by the absorbance maxima at 554 nm (α-band), 524 (β- band) and 421 nm (Soret band). According to these spectra, the concentration of P.hollandica cyt f was calculated from the α-band of the reduced protein (Figure 11). The oxidized spectrum of cyt f yielded s blue shift of Soret band from 421 nm to the 410 nm (Figure 12)(69).

32 A B

3.0 0.8

2.5 0.7

0.6 2.0 0.5 1.5 0.4 Absorbance 1.0 0.3 Absorbance 0.2 0.5 0.1

0.0 0.0

500 520 540 560 580 600 350 400 450 500 550 600 Wavelength, nm Wavelength, nm

Figure 11 (A) Visible absorption spectrum of reduced cyt f in 10mM phosphate buffer, 1mM sodium ascorbate, pH 6.0; Black line, cytochrome f from P.hollandica; Red line, Cytochrome f from P.laminosum; Green line, cytochrome f from turnip (gift from Prof. W.A. Cramer. Purdue

University). (B) Enlargement of absorption spectrum of α- and β-peaks.

2.2

2.0

1.8

1.6

1.4

1.2

1.0

0.8 Absorbance 0.6

0.4

0.2

0.0

-0.2 350 400 450 500 550 600 650 Wavelength, nm

Figure 12. Absorption spectra of oxidized (dash lane) and reduced (solid lane) P.hollandica cytochrome f from 610 nm to 350 nm. Eleven micromolar cytochrome f in 10mM sodium phosphate buffer pH 7.5 with 1mM sodium ascorbate (reduced) and 5 mM potassium ferricyanide (oxidized).

The extinction coefficient of P.hollanica cyt f was determined by converting the cytochrome heme to the pyridine hemochrome as described above. Using the Beer-Lambert law and the

-1 -1 millimolar extinction coefficient of the pyridine hemochrome (∆ε554 = 24.3 mM cm ), the concentration of the heme in the sample was calculated from the reduced minus oxidized absorption spectrum of the pyridine hemochrome (Figure 13). The absolute reduced spectra of

P.hollandica cyt f was recorded in the α-band region at the same heme concentration as in Fig.

-1 -1 12, and the absolute absorption coefficient was calculated as ε554 = 28.5 ± 1.5 mM cm . By comparison to turnip and P.laminosum cyt f, the P.hollandica coefficient it is slightly higher

(Table 3).

oxidized cytochrome f reduced cytochrome f 0.09 reduce - oxidized

0.08

0.07

0.06

0.05

0.04 Abcorbance 0.03

0.02

0.01

0.00 520 530 540 550 560 570 580 Wavelength,nm

Figure 13. Reduced minus oxidized absorption spectrum (green line) of the pyridine hemochrome produced from treatment of P.hollandica cytochrome f. Oxidized cytochrome f spectrum (black line), Reduced cytochrome f spectrum (red line).

The secondary structure of P.hollandica cyt f was determined by far-UV CD spectroscopy and such spectra show similarity to turnip cytochrome f(36) (Figure 14). These spectra

34 demonstrate the intense band around 198 – 200 nm that is characterized by the presence of β- sheet structure, and in each case the CD spectrum indicates almost the same secondary structure:

8 – 9 % α-helix, 42 – 48 % β-sheet and 44 – 48 % random coil. This is consistent with the known crystal structure of cyt f from plants and cyanobacteria.

A B

2000

-2000

-4000 9 % - α-helix 42 % - β-sheet -6000 48 % random coil Molar ellipticity (degree ellipticity cm2 Molar dmol-1) -8000

-10000

180 190 200 210 220 230 240 250 260 270 Wavelength, nm

Figure 14. CD spectra of P.hollandica cytochrome f. CD spectra of 40uM solutions of cytochrome f at 25°C

(A) CD spectra of wild-type and mutants of turnip cytochrome f from Gong et al.(36) .

(B) CD spectra of P.hollandica cytochrome f

The secondary structure content was estimated using the program k2d(60).

Thus, our cyt f CD spectra look similar to CD spectra of several cyt f derivatives turnip (Fig

14A), proving that our expression system yields a correctly assembled and folded cyt f preparation.

Determination of redox potential of cytochrome f. The midpoint redox potential of the P.hollandica cytochrome f was measured by titration

(Figure 15) with ferricyanide.

reduced cytf ratio 30 0.18 ratio 25 ratio 20 ratio 15 0.16 ratio 10 ratio 7.5 0.14 ratio 5 oxidized cytf 0.12

0.10 Intensity

0.08

0.06

0.04 546 548 550 552 554 556 558 560 Wavelength, nm Figure 15. Spectrophotometric titration of cytochrome f from P.hollandica.

An average midpoint redox potential of + 350 mV for P.hollandica cytochrome f was calculated from the plot shown in Figure 16, assuming the value of + 430 mV for the midpoint potential of ferricyanide/ferrocyanide couple and Nernst equation Eh = Eo + RT/nF ln([oxidized]/[reduced]). The P.hollandica cytochrome f redox potential is slightly higher then

P.laminosum cytochrome f according to Schlarb-Ridley et al and slightly lower than turnip cytochrome f(36) (Table 3).

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2 log (ferrocyanideferricyanide)/ 0.0

-0.2 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 log (reduced cytf / oxidized cytf)

Figure 16. Redox titration of P.hollandica cytochrome f. A 6 µM solution of cytochrome f in 10 mM phosphate buffer pH 6.0 with 90 mM NaCl containing 2 mM potassium ferricyanide was titrated with 400 mM potassium ferrocyanide at 22 °C. After each addition the absorption spectrum was recorded.

Parameters of cyt f P.laminosum(98) Turnip(36) P.hollandica

Midpoint redox 332 364 350 potential, mV Molecular mass 27212 28133 27371 calculated, Da Molecular mass 27213 ± 5 28164.9 ± 5.2 27363 ± 8 measured, Da Extinction 31.5 26.1 ± 0.25 28.5 ± 1.5 coefficient at 554 nm mM-1cm-1

Table 3. Physicochemical parameters of cytochrome f.

In conclusion, a novel recombinant protein expression system was developed for the expression of heterologous cytochrome f. Spectral and physicochemical studies indicate that folding and heme ligation of the recombinant P.hollandica cytochrome f is identical to native cyts f. Thus, this protein behaves and functions similarly to other cyt f homologues. The following chapter examines the reactivity of P.hollandica wild type and mutant cyt f with several

P.hollandica plastocyanin derivatives.

CHAPTER III PROTEIN-PROTEIN INTERACTIONS AND ELECTRON TRANSFER BETWEEN PROCHLOROTHRIX CYTOCHROME F AND PLASTOCYANIN – MUTAGENESIS OF A CYT F LOOP REGION

Introduction

The cytochrome f / plastocyanin interaction is an excellent system for the study of interprotein electron transfer because of its obvious physiological relevance and its high reaction rate (for higher plants ~ 2 × 108 M-1s-1 at 25 ºC, 100 mM ionic strength, pH 6.0), despite the small driving force of about 20 mV and a modest binding constant of about 7 mM-1 (67). Thus, it is one of only a few transient biological complexes extensively studied in vitro and in vivo(118).

Cytochrome f is a largely soluble component of the cytochrome bf complex, which is anchored to the membrane and has a covalently bound c-type (Cys-X-Y-Cys-His sequence) heme group in the middle of two β sheet domains. The heme group is partially covered by two short helices and an intervening loop (residues 1-25) that provide the covalent heme attachement. The heme is coordinated by Cys 21 and Cys 24, His25 and Tyr1 (an unusual c-type heme ligand). Thus, the heme environment is essentially hydrophobic. There is an elongated patch of positively charged residues on the small domain: Lys187, Arg209 and: Lys66, Lys65, Lys 58 on the larger domain that are typical for chloroplast cyt f.

The cyt f redox partner is the soluble copper protein plastocyanin, with the type I copper center that has a distorted tetrahedral structure composed of Cys84, His87 and His 37 and Met92 metal ligands. The secondary structure of plastocyanin consists of 8 β strands that form two twisted β-sheets to surround a hydrophobic interior. Although the copper ion is shielded from

39 solvent, one of the histidine ligand residues is on the surface of the protein and is surrounded by hydrophobic residues. This hydrophobic surface patch has been implicated as a major binding site for electron transfer partners. Two anionic patches have also been identified: one of them

Asp42, Glu43, Asp44, Asp51 and another one Glu59, Glu60, asp61, Glu68 that are also typical for chloroplast plastocyanin.

Before the cyt f structure became available(75) in 1994, it was not obvious the location of electron transfer site and results of site-directed mutagenesis studies were contradictory(12, 48).

Combining cyt f and PC crystal structures(75) together with the electron transfer pathway calculations(84) strongly support the view of close contact between the heme area and the hydrophobic patch of plastocyanin, where copper ligand His-87 is in Van der Waals contact with

Tyr-1 of the heme ligand of cyt f (Fig.17). The best electron transfer pathway (shortest distance) highlighted as thick green line(84). The Cu-Fe distance is 10.7 – 12.2 Ǻ depend on the analysis method.

11Ǻ

Figure 17. A stick diagram revealing the details of the complex interface(84).

Electron transfer between Pc and cyt f is mediated via short-lived protein interactions. An essential feature for the transient protein interactions is fast dissociation (rate of dissociation

2 4 -1 koff = 10 – 10 s ), which ensures a limited lifetime for the complex. The rate of association kon

7 9 -1 -1 is also high, in the range of 10 – 10 M s . Thus, binding constant Ka = kon / koff will be in the range 103 – 106 M-1.

Oppositely charged surfaces on the partner proteins are largely responsible for enhancing kon, since Columbic attraction provides long-range recognition and can pre-orient the diffusing proteins towards productive binding. Whereas, electrostatics can promote association, specificity and reactivity are determined by short-range hydrophobic interactions that bring the proteins into reactive conformations.

The kinetic model used for the interaction between cyt f and PC is given on scheme 1 and eq 7 is the formula derived from it assuming steady-state conditions.

Scheme 1

kon ket

A+B (AB) (A-B+) A- + B+

koff k-et

k2 = konkf / (koff+kf) (Eq 7)

where k2 is the second order rate constant of the overall reaction; kon – the rate constant of complex formation; koff the rate constant of dissociation before electron transfer has taken place; kf is related to ket, the intrinsic rate constant of electron transfer, and would become equal to ket if the driving force were large enough. It also has been demonstrated that for the reaction

between PC and cyt f, kf >> koff and thus k2 = kon. A consequence of this kinetic model is that variations in the reaction potentials of the PC should not influence k2(97). The overall rate constant of reaction, k2 can be measured by stopped-flow spectrophotometry method or by laser flash photolysis.

The first stopped –flow experiments with cytochrome f were carried out in 1974 for the parsley cyt f and plastocyanin by Paul Wood(125). Next, the reactivity of Brassica cyt f with plastocyanin was reported by Takabe in 1980(86). This method is widely used for the electron transfer rate measurements and based on a very quick mixing step followed by a fast oxidation- reduction reaction when the rate of oxidation or reduction can be monitored. For example, the cyt f to PC electron transfer rate can be monitored by measuring the increasing absorption band of oxidized cyt f at 410 nm or by the decreasing absorption band of reduced cyt f at 421 nm.

-1 From the exponential decay traces of absorbance changes, observed rate constant kobs in s can be detected and plotted against increased PC concentration. The slope of the linear fit curve will be the second-order rate constant k2.

Laser flash photolysis method is one of the indirect methods for measurement the kinetics of intracomplex electron transfer between reduced and oxidized proteins and pretty rarely used for in vitro studies of cyt f to PC electron transfer. One of these experiments was described by Kostic

(91, 93), where excited flavin semiquinone (FH*) was used as a strong reductant for the oxidized cyt f or PC. Thus, for the single proteins as well as for the cross-linked complex, cyt f-

PC reduction and reoxidation rates were determined by monitoring exponential decay at 554 nm.

In other words, cyt f reduction by flavin semiquinone happens in the complex cyt f (Fe+3) –

PC(Cu+2) (eq 8) followed by an intracomplex (unimolecular) reoxidation reaction or electron transfer from cyt f (Fe+2) to PC(Cu+2) (eq 9).

kcyt Cyt f (Fe+3)/PC (Cu+2) +FH* Cyt f (Fe+2)/PC (Cu+2) (eq 8)

ket Cyt f (Fe+2)/PC (Cu+2) Cyt f (Fe+3)/PC (Cu+1) (eq 9)

krev

Thus, kobs will be detected and plotted against different flavin semiquinone concentrations. The slope of this line will be the second order rate constant.

Protein protein contacts between PC and cyt f.

The overall molecular structure of plastocyanin from different organisms is very similar.

However, the surface properties are not conserved. There is a big difference between the surface electrostatics in plant and cyanobacterial plastocyanin. Higher plant plastocyanins are characterized by negative net charge (~ 9) at pH 7.0, whereas, cyanobacterial plastocyanins are widely variable, ranging from weakly acidic to highly basic. Thus, chloroplast plastocyanin exhibit two distinct patches: an acidic or so-called ‘eastern’ patch and a hydrophobic or

‘northern’ patch. Cyanobacterial PC only has a conserved hydrophobic patch. These differences in the surface charge properties have a significant influence on the kinetics of the interactions between cyt f and PC(47, 97)

Complex formation between cyt f and PC has been studied by several different computational methods. Manual docking was the first method that use information derived by mapping the electrostatic field on the protein surface(89, 106). Next, molecular dynamics(121) and Brownian dynamics(90) were used. The structures calculated using these pure computational methods suggest the existence of several different orientations used by two proteins to approach

43 each other. NMR spectroscopy in combination with restrained rigid body molecular mechanics gives more details for the transient complex formation between these proteins.

For example, the structure between spinach PC and turnip cyt f reveal van der Waals interactions between the heme region of cyt f and the hydrophobic region of PC that brings the two metal ions, iron and copper, together within ~ 11 Å of each other. At the same time additional interaction occur between basic and acidic patches of cyt f with the PC surface respectively(13, 30). This complex involves a close contact between the PC eastern patch and the small β-sheet domain of cyt f (Fig 18A).

The structure of the transient homologous complexes formed by cyt f and PC from the cyanobacteria P.laminosum and P.hollandica were also determined by the same combination of

NMR and computational methods(21, 23). These complexes are less well defined compared to the higher plant structure due to increased mobility and the lower number of restraints used in calculations. In the cases of these cyanobacterial complexes, none of the lowest energy structures was similar to the higher plant analogues, due to different surface characteristics of cyanobacterial and plant redox partners. This fact was also confirmed by NMR studies that show the absence of electrostatics involved in cyanobacterial complex formation, instead the partners interact mainly through hydrophobic contacts. Thus, the plastocyanin – cytochrome f complex from P.laminosum demonstrate a “head on” plastocyanin orientation perpendicular to the cyt f heme plane. This structure and the affinity of the reaction partners is independent of ionic strength and confirms that there are few electrostatic interactions. In sum, complex formation is predominantly controlled by hydrophobic interactions in cyanobacteria (21) (Fig 18B)

The structure of the plastocyanin-cytochrome f complex from higher plants represent on

Fig.18A, were both the hydrophobic and acidic patches of plastocyanin make contact with the

44 cyt f surface, with the total interface size of 400 – 500 Å. Plastocyanin residues Asp42, Glu43 and Asp44 contact cyt f residues Arg209, Lys187 and Lys185, respectively and residues Glu59 and Glu60 contact Lys65and Lys58, respectively. In plastocyanin, hydrophobic patch residues:

Gly10, Leu12, Phe35, Pro36, Leu62, Asn62 and Ser85-Ala90 are close (< 4 Å) to cytochrome f side chains near the heme site, yielding a copper-iron distance range from 10.7 – 11.3 Å.

A B

Figure 18. The structure of the plastocyanin/cytochrome f complex from (A) chloroplast (120) and (B) cyanobacteria (21).

Overall, the difference seen between the cyanobacterial and higher plant cyt f/PC complexes mirrors what has been documented fro PC/PSI interactions. In chloroplast systems, electrostatics plays an important role in the so-called Type III mechanism(27, 49). In cyanobacteria, complex formation in a Type II reaction mechanism is mediated by hydrophobics almost exclusively.

As was mentioned before, the cytochrome f (cyt f) – plastocyanin (PC) complex in the cyanobacterium Phormidium laminosum (Fig. 18 B), involves a ‘head on’ contact between the hydrophobic (northern) patch of PC with the hydrophobic surface surrounding the cyt f heme, with an average Cu-Fe separation of 11-15Å. At the same time, this model shows an additional interaction of Pc with a flexible loop of cyt f forming a ‘pocket-like’ region in the vicinity of cyt f residues 99 – 104 (red arrow on Fig 18B). There are two aromatic amino acids, Tyr and Phe among them, that face toward the PC and are possibly involved in protein – protein contacts.

This loop region aligned for the plants, algae and cyanobacteria are highlighted in green in Fig. 8 of Chapter II.

Other studies from our lab have employed the divergent cyt f from the cyanobacterium

Prochlorothrix as a natural mutant to define better the structural requirements for transient complex formation. Previous work revealed that PC from Prochlorothrix and cyt f from

P.laminosum forms a similar hydrophobic complex as the homospecific P.laminosum complex, despite large differences in the conformation of the Prochlorothrix and Phormidium PC hydrophobic patches(23). Also, the binding constant for the P.hollandica PC/cyt f complex is 20 – fold higher than that for the physiological P.laminosum complex. Moreover, PC from

P.hollandica is positively charged and weakly interacts with negatively charged cyt f from

P.laminosum. Thus, ionic strength likely affected this heterospecific complex much more than the native P.hollandica PC/cyt f complex(23). So, the missing piece of the puzzle is the lack of information about homologous complex formation and electron transfer between cyt f / PC from

P.hollandica.

In addition, theoretical analysis of this complex model shows the structurally unique

Prochlorothrix cyt f ‘pocket-like’ region may be involved in the PC/cyt f complex formation.

Thus, I propose that Tyr102 and Phe100 are actively involved in complex formation between cyt f and PC to minimize distance between reaction partners. Thus, by altering these residues by mutagenesis, the Cu-Fe distance increases, slowing electron transfer rates.

Materials and methods

Expression, purification and characterization of cytochrome f mutants were performed as described above in Chapter II.

Plastocyanin expression, purification and reconstitution.

P.hollandica plastocyanin wild type and mutants were expressed in E.coli strain

BL21(DE3) pLysS (Invitrogen, Carlsbad, CA) bearing PC expression plasmid pVAPC10.

Transformed cells were incubated overnight at 37ºC at continuous shaking (200 rpm) in 10 ml of

LB media supplemented with ampicillin (50 µg/ml) and chloramphenicol (34 µg/ml). The following morning, 500 ml of LB media in a 1 L flask, with the same antibiotic content, was inoculated by 10ml of overnight stock culture and grown to an optical density at 600 nm of 0.5 –

0.7. To induce the expression of PC, isopropyl-ß-D-thiogalactopyranosid (IPTG) was added to a final concentration of 1mM and the culture was incubated for another 8 – 10 h. Cells were collected by centrifugation and resuspended in 10 ml of lysis buffer (50 mM Tris-HCl, 10 mM

EDTA, 100 mM NaCl pH 8.0). Lysozyme was added to a final concentration of 1 mg/ml and incubated for 30 min at room temperature. In order to increase cellular lysis, 10 % Triton X-100 was added to a final concentration of 0.2 % and incubated at room temperature until viscous.

DNAse I was added to the final concentration of 30µg/ml together with MgCl2 (final concentration 20 mM) to support enzyme activity. The sample was incubated at 37 ºC until it was no longer viscous. The insoluble PC inclusion bodies were collected by centrifugation and then resuspended in 10 ml of lysis buffer containing 1 % Triton X-100 by brief pulses with a F60

Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) and recentrifuged. This, protein purification cycle was repeated three times with lysis buffer followed by a final cycle with distilled water to remove traces of Triton X-100. For protein refolding, washed inclusion bodies

48 were dissolved in 10ml of reconstitution buffer (0.2 M Tris-HCl, 6 M guanidine chloride, pH =

8.5) or urea buffer (0.2 M Tris-HCl, 6 M urea pH = 7.2) depending on the experiment, for 2 hr at room temperature with continued shaking. Next, β-mercaptoethanol was added to a final concentration of 25 mM and the pH of the sample was adjusted to 7.0 or another pH depending on the experiment. The resulting solution was diluted by 20 ml of freshly prepared Cu buffer (0.2

M Tris-HCl, 1 mM CuCl2, 25 mM β-mercaptoethanol, pH 7.0 and mixed overnight at 4 ºC with continued shaking. In order to refold protein and remove solvent, dialysis at 4 ºC was done four times against 4 L of dialysis buffer (first and second changes: 50 mM Tris-HCl, 1 mM CuCl2, 25 mM β-mercaptoethanol pH 7.0; third and fourth: 50 mM Tris-HCl pH 7.0. For the reconstitution of plastocyanin mutants, dialysis was done for at least 5 times against buffers containing a gradually decreasing concentration of guanidine chloride. This first buffer contained

1 M guanidine chloride; the second, - 0.5 M; the third - 0.25 M and the fourth and fifth changes were against 50 mM Tris-HCl buffer. The dialysis was performed by using Spectra/Por Cellulose

Ester Membrane, with a 1 – 3 k MWCO (Spectrum Laboratories Inc., Rancho Dominguez, CA).

The solution was concentrated by reverse dialysis against high molecular weight Polyethylene

Glycol (Sigma-Aldrich, St. Louis, MO, Cat # P3640). The fraction of protein that was misfolded and precipitated during the procedure, was removed by centrifugation. The final volume, 2 – 4 ml of purified reconstituted proteins, was achieved by using a Centriplus YM-3 centrifugal filter device with a MWCO 3,000 (Millipore, Bedford, MA).

Plastocyanin characterization

Plastocyanin preparation was analyzed by visible absorption and far-UV circular dichroism

(CD) spectroscopy as was published before(6) and the concentration was calculated from the

-1 -1 molar extinction coeffitient of fully oxidized PC ε600 = 4.9 mM cm . The absorption spectra

49 were obtained with an HP8453 UV/Vis spectrophotometer (Hewlett-Packard Company, Palo

Alto, CA). In order to obtain fully oxidized plastocyanin, potassium ferricyanide was added to the protein solution until the absorption band at 600 nm no longer increased. The purity of folded protein remaining in the solution was monitored by the ratio A278/A600. Typically we achive a ration of 3.2.

Site-directed mutagenesis.

PCR-mediated mutagenesis by the QuickChange PCR method (Stratagene, La Jolla, CA) was performed on the P.hollandica cyt f expression plasmid pBAR, carrying a truncated petA gene that encodes wild type P.hollandica cyt f . Custom mutagenic primers were HPLC purified and obtained from Invitrogen Life Technologies, Inc. The primer sequences directing the following mutations were obtained (changed codons are highlighted in red).

Y102G 5’- GAG GAA GTG GGC GAC TTC TAC GGT CTG GTC ACC CCC TAC AGC- 3’

F100S 5’- GAG GAA GTG GGC GAC TTC TAC GGT CTG GTC ACC CCC TAC AGC- 3’

Y102G/F100S 5’- GAG GAA GTG GGC GAC TTC TAC GGT CTG GTC ACC CCC TAC

AGC- 3’

After 18 cycles of amplification with Pfu Turbo DNA polymerase, the plasmids were treated with DpnI restriction enzyme in order to digest the methylated template plasmid leaving only copies with introduced mutations. The amplified plasmids were transformed into XL1-Blue supercompetent cells and isolated using the QIAprep Spin Miniprep Kit (QIAGEN, Valencia,

CA).The mutations were confirmed by DNA sequencing at University of Chicago Cancer

Research Center, DNA Sequencing Facility, Chicago, IL.

Kinetic measurements and binding constant.

For the measurements of k2, the second order rate constant of electron transfer from cytochrome f to plastocyanin, reduced cytochrome f and oxidized plastocyanin were prepared by adding 10-fold excess of sodium ascorbate and potassium ferricyanide, respectively. Protein samples were dialyzed for a 6 – 8 hours vs 10 mM phosphate buffer, pH 6.0, 90 mM NaCl.

Electron transfer was monitored at 421 nm with a stopped-flow spectrophotometer HiTech

SF61DX2 in Dr Lucy Waskell’s lab at University of Michigan, Ann Arbor, MI. The cytochrome f concentration was held constant at 0.15 µM, while the plastocyanin concentration was varied between 1.0 – 3.0 µM. The observed pseudo-first-order rate constant was plotted against the concentration of plastocyanin and a curve was fitted by linear regression. The slope of the linear curves gave the second-order rate constant k2.

The binding constant for cytochrome f and plastocyanin was measured taking advantage of the absorbance enhancement at 410 nm when oxidized cytochrome f binds to oxidized plastocyanin(48). Oxidized cyt f (10 µM) and oxidized plastocyanin (0 – 100 µM) were dissolved in 10 mM phosphate buffer with 90 mM NaCl. The absorbance at 410nm was measured after each addition against the cuvette containing the same concentration of oxidized cyt f. The data are presented as the ratio of the observed absorbance change to the absorbance change at infinite plastocyanin concentration plotted as a function of the ratio plastocyanin to cyt f concentration(32, 48) and KA was calculated by using equation (Eq 10),

1/2 ∆A=∆A∞/2C[P+C+1/KA-{(P+C+1/KA)24PC} ] (Eq 10)

where ∆A∞ is the value of ∆A with excess plastocyanin, which was determined directly; P and C represent the concentrations of plastocyanin and cytochrome f respectively.

Molecular structures The cytochrome f structure from P.laminosum was obtained from the Protein Data Bank

(PDB)(63) with PDB ID number 1CI3. The P.hollandica cyt f structure is not yet determined, so a model was build in Swiss-MODEL(43) using the X-ray structure of P.laminosum cyt f as a template.

Results and Discussion Protein modeling The protein sequences for the truncated petA genes from P.laminosum and P.hollandica share

60 % identity and shown on Fig.19.

P.hollandica MLMKMHSSMAHLQSQFRSLSQVVLVAIAALTLWVGLDTLVPQSAAA WT P.laminosum MNF KVC SFP S RRQS –IAAFVRVLMVILLTLGAL VSSDVLLPQP AAA

P.hollandica YPFYAQYNYDS PREATGKIVCANCHLAKKTVEIEVPQAVLPDTVFKAVV WT P.laminosum YPFWAQQNYANPREATGRIVCANCHLAAKPAEIEVPQAVLPDSVFKAVV

P.hollandica KVPYDLDIQQVQADGSPSGLNVGAVLMLPEGFKLAPPERVDEELMEEVGDF WT P.laminosum KI PYDHSVQQVQADGSKGPLNVGAVLMLPEGFTIAPEDRI P EEMKEEVGPS

P.hollandica YYLVTPYSETD ENIL LAGPLPGEDYQEMIFP I LSPNPATDAGVYFGKYSIH WT P.laminosum Y- LFQP YADDKQNIVLVGPLPGDQYEEI VFPVLSPNPATNKSVAFGKYSIH

P.hollandica LGGNRGRGQVYPTGELSNNNAFSAS I AGT IAAIEDNGFG F -DVTIQPEDG WT P.laminosum LGANRGRGQ IYPTGEKSNNAVYNASAAGVITAIAKADDGSAEVKIRTEDG

P.hollandica DAVVTSIL PGPELIVAVGDTVEAGQLLTTNPNVGGFGQMDSEIVLQSS SR WT P.laminosum TT I VDKIPAGPELIV SEGEEVAAGAALTNNPNVGGFGQKDTEIVLQSPNR

Figure 19. Sequence alignment of cyanobacterial cytochrome f from Phormidium laminosum and Prochlorotrix hollandica.

The cleavable cyt f leader peptide sequences that are responsible for the mature protein transit into the thylakoud lumen are underlined on Fig. 19 and have 35% identity between P.hollandica

53 and P.laminosum. The ‘pocket-like’ loop region potentially involved in PC interactions is highlighted in yellow and shows that P.hollandica has an extra aromatic residue, Tyr-102 that might be involved in PC/cyt f complex formation. Since Prochlorothrix PC has a structurally unique hydrophobic patch that likely comes in contact with this loop region of cyt f, I propose that this unique Tyr 102 is a co-adaptation existing to assist in docking the P.hollandica PC hydrophobic patch.

In order to show the difference in the loop region structure, 3D models for P.hollandica vs.

P.laminosum cyt f were presented on Fig. 20 and 21. The ‘pocket-like’ region highlighted in green and enlarged on Fig. 20 C.

A B C

Tyr 102

Tyr 102* Phe 100

Tyr 102 Tyr 102

Tyr 102*

Figure 20. 3D structure of cytochrome f from P. laminosum (A) and P. hollandica (B). Enlarged

“pocket-like” region of P.holladica cyt f (C).

To understand how this pocket-like loop influences PC/cyt f complex formation and efficiency of electron transfer, three mutants were prepared. The Y102G mutant lacks only the Tyr residue.

The bulky Phe100 residue was replaced by Ser100 in the F100S mutant. In addition, a double

Y102G/F100S mutant lacking both residues Tyr102 and Phe100 was constructed, yielding a loop that looks more like the P.laminosum structure, which is less hydrophobic and less likely protruded toward the PC interaction site.

Both Y102G and Y102G/F100S mutants were expressed and characterized as well as the wild- type and comparison data are presented below. However, the F100S mutant is still in preparation.

A B

Loop region

P. laminosum PSY- L FQ

P. hollandica DFYYLVT

“pocket like” region

Figure 21. Ribbon diagram of P.laminosum (A) and P.hollandica (B) cyt f .The ‘Pocket-like’ region indicated with the red arrow. Amino acid residues that were mutated highlighted in red.

First of all, in order to prove that mutations are properly expressed and have the correct molecular masses, we performed MALDI-TOF MS of the pure proteins. These data are

55 represented in Table 4 and Figures 22 and 23. As far as we can see the difference for the wild- type and Y102G mutant molecular masses are within the machine error, which is 10 Da.

However, for the Y102G/F100S cyt f mutant difference is slightly higher (42 Da) and might be associated with two sodium ions left over following sodium ascorbate reduction of the cyt f preparation in addition to machine error ± 5Da.

Protein Calculated mass Measured mass Difference (Da) (Da) (Da) WT 27371 27363 -8 Y102G 27265 27257 -8

Y102G/F100S 27205 27247 +42

Table 4. Molecular masses of wild type and mutant cyt f holoproteins.

On Fig. 22 we can see that the major peaks are around 27 kDa and the enlargement of these spectra on Fig. 23 show the accurate molecular mass for the WT and mutants. At the same time, we observed a peak around 54 kDa, which might represent stable cyt f dimers that survived the ionization process. This fact also strongly suggest that an extra minor band on the SDS-PAGE gel (Fig. 9 Chapter II) is a cyt f dimer.

Second, the visible spectra of the mutants were identical to those of the wild type (data not presented). These results indicate that the mutant proteins were properly expressed and have similar heme coordination and folding. Circular dichroism data also support the correct following of the expressed proteins. Third, extinction coefficients for the mutants and WT were calculated as described before (see Chapter II), and the data are present in Table 5. The ε554 for the three proteins are virtually identical.

Figure 22. Partial MALDI-TOF data of WT and mutants of P.hollandica cyt f.

Figure 23. MALDI-TOF data of enlarged region around 27kDa for the WT and mutants of

P.hollandica cyt f.

Midpoint redox potentials were determined as described before (see Chapter II) and the data presented in Table 5 and Fig. 24. Logarithmic plots the ferri/ferrocyanide ratio versus reduced/oxidized PC yielded a slope for all samples at 45 degrees, indicating single-electron reduction of PC. At the same time, ferri/ferrocyanide ratios, which corresponded to the point at which ratios of reduced/oxidized PC equal one, are virtually identical for the cyt f WT and the mutants. By comparison to the higher plant cyts f, these redox potentials are slightly higher, whereas the potential for P.laminosum cyt f is slightly higher than P.hollandica cyt f.

1.6

1.4 1.2

1.0

. 0.8

0.6

0.4

0.2

ferricyanide) / (ferrocyanide log 0.0

-0.2 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 log (reduced cytf / oxidized cytf)

Figure 24. Redox titration of P.hollandica cytochrome f WT(black line), Y102G mutant (red line ) and Y102G/F100S mutant (green line). A 6µM solution of cytochrome f in 10mM phosphate buffer, pH 6.0 with 90 mM NaCl, containing 2 mM potassium ferricyanide was titrated with 400 mM potassium ferrocyanide at 22 °C. After each addition, the absorption spectrum was recorded.

However, driving force between physiological partners PC and cyt f should not be affected as far as PC redox potential is also varies from 355 – 370 mV from species to species. In the case of

P.hollandica, the E of PC (+365)(6) yields a modest favorable driving force.

-1 -1 Cytochrome f ε554, mM cm E, (mV) WT 28.5 350

Y102G 28.0 350

Y102G/F100S 28.3 348

Table 5. Midpoint redox potentials and extinction coefficients for the WT and mutants of P.hollandica cyt f.

Finally, in order to check proper protein folding in comparison to the wild-type, circular dichroism spectra were performed as described earlier (Chapter II) and the data presented in

Fig. 25.

) 4 -1 2 dmol

. 0

-2

-4

-6 WT -8 Y102G cm (degrees ellipticity Molar Y102G/F100S -10

-12

180 190 200 210 220 230 240 250 260 270 Wavelength, nm

Figure 25. CD spectra of P.hollandica cytochrome f wild type and mutants. CD spectra of 40

µM solutions of cytochrome f in 10mM phosphate buffer pH 6.0 at 25°C.

Aromatic amino acids significantly influence CD spectra, so the spectral shifts observed with the mutant protein are most likely associated with the lack of one or two Tyr/Phe chromophores in the pocket-like domain(108). At the same time aromatic rings, in close contact may cause strong coupling and affect the intensity of the CD spectra especially in the wild type(126).

Kinetic measurements

The rate constant for electron transfer from reduced P.hollandica cytochrome f to oxidized wild-type and the Y12G/P14L mutant of P.hollandica plastocyanin was determined by stopped- flow spectrophotometry by following the oxidation of cytochrome f at 421nm under pseudo-first- order conditions in the presence of increasing plastocyanin. The temperature for this experiment was set to 5 ºC to slow down the kinetics relative to the room temperature due to the very fast process. The time course of the absorbance changes have been fitted to the single exponential decay.

We chose the PC Y12G/P14L mutant for kinetic studies by comparison with WT PC because virtually all plastocyanins have a glycine at position 12 and leucine at position14. These are conserved amino acids in the hydrophobic patch known to be involved in the formation of the transient docked complex. By contrast, P.hollandica PC has a tyrosine and proline at posions 12 and 14 respectively. We are interested whether this alteration in a functionally important part of the protein dictates its species specificity. The observed pseudo-first rate constant was plotted against the plastocyanin concentration. The slope of these lines gave the second order rate constant k2.

800

700 A 600

500

-1

,s 400 obs k 300 200

100 WT cyt f + WT PC WT cyt f + Dbl PC 0

1.0 1.5 2.0 2.5 3.0 140 Plastocyanin concentration, µµµM

120 B 100

80 -1 , s ,

obs 60

k 40 20 Y102G cytf + WT PC Y102G cytf + Dbl PC 0

1.0 1.5 2.0 2.5 3.0 Plastocyanin concentration, uM C 45

40 35 30 -1

s , 25 obs k 20 15 Y102G/F100S cytf + WT PC Y102G/F100S cytf + Dbl PC 10

5 1.0 1.5 2.0 2.5 3.0 Plastocyanin concentration uM

Figure 26. Rates of electron transfer from cytochrome f to wild-type and the Y12G/P14L(Dbl) mutant of plastocyanin both from cyanobacteria Prochlorotrix hollandica. Electron transfer from reduced cytochrome f (0.15 µM) to oxidized plastocyanin was monitored at 421 nm. The reactions were carried out at 5°C in a buffer consisting of 10 mM potassium phosphate pH 6.0 and 90 mM NaCl; (A) Oxidation of WT cyt f by WT PC (black ) and Dbl mutant PC (blue)

(B) Oxidation of Y102G cyt f mutant by WT PC (red) and by Dbl mutant PC (cyan)

(magenta)

The native (wild type) plastocyanin has almost a two fold faster electron transfer from

8 -1 1 cytochrome f than for the Y12G/P14L (Dbl) mutant: k2 = 3.28 ± 0.27 × 10 M s versus k2 = 2.0

± 0.12 × 108 M-1s-1 (Figure 26A). The same pattern was observed for the Y102G and

Y102G/F100S cyt f mutants, where reaction with Dbl PC mutant yielded rate half that of WT PC

(Figure 26 B,C and Table 6). This is good evidence showing the potential importance of PC surface conformation in influencing electron transfer or binding affinity to cytochrome f.

At the same time, if we compare cyt f WT and the mutant cyts f surface differences and approach the second-order rate constant, we observed that removing the hydrophobic aromatic residues Tyr 102 and Phe 100 from the loop region decreased the rate of electron transfer more than 10 times. These data presented on Figure 27 and Table 6 demonstrates that the cyt f loop conformation significantly affects electron transfer with PC. Moreover, electron transfer from cyt f to PC WT and Dbl mutant yielded similar rates (Fig. 27). Error bars were determined as a standard deviation of three experiments.

800 450 B

700 A 400 600 350

500 300 -1

-1 250

400 ,s , s , obs obs 200 k

k 300 150 200 100

100 50

0 0

1.0 1.5 2.0 2.5 3.0 1.0 1.5 2.0 2.5 3.0 Plastocyanin concentration, uM Plastocyanin concentration, uM

Figure 27. Rates of electron transfer from cytochrome f WT and mutants to PC wild-type (A); and from cyt f WT and mutants to PC Y12G/P14L mutant (B). Line colors are coded as on

Fig.26.

All kinetic data are summarized in Table 6.

8 -1 -1 8 -1 -1 Cytochrome f k2 (x 10 M s ) of PC WT k2 (x 10 M s ) of Y12G/P14L PC mutant

WT 3.28 ± 0.27 2.01± 0.12

Y102G 0.54 ± 0.06 0.29 ± 0.04

Y102G/F100S 0.15 ± 0.01 0.12 ± 0.06

Table 6. The second order rate constant for electron transfer to WT and Y12G/P14L mutant of plastocyanin.

Binding constant determination.

To determine whether changes in the rates of electron transfer from the mutants and WT of cyt f to plastocyanin were due to changes in complex formation or the intrinsic rate of electron transfer, the binding constant KA was determined. KA was measured by taking advantage of the increased absorbance of the Soret band (410 nm) of oxidized cyt f on binding to plastocyanin and values of KA were calculated as described before (32, 48). The KA values obtained for the homospecific interaction of cyt f/PC from P.hollandica are two orders of magnitude higher than for P.laminosum cyt f / P.hollandica PC(23) and for the higher plant complexes. However, obtained Ka is within the range for the transient complexes(36) binding constants, which is 103 –

106 M-1. Errors presented as a standard deviation for the three independent measurements.

64 A B 1.0

1.0 0.8

0.8

0.6 0.6 00 00 0.4 A/A A/A 0.4 WT cytf + Dbl PC 0.2 Y102G/F100S cytf + Dbl PC 0.2 Y102G/F100S cytf + WT PC WT cytf + WT PC Y102G cytf + Dbl PC Y102G cytf + WT PC 0.0 0.0

0 1 2 3 4 5 6 0 1 2 3 4 5 6 [PC]/[cytf] [PC]/[cytf]

Figure 28. Binding curve for the interaction of (A) WT plastocyanin with WT and mutants of cyt f; (B) Dbl mutant PC with WT and mutants of cyt f. The increase in absorbance at 410nm was measured when plastocyanin was present at concentrations between 0 and 60 µM. Proteins were dissolved in 10mM phosphate buffer, 90 mM NaCl, pH 6.0 at 25 ºC. Color code is the same as for Fig.16 and 17.

No large differences in KA were observed between the WT and Y102G mutants of cyt f in binding with the WT of PC. However, absence of both residues Tyr 102 and Phe 100 on the cyt f loop surface, significantly affected binding affinity (64 % lower) (Fig 28A and Table 7) with WT

PC. It is hard to explain how by second-order rate constant a 10-fold decrease in binding constant could increase or stay the same. At the same time, the KA for the Y102G cyt f mutant drops down almost 35% in comparison to WT by the interaction with the PC Dbl mutant.

However, Y102G/F100S cyt f mutant binding affinity to Dbl PC mutant is almost the same as for the WT cyt f (Fig. 28B and Table 7).

Cytochrome f Plastocyanin Ka (x 105 M-1)

WT WT 5.8 ± 0.7

Y102G WT 5.2 ± 0.3

Y102G/F100S WT 9.1 ± 0.6

WT Y12G/P14L 4.3 ± 0.6

Y102G Y12G/P14L 1.5 ± 0.3

Y102G/F100S Y12G/P14L 3.8 ± 0.2

Table 7. Binding constant, KA for the interaction of cytochrome f and plastocyanin from the cyanobacteria P.hollandica.

From these data we can conclude that two Prochlorothrix cyt f mutants, Y102G and

Y102G/F100S, yielding a modified pocket region, were expressed and folded properly. Their physicochemical characterizations are similar to the wild-type. Removal of aromatic residues Tyr

102 and Phe 100 from the loop region of P.hollandica cyt f modestly affected binding affinity with WT plastocyanin as well as with the Y12G/P14L mutant. More importantly was the profound effect of these mutations on k2. These data suggest that complex formation occurs between all cyt f and PC reaction pairs, but the cyt f loop mutants yield complexes less reactive than wild type. Thus, we suggest that the ‘pocket like’ loop region is actively involved in complex formation leading to productive electron transfer between cyt f and PC in P.hollandica.

Concluding remarks

Analysis of PC and cyt f amino acid sequence from cyanobacteria suggest a co adaptation of the PC hydrophobic patch to the ‘pocket-like’ loop region. We suggest that the unique PC hydrophobic patch likely co-evolved with the cyt f loop region to yield a species-specific contact point. It is possible that all cyanobacterial cyts f and PCs yield a similar or identical contact in mediating complex formation prior to ET. Future mutagenesis studies on Phormidium cyt f / PC and those of higher plants will help resolve this issue. We would predict from our studies that mutagenesis of the Phormidium proteins would yield changes in k2 and maybe KA, but in chloroplast proteins, a mutation in the loop region would have no effect, due to the different structures of the complex (see Fig.18 A, B).

CHAPTER IV

STUDIES OF MUTANT P. HOLLANDICA PLASTOCYANIN INTERACTIONS WITH PHYSIOLOGICAL AND NON-PHYSIOLOGICAL PARTNERS. (TRIALS, ADDITIONAL STUDIES AND FUTURE PERSPECTIVES)

Introduction

Blue copper proteins are a large class of metalloproteins and their functions are very important in such metabolic processes as photosynthesis, respiration, detoxification of harmful compounds among others(83)

For example, azurins participate in deamination of primary amines and in denitrification processes by shuttling electrons from aromatic amine dehydrogenase to cytochrome oxidase and from some c-type cytochromes to nitrite reductases(29). Pseudoazurin also participates in the denitrification process and plays the role of electron donor to nitrous oxide reductase from nitrite reductase. Pseudoazurin takes part in a wide range of electron transport reactions and is able to interact with structurally different proteins like cytochrome oxidase and cytochrome cd1(72).

Amicyanins shuttle electrons from methylamine dehydrogenase to a c-type cytochrome

(cytC551i) in the methylamine-oxidizing chain of methylotrophic bacteria(18).

Finally, plastocyanin (PC) is the well-studied small copper protein involved in photosynthetic electron transfer. It is composed of eight anti-parallel β-sheets, having a molecular weight around

10 kDa. The overall dimensions of the molecules are approximately 40 × 32 × 28 Å.

Plastocyanin, like other blue (type I) copper proteins (azurin, stellacyanin, amicyanin) is characterized by an intense ligand-to-metal-charge-transfer (LMCT) absorption band around 600 nm and unusually high reduction potential ~ 355 mV. The spectral characteristics of blue-copper protein are related to the structure and geometry of the copper site. The Cu(I) has a distorted

68 tetrahedral geometry which is formed by two histidine (His) nitrogens with typical a Cu-N bond

( 2.0 Å) and one highly covalent cysteine (Cys) sulfur ligand forming an unusually short Cu-S bond (2.1 Å). It is also weakly coordinated by a methionine (Met) sulfur ligand atom (2.9 Å)(34,

70) (Figure 29).

Figure 29. X-ray structure of the Cu center from poplar plastocyanin(105).

The normal tetragonal geometry of Cu(II) has a distorted tetrahedral structure which gives the unusual spectral features to the blue copper protein(105). The intense absorption band at 600nm corresponds to the SCys π→ Cu dx2-y2 charge transfer transition and the SCys σ→ Cu dx2-y2 charge transfer transition is a weak band of high energy. This is very unusual, because normally σ charge transfer is intense and π charge transfer is weak. The intensity of charge transfer depends on the overlap of the orbitals involved in the transition and the dx2-y2 orbital is rotated in the xy plane(104). These explanations are presented according to the Cu-cysteine bonding interaction

(Fig. 30) in the case of plastocyanin. In a tetrahedral coordination of Cu(II), the dx2-y2 orbital is oriented along the ligand-metal bond and hence the ligand σ orbital is overlapped more and has an increased charge transfer transition than the cysteine π orbital. A different situation can be observed in the case of plastocyanin, where the dx2-y2 orbital is rotated to 45° due to the distorted geometry, thus overlapping with a π antibonding orbital, which becomes much stronger(105).

These unique spectral features are accessible only for oxidized blue copper, while for the reduced copper with the d10 closed shell configuration they are inaccessible.

Figure 30. Cu-cysteine bonding interactions. (A) ‘Normal’ bonding with weak π and strong σ transfer transition; (B) Strong π and weak σ charge transfer spectra(105).

These electronic structures of the blue copper site are responsible for its function and reactivity. The main function of PC is shuttling electrons from the membrane-bound cytochrome

+ b6f complex to the membrane-bound chlorophyll-protein complex, P700 , in photosystem I.

Prior to electron transfer, PC typically forms a transition complex characterized by low binding affinity (103 – 10 6 M-1) and lifetime (millisecond time scale), but the high specificity of binding allows electron transfer to occur efficiently. This means that the process of interaction and recognition between proteins surfaces is both very specific and transient(23). There are two distinct features on the surface of PC: the ‘eastern’ and ‘northern’ patches. PC from higher plants has two acidic patches in the ‘eastern’ part and a hydrophobic surface in the ‘northern’ that surrounds the Cu site (Fig. 31). Three residues in the large acidic patch are conserved in all PC from the high plants and algae:Asp42, Asp44 and Glu43. The small negative patch consists of

70 residues 59 – 61 in PC from higher plants. In algal PC, the acidic region appears to be slightly different; instead of acidic residues at positions 57 – 60 there are usually two negative side chains present at positions 53 and /or 85(102).

‘Northern’ patch

‘Eastern’ patch

Figure 31. Structure of poplar plastocyanin(67). Copper highlighted with the blue arrow.

Alternatively, cyanobacterial PC lacks negatively charged regions on the surface, but the big hydrophobic patch on the ‘northern’ part is shared among all PCs. This variation of PC surface structure plays an important role in their interactions with biological and non-biological partners.

Cytochrome f is one of the biological partners of PC. Protein-protein complex formation and electron transfer was discussed before in Chapter 3.

Another biological partner of PC is photosystem I (PSI), which consists of 96 chlorophyll a molecules, 22 β-carotenes, two phylloquinones, 4 lipids, 3 [4Fe-4S] clusters and 11 – 12 protein subunits (PsaA, B, C, D, E, F, I, J, K/K2, L, M, U)(127). The PsaF subunit in higher plants is involved with electrostatically-driven docking of PC to PSI, hence the recognition between them

71 should be very specific. Thus, interaction is involved in the so-called Type III reaction mechanism that occurs in chloroplast systems. Two negative patches of PC the ‘eastern’ face interact with lysine residues in the N-terminal domain of PsaF in higher plants.

The interaction of PC with non-physiological reaction partners can also yield information about events leading to reactivity. For example, studying the interactions between PC and a synthetic lysine peptide (Lysptd) helped in the understanding of the structural basis for both binding and electron transfer(54); any charged peptides could be useful for studying the molecular recognition characteristics of protein and their structural changes during interactions because they lack any visible absorption(55). The structure of this electrostatically mediated complex was as predicted in Figure 32. Early studies show that the negative patch on the surface of PC is very important for molecular recognition. It is well known that chloroplast PC and cyt f recognize each other by electrostatic interactions prior to electron transfer. Hirota et al established that Lysptd competitively interacts with PC and in this process neutralizes the negative patch by forming the PC-Lysptd complex; hence, it inhibits the electron transfer from reduced cyt c to oxidized PC(54).

Figure 32. Scheme of interaction between poplar Pc and Lys peptide(54).

Some mutational studies show that as the charge of the negative patches decreases the rate of electron transfer from cyt f to PC also decreases(53). These two proteins can thus bind and react in different configurations that result in gated electron transfer. This process is limited by reorganization or electron coupling as described by the Arrhenius or Eyring equations,(25) whereas the actual electron transfer rate is determined by the Marcus equation(74).

Kostic and co-workers also studied structural rearrangements leading to electron transfer between plastocyanin and cyt c. They established that the gating process could be described as a configurational fluctuation of the diprotein complex, where by proteins docked at the same orientation slide on each other’s surface(19). Kinetic analyses revealed a trajectory for the cyt c on the surface of plastocyanin, thus the dynamic process of gating was studied at high structural resolution.

In conclusion, analysis of interactions between plastocyanin and their partners (cyt f, PSI, charged peptides, metal complexes) provides various models for studying and understanding mechanisms of the reactions and the specificity of their interactions.

The purpose of this project: firstly, is to study interactions of mutant P.hollandica PCs with their biological partners: cytochrome f and PSI. Their mutants will contain increasing amount of acidic residues to mimic the higher plant PC. Thus, the role of each individual negative charge can be assessed in the docking of PC to cyt f and PSI. Secondly, to examine the interaction of PC with non-physiological synthetic peptides to reveal the dynamic events underlying gated electron transfer.

Materials and Methods

Expression plasmid pVAPC10 (Fig. 33 ) an E.coli vector for the high level production of P.hollandica PC was constructed as described before(6) and was used for the PC expression in our lab.

NheI - 175

T7 prom BamHI - 462 oter ri pe o tE

pVAPC 3441 bp

Am p

Figure 33. Expression plasmid pVAPC10

Site-directed mutagenesis was done as described in Chapter 3 (Materials and Methods).

The truncated petE gene, encoding PC and cloned into the pVAPC expression plasmid was subjected to site-directed mutagenesis. The primer sequences directing the following mutations are presented below and the altered codons are highlighted in red. Five different mutations were obtained by combination of these primers.

K45E-F 5’ - CAACGTGATCTTTGATGAGGTTCCCGCCGGT – 3’

D+F 5’ - CAACGTGATCTTTGATGAGGATGTTCCCGCCGGT – 3’

D57/59 REV 5’ – AGCGCCCCTGCTCTGTCCGACACCGAGTTGGCTATCGCTCCG – 3’

T58D5’ 5’ – AGCGCCCCTGCTCTGTCCGACGACGAGTTGGCTATCGCTCCG – 3’

The mutations were confirmed by sequencing analysis at University of Chicago Cancer Research

Center, DNA Sequencing Facility, Chicago, IL.

Plastocyanin expression, purification and characterization was done as described before in

Chapter 3 (Materials and Methods).

Western blot.

Proteins were resolved by 15 % SDS-PAGE using SDS-Tris buffer(71) in a Mini-PROTEIN II gel electrophoresis system (BioRad) prior to electrophoretic transfer to a PVDF membrane

(HybondTM –P 0.45 µm, Amersham Biosciences , Piscataway, NJ) in Tris –Gly buffer (25 mM

Tris, 192mM Glycine) containing 20 % methanol at pH 8.3(116). The membrane was then incubated with blocking solution (20 mM Tris/HCl pH 7.5, 0.5 M NaCl, 0.1 % (v/v) Tween 20, 5

% dry milk), primary antibody (rabbit anti-P.hollandica plastocyanin serum Tris/NaCl/Tween) and secondary antibody (anti-rabbit IgG (Fc) Alkaline Phosphatase Conjugate (Promega,

Madison, WI) in Tris/NaCl/Tween). After each incubation step the membrane was washed twice with Tris/NaCl/Tween buffer. To visualize conjugation, BCIP/NBT color development substrate

(Promega, Madison, WI) was used.

2+ Kinetic analysis of the bimolecular reaction between [Ru(bpy)3] and PC was done using a nitrogen-pumped broadband dye laser (2 – 3 nm fwhm) from PTI (GL-3300 N2 laser, GL-301dye laser). The N2 fundamental (337.1 nm) as well as Coumarin 460 (440 – 480 nm) and BPBD (350

– 400 nm) dyes were used to tune the unfocused excitation. Pulse energies were typically attenuated to ~ 100 µJ/pulse, measured with a Molectron Joulemeter (J4 – 05). The excitation

75 wavelength was set to 458 nm using a Coumarin dye. The ruthenium emission was monitored at

2+ 610 nm. Measurement were performed on argon-saturated solutions of the [Ru(bpy)3] complex with PC dissolved in 100 mM Tris buffer. The emission was detected with a Hamamatsu R928

PMT. The PMT signal was terminated through a 50 ohm resistor to a Tektronix TDS 380 digital oscilloscope (400 MHz).

2+ Kinetic analysis of the bimolecular reaction between Ru(bpy)3 and PC was concluded

(according to Scheme 2)

hν 2+ 2+ * 2+ 2+ K1 3+ +1 Scheme 2: Cu + [Ru(bpy)3] [Ru(bpy)3] +Cu [Ru(bpy)3] +Cu

Molecular biology - expression plasmid construction.

By analogy to higher plants, cyanobacterial plastocyanin is expected to have a functional location in the thylakoid lumen. This would require translocation of PC across the cyanobacterial thylakoid membrane from its presumed site of synthesis in the cytoplasm that can be done in

E.coli by presence of petE gene N-terminal bacterial leader peptide that cleaved from the mature polyptide since periplasmic space is reached.

In order to introduce the P.laminosum PC leader peptide coding sequence 5’ in frame with the truncated P.hollandica mutant petE gene in the pVAPC plasmid the pBAR plasmid (Chapter 2) was used as a template for the PC leader amplification. Primers were designed in order to have

NheI restriction sites that allow ligation to the NheI site of the pVAPC plasmid. Moreover, the 3’ primer modified the initial PstI site into a NheI site during the PCR process. PST-NHE3’ and

PVAPCF primers are listed below and NheI restriction sites highlighted with yellow:

PVAPCF 5’-GAAGGAGATATACATATGGCTAGCATGAAG- 3’

PST-NHE3’ 5’- GTAGAAGGGATAGGCTAGCAGCAGGAGACATC -3’

The resulting PCR product was inserted into the 2.1-TOPO vector (Invitrogen, Carlsband, CA), digested by NheI and purified by agarose gel electrophoresis. The pure P.laminosum PC leader peptide was ligated into the pVAPC plasmid previously digested with NheI endonuclease, yielding plasmid pVAPC+PC+Mut4 (Fig. 34 A).

A B NheI - 175

PC leader T7 w petE ho 7 m le T u p BamHI - 610 t e 4 tE

pVAPC+PC+Mut4 pVAPC +whole petE(PCX) 3577 bp 3541 bp

A m Am p p

Figure 34. Protein expression vectors. A, pVAPC plasmid with 4 mutations of petE gene fused together with P.laminosum PC peptide leader. B, pVAPC vector with the fused whole natural petE gene together their own promoter and PC leader peptide.

Another PC expression plasmid, pVAPC whole petE (PCX)(Fig. 34 B) was designed based on the same expression plasmid pVAPC. Total P.hollandica DNA was isolated from the cells and the whole petE gene (including the native leader peptide coding sequence) was amplified by using primers NheI5’ and BamHI3’ that engineered appropriate restriction sites to the flanking

DNA sequence. Primers are presented below:

NHE5’ 5’ – CTTACTACGGCTAGCCGAAACAACGAC – 3’

BAMH3’ 5’ - GCATCTAAACTCTGCTGGATCCAGAC – 3’

The fresh PCR product was inserted into the 2.1 TOPO vector (Invitrogen, Carlsband, CA) and double digested with NheI and BamHI restriction enzymes. The whole petE gene was purified by agarose gel electrophoresis and ligated into the pVAPC plasmid lacking the petE truncated gene under the control of the T7promoter. Both plasmids was transformed into BL21(DE3) pLysS

E.coli strain for further protein expression.

Results and discussion

Mutagenesis of Plastocyanin

The first step of this project was the modification of electrostatically slightly basic PC from P. hollandica to an acidic (-5) PC in order to make it structurally more similar to the plant homolog. Mutants of increasingly acidic isoelectric point (pI) could be tested with cyanobacterial and higher plant PSI to determine precisely the contribution of electrostatics in PC/PSI interactions. Specifically, negatively charged residues were added in the ‘eastern’ face of P. hollandica PC (Fig. 35).

The amino acid sequence alignments of P.hollandica and spinach plastocyanin were compared (Table 8). Residues in red indicate the likely functionally important acidic amino acids in the spinach sequence alignment. Yellow designates the polar glutamine 86 in high plant PC and the corresponding arginine residue in the case of cyanobacteria.

P.hollandica ASVQIKMGTDKYAPLYEPKALSISAGDTVEFVMNKVGPHNVIFDK--VPA

Spinach --VEVLLGGDDGSLAFLPGDFSVASGEEIVFKNNAGFPHNVVFDEDEIPS

P.hollandica GESAPAL-SNTKLAIAPGSFYSVTLGTPGTYSFYCTPHRGAGMVGTITVE

Spinach GVDAAKIMSEEDLLNAPGETYKVTLTEKGTYKFYCSPHQGAGMVGKVTVN

Table 8. Amino acids sequence alignment of plastocyanin from P.hollandica and spinach.

According to these alignments, amino acids from P. hollandica were substituted by negative charges provided by aspartic acid or glutamic acid in the proper relative positions. As starting material, we initiated these substitutions from the previously constructed P.hollandica mutant protein R86Q(50), as Q86 is the conserved residue in plants. The P.hollandica PC protein

79 expression system, pVAPC10 was subjected to site-directed mutagenesis. These constracts are presented in Table 9. “Northern” face “Northern” face A B - + - - “Eastern” face “Eastern” face - +

- Figure 35. P.hollandica plastocyanin structure. A. Wild-type- plastocyanin B. Mutant plastocyanin with the negative amino acids on the surface. Blue indicates original amino acids,

Red – new involved negative amino acids. Green – initially mutated arginine to glutamine.

Species Amino acid sequences Charges

WT D44-K45-V46 // S56-N57-T58-K59-L60 // H85-R86-G87 0

RQ D44-K45-V46 // S56-N57-T58-K59-L60 // H85-Q86-G87 0

K45E D44-E45-V46 // S56-N57-T58-K59-L60 // H85-Q86-G87 -1

N57D // K59E D44-K45-V46 // S56-D57-T58-E59-L60 // H85-Q86-G87 -2

K45E //N57D //K59E D44-E45-V46 // S56-D57-T58-E59-L60 // H85-Q86-G87 -3

K45E// N57D //K59E D44-E45-V46 // S56-D57-D58-E59-L60 // H85-Q86-G87 -4 T58D

K45E //N57D //K59E D44-E45-D -V46 // S56-D57-D58-E59-L60 // H85-Q86-G87 -5 T58D //45D46 Table 9. P.hollandica amino acid sequences of the negative patch for wild-type and mutant PCs

Plastocyanin mutants expression and reconstitution

The WT and mutant plastocyanins were expressed in BL21(DE3)pLysS E.coli strain according to the protocol developed in our lab(6) and proteins were purified as denatured inclusion bodies, solubilized in guanidine chloride and dialyzed against 50 mM Tris-HCl buffer with 1 mM CuCl2. This expression gave very high yield of 8 – 16 mg from 1 L of culture for the wild type and the initial R86Q mutant, but yielded very poor expression for all five mutants presented in Table 9. The mutant proteins were subject to precipitation during dialysis, resulting from the misfolding of new mutant proteins. According to our procedure, the guanidine chloride solvent was removed from samples very slowly to allow efficient refolding of the proteins.

Additionally, the pH of dialysis buffers as well as samples was likely very important. Thus, we tried dialyzing the proteins at different pH in the range of 5.0 to 8.0. Another parameter that was changed from the regular procedure was reconstitution buffer. Instead of guanidine chloride we used urea buffer at the same pH range. Unfortunately, all these manipulations did not increase yield for the new mutant PCs. In order to prove that there are no problems with petE gene expression we electrophoresed proteins from inclusion bodies having two (N57D/K59E) and five

(K45E/N57D /K59E/T58D/45D46) acidic residues in guanidine chloride (lanes 1 and 3 Fig. 36) and obtained bands around 10kDa that indicated the presence of denatured PC in our sample.

Further dialysis of these samples did not show any presence of this protein on the gel (lanes 1 and 6 Fig. 36). The appearance of double bands in lanes 3 and 4 could be associated with the folded/ unfolded form of the protein in guanidine chloride buffer.

51 36 28 20

6.5

Figure 36. SDS-PAGE of PC WT and mutants. Coomassie staining.

Lanes: 1- PC with 5 mutations (K45E/N57D/K59E/T58D/45D46) in urea buffer

2 – PC with 2 mutations reconstituted from guanidine chloride buffer

(N57D/K59E)

3 – PC with 2 mutations in guanidine chloride buffer

4 – WT PC in guanidine chloride buffer

5 – WT PC reconstituted from guanidine chloride buffer

6 - PC with 5 mutations reconstituted from urea buffer

CD spectra of WT and mutants were recorded to detect proper protein folding (mostly β-

sheets). Occasionaly, WT apoplastocyanin lacking Cu was obtained and CD spectra recorded

(black lane Fig 37). The mutant PC preparations commonly lacked Cu centers, yielding proteins

lacking color and the absorption band at 600 nm. Moreover, in most cases the mutant PCs were

not properly folded and yielded a random coil structure by CD spectroscopy (blue and cyan

curves Fig 36). However, some preparations exhibited correct folding similar to WT PC (green

lanes Fig.36). Another phenomenon was noticed for the PC 2 mutant; a freshly prepared sample

looked slightly blue and had the proper CD spectra (red curve Fig 36.). However, after 48 hr at 4

ºC, the solution became purple and yielded a CD spectrum suggestive of denaturation (cyan Fig.

37). Thus, we can conclude that mutant PC could not be reconstituted and/or were more unstable under our standard storage conditions (4 ºC over a week).

-2 -4

Elipticity -6 PC WT no copper -8 PC 2 mut with copper initial PC 2 mut no copper -10 PC 5 mut no copper PC 2mut with copper after 48hr -12

190 200 210 220 230 240 250 260 Wavelength, nm

Figure 37. CD spectra of WT and mutants of plastocyanin. All samples were in 50 mM

Tris/HCl buffer

P.hollandica wild type plastocyanin and their mutants were expressed under the control of very strong phage T7 promoter. This bacteriophage T7 DNA-dependent RNA polymerase expression system has been extensively used in E.coli cells for the last decade(36, 65, 77, 98).

The T7-based system is usually characterized by very high protein yield that often cause misfolding and aggregation of proteins in inclusion bodies. In many cases, the insolubility may be advantageous due to minimization of purification steps, simplicity of collection material from bacterial lysates, and strong protection of the protein against proteolytic degradation by host

83 proteases. Inclusion bodies may be “heaven or hell” for the protein production(33). Usually, it is possible to resolubilize, renaturate, and further purify protein from these inclusion bodies. In other cases, proteins in inclusion bodies are difficult to renaturate or do not fold properly. In this case the most efficient way to avoid inclusion bodies is to change the expression system to prevent intracellular accumulation of the protein (33). Thus, since reconstitution from inclusion bodies yielded only a low amount of protein, another method to translocate soluble PC into the

E.coli periplasmic space was developed. Some evidence shows that for some proteins, the introduction of leader peptides helps to increase the protein yield by delivering correctly folded protein to the periplasmic space(52, 98, 122). To test this, we engineered plasmids with the PC gene (petE) fused to the sequence encoding the P.laminosum PC leader peptide (Figure 34 A) and plasmids with the natural P.hollandica PC leader peptide together with the whole petE gene

(Figure 34 B).

36.4

28.4 19.7

9.0

Figure 38. SDS-PAGE Western blotting of E.coli BL21(DE3)pLysS strain periplamic fraction.

IPTG was added to the final concentration 0.5 mM. Total protein concentration 1 µg/µl

Lanes: 1 – Control (pure PC WT reconstituted from inclusion bodies)

2– No expression plasmid transformed

3 – TOPO + whole petE plasmid transformed

4 – pVAPC +PC +4Mut plasmid transformed.

5 – pVAPC plasmid with truncated petE gene transformed.

These two plasmids, as well as the PC expression plasmid pVAPC10 were transformed into

BL21(DE3)pLysS E. coli strain and the periplasmic fraction was extracted. Protein overexpression was checked by Western blotting analysis (Fig.38).

Unfortunately, the antibody was not specific enough to yield distinct bands, but from this gel it is evident there was no difference between the expression of total periplasmic proteins in the plasmidless control E.coli strain BL21(DE3)pLysS (lane 2 Fig. 38) and the strains transformed with plasmids pVAPC+PC+4Mut (lane 4) and TOPO+whole petE (lane 3). Thus, this strategy was abandoned for mutant PC expression.

2+ Kinetic studies of plastocyanin [Ru(bpy)3] complex

To determine the reactivity of the succesfully reconstituted PC preparations, the next step was to study interactions and electron –transfer process between PC and tris(2,2’-

2+ bipyridine)ruthenium(II) (Ru(bpy)3 ). This ruthenium complex has an excited-state reduction

potential of E˚=-0.84V(31) and PC has a reduction potential is E ْ = 0.36V(105). Hence, the driving force is rather large, ∆E˚= -1.2V, and this electron transfer process is allowed.

The dependence of the observed rate constant (kobs) on the Prochlorothrix hollandica wild type plastocyanin is presented in Figure 39. The fact that kobs did not saturate with increasing PC concentration indicates no complex formation; therefore only the bimolecular rate constant could be calculated for a type1 (collisional) reaction mechanism.

3.2

3.0

2.8

2.6 ) -1 s

6 2.4 (10

obs 2.2 K 9 -1 -1 2.0 k2 = 8.89×10 s M -second order rate constant ’ 5 -1 1.8 k1 =4.5×10 s –pseudo first rate constant

1.6

0 20 40 60 80 100 120 140 160 180 [PC]µµµM

Figure 39. Dependence of kobs on PC concentration in electron transfer from

* 2+ 2+ [Ru(bpy)3] to PC in 100 mM Tris buffer with [Ru(bpy)3] = 50µM

In summary, the negatively charged P.hollandica mutant PC mimicking higher plant plastocyanin did not fold efficiently, but we are continuing to search for the appropriate conditions for protein refolding from inclusion bodies.

FUTURE PERSPECTIVES

A novel efficient P.hollandica cyt f expression system has been developed that yielded a high level of protein production (4.6 mg/L) allowing studies of homospecific interactions with

PC from P.hollandica. Kinetic measurements show that by removing Tyr102 residue along or together with Phe100 residue from the ‘pocket-like’ region of Prochlorothrix cyt f will decrease k2 10 times to both WT and the Y12G/P14L mutant of Prochlorothrix PC. At the same time it was established that binding affinity will also depend on absence of these two bulky aromatic residues (Tyr102 and Phe100) that protruded towards PC docking site. By this study, we can conclude the importance of the cyt f loop region in PC/cyt f interactions, likely bringing Cu and

Fe centers closer for faster electron transfer.

Thus, in order to map protein interaction surfaces and establish minimal structural requirement for complex formation samples were sent to Dr Marcellus Ubbink for HSQC NMR studies (Leiden University). At the same time P.hollandica cyt f samples are being investigated now by X-ray crystallography at University of Toledo by Dr Ronald Viola’s research group.

Another cyt f mutant, F100S, that lacks the Phe100 residue only, is still in preparation and should be completed soon. This mutant will give more information about importance of the loop region of cyt f.

For the future, P.hollandica PC mutants with acidic residues at the eastern patch could be studied for protein-protein interactions with physiological partners: cyt f and PSI and non physiological reactants such as Lysine peptide in order to understand the minimal structural requirements necessary for these interactions, and how these interactions will be affected by changes in ionic strength and pH.

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