Ph.D. thesis Electron microscopic studies of ...

The University of South Bohemia Institute of Physical Biology, Nové Hrady

Ph.D. thesis

Electron microscopic studies of photosynthetic membranes and their pigment-protein

complexes

Zdenko Gardian

Supervisor: Doc. RNDr. František Vácha, Ph.D.

Institute of Plant Molecular Biology, Biology Centre ASCR, v.v.i. Branišovská 31, 370 05 České Budějovice

České Budějovice 2009

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Gardian Z. (2009) Electron microscopic studies of photosynthetic membranes and their pigment-protein complexes. Ph.D. thesis, Pp 82. Institute of Physical Biology, The University of South Bohemia, Nové Hrady, Czech Republic.

This thesis is based on the following publications: Zdenko Gardian, Ladislav Bumba, Adam Schrofel, Miroslava Herbstova, Jana Nebesarova and Frantisek Vacha, Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts. Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1767, Issue 6, June 2007, Pages 725-731. Ulf Dühring, Roman Sobotka, Josef Komenda, Enrico Peter, Zdenko Gardian, Martin Tichy, Bernhard Grimm, Annegret Wilde, Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes. JBC, 283, NO. 38, September 2008, Pages 25794 –25802. Zdenko Gardian, Frantisek Vacha, Structural characterization of Photosystem I, Photosystem II and FCP complexes from chromophytic alga Xanthonema debile. To be submitted. Annotation: The overall structure of photosynthetic pigment-protein complexes and thylakoid membranes of various photosynthetic organisms was studied using electron microscopy. Anotace: Pomocí elektronové mikroskopie byla studována celková struktura fotosyntetických pigment-proteinových komplexů a tylakoidných membrán různých fotosyntetických organismů.

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Declaration

I declare that my role in preparation of publications was following: Paper 1 Main author – isolation and purification of photosynthetic complexes, their analysis by electrophoresis and spectroscopic measurements, EM and single particle analysis of PSI complexes, preparation of the manuscript, the estimated overall contribution to the publication 75% Paper 2 Co-author – preparation and electron microscopy of the ultra thin specimens of Synechocystis strains, the estimated overall contribution to the publication 15% Paper 3 Main author – isolation and purification of photosynthetic complexes, their analysis by electrophoresis and spectroscopic measurements, EM and single particle analysis of PSI, PSII, FCP complexes, preparation of the manuscript, the estimated overall contribution to the publication 95% On behalf of the co-authors, the above mentioned declaration was confirmed by:

František Vácha.……………………………… supervisor and co-author of the papers 1 and 3

Prohlašuji, že jsem svoji disertační práci vypracoval samostatně pouze s použitím pramenů a literatury uvedených v seznamu citované literatury. Prohlašuji, že v souladu s § 47b zákona č. 111/1998 Sb. v platném znění souhlasím se zveřejněním své disertační práce, a to v úpravě vzniklé vypuštěním vyznačených částí archivovaných fakultou elektronickou cestou ve veřejně přístupné části databáze STAG provozované Jihočeskou univerzitou v Českých Budějovicích na jejích internetových stránkách. České Budějovice, 26.02.2009 Zdeno Gardian.……......……………….....……

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Curriculum Vitae

ZDENKO GARDIAN 27. 4. 1979 SNP 1445/38, 017 01 POVAZSKA BYSTRICA, SLOVAK REPUBLIC E-MAIL: [email protected] EXPERIENCES • 2004 – now, Institute of Plant Molecular Biology, Academy of Sciences, Ceske Budejovice, Czech Republic

Area of interest: Transmission electron microscope, experiments with protein complexes, image analysis

• 2007 – february 2008 BioTech a.s., Praha, Czech Republic Product manager: Distribution of reagents for molecular biology • 2003 – 2004 Summer academic courses in Nove Hrady, South Bohemia, Czech Republic Supervisor of the project: Optimalization of measurements of chlorophyll fluorescence • 2003 – March 2004 Institute of Microbiology, Academy of Sciences, Trebon, Czech Republic Area of interest: Chlorophyll fluorescence, fluorescence microscope. • 2002 – September 2003 Institute of Physical Biology of the University of South Bohemia, Nove Hrady, Czech Republic

Area of interest: Continuous cultivations, substrate limitation, forced metabolic oscillations EDUCATION • 2003 – 2009 Ph.D. degree at the Institute of Physical Biology of the University of South Bohemia, Nove Hrady, Czech Republic

Doctoral thesis: Electron microscopic studies of photosynthetic membranes and their pigment-protein complexes

• 1997 – 2002 engineer's degree at the Faculty of Agriculture, The Slovak University of Agriculture in Nitra, Slovak Republic Specialization: Applied biology

Diploma thesis: Prepare of data for image database GENOTYPDATA PHASEOLUS

• 1993 – 1997 Gymnazium [High School with general education], Povazska Bystrica, Slovak Republic GRANTS • 2005 University development foundation of the Ministry of Education

Project: Changes of metabolic feedback regulations of algae, induced with heavy metals

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PUBLICATIONS Zdenko Gardian, Ladislav Bumba, Adam Schrofel, Miroslava Herbstova, Jana Nebesarova and Frantisek Vacha, Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts. Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1767, Issue 6, June 2007, Pages 725-731. Ulf Dühring, Roman Sobotka, Josef Komenda, Enrico Peter, Zdenko Gardian, Martin Tichy, Bernhard Grimm, Annegret Wilde, Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes. JBC, 283, NO. 38, September 2008, Pages 25794 –25802. Zdenko Gardian, Frantisek Vacha, Structural characterization of Photosystem I, Photosystem II and FCP complexes from chromophytic alga Xanthonema debile. To be submitted. SELECTED POSTER PRESENTATIONS:

• Z. Gardian, M. Koblizek, D. Stys: Continuous cultivation of Synechococcus bigranulatus: substrate limitation monitored by forced oscillations: Plant Physiology Conference of Ph.D. Students, Brno, Czech Republic, 2003.

• M. Sergejevova, M. Sekerkova, Z. Gardian, J. Cerveny, J. Kopecky, J. Masojidek: Cultivation of the alga Pleurastrum sarcinoideum in close solar photobioreactor: biomass productivity and carotenoid content under high irradiance: 5th Europen workshop Biotechnology of Microalgae. Bergholz-Rehbrucke, Germany, 2003.

• I. Setlik, E. Setlikova, Z. Gardian, R. Kana, J. Komenda, V. Kasalicky, O. Prasil: New approaches to studies of cell cycle regulation of photosynthesis in algae: 13th International Congress on Photosynthesis, Montreal, Quebec, Canada, 2004.

• Z. Gardian, L. Bumba, F. Vacha: Electron microscopy in structural studies of Photosystem I from the red alga Cyanidium caldarium: Mikroskopie 2006, Československá mikroskopická společnost, Nove Mesto na Morave, 2006.

• J. Vanek, J. Urban, Z. Gardian: Automatic detection of Photosystems II in electron microscope photographs: International Symposium Technical Computing Prague 2006.

• Z. Gardian, L. Bumba, F. Vacha: Electron microscopy in structural studies of Photosystem I from the red alga Cyanidium caldarium: 12th International Symposium on Phototrophic Prokaryotes (ISPP2006), Pau, France, 2006.

OTHER ABILITIES • computer skills on an advanced user level, OS Windows • general knowledge of MS Office [Word, Excel, PowerPoint] • graphics editor Adobe Photoshop Adobe Illustrator, Zoner media, Corel… • general knowledge of internet search engines, regular searching in scientific databases • English on an upper intermediate level • interest in digital photography • driving licence A, B

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Acknowledgement

I would like to express my gratitude to my supervisor František Vácha for his support and guidance. My thanks also belong to Ladislav Bumba and Zbyněk Halbhuber who introduced me to the scientific work and life.

I want to thank the members of the Electron Microscopy lab and the Department of Photosynthesis of the Institute of Plant Molecular Biology for their assistance with my work. Especially, I would like to give my thanks to Táňa for correcting english grammar and for stimulating support.

I am also grateful to all my friends and to our collective friend Mr. Nezmar who gave me the power to complete this thesis.

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Abbreviations

2-D Two-Dimensional 3-D Three- Dimensional A0 Chlorophyll molecule A1 Phylloquinone ATP Adenosine Triphosphate Car Carotenoid Chl Chlorophyll DM n-dodecyl-α-D-maltoside EM Electron Microscopy FA Iron sulphur cluster FA of Photosystem I FB Iron sulphur cluster FB of Photosystem I Fx Iron sulphur cluster FX of Photosystem I FCP Fucoxanthin-chlorophyll protein LHCI Light Harvesting Complex I LHCII Light Harvesting Complex II LM Light Microscope NA Numerical Aperture

NADPH Nicotinamide Adenine Dinucleotide Phosphate NMR Nuclear Magnetic Resonance OEC Oxygen-Evolving Complex P680 Primary electron donor absorbing light at 680 nm P700 Primary electron donor absorbing light at 700 nm PSI Photosystem I PSII Photosystem II RC Reaction Center QA Primary stable quinone electron acceptor in PSII QB Secondary stable quinone electron acceptor in PSII SEM Scanning Electron Microscope TEM Transmission Electron Microscope

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Contents

Chapter 1 10 Introduction 1. Electron microscopy 11 2. Electron microscopy and macromolecular structural studies 16 3. Electron microscopy and photosynthetic pigment-protein structural studies 18 Chapter 2 30 Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts Chapter 3 36 Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes Chapter 4 40 Structural characterization of Photosystem I, Photosystem II and FCP complexes from chromophytic alga Xanthonema debile Reference List 51 Summary 56

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Chapter 1

Introduction

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1. Electron microscopy Introduction to electron microscopy

The human eye can recognize two objects if they are not closer than 0.2 mm at a normal viewing distance of 25 cm. Any other details between these objects can be recognized by the eye only if the objects are enlarged. The enlargement can be done by the use of optical instruments, such as hand lenses or microscopes.

The first compound light microscope was developed in 1590 by the Janssen brothers and led to a rapid rise of interest in microscopic life forms and microscopic structures found in multicellular organisms. By the 1800s, the optical microscopes had been improved in many ways and important discoveries concerning cell structures had been reported. Brown had identified the nucleus of eukaryotic cells (Brown 1866), Schleyden and Schwann had independly posited that the whole life was made up of cells, which was the basis of the cell theory (Schwann and Schleyden 1847). In 1886 Carl Zeiss and Ernest Abbe produced compound light microscope that was corrected for spherical and chromatic aberration and the maximum resolution with glass optic and visible light had been achieved. The resolution capabilities of the microscopes were improved upon the Louis de Broglie advanced his theory that the electron has a nature with characteristics of a particle or a wave.

The first electron microscope was described in 1932 by Knoll and Ruska (Knoll and Ruska 1932) and commercial development followed in 1939. The introduction of the electron microscope brought about a complete reappraisal of the micro-anatomy of biological tissues, and many previously unimagined structures in cells were revealed. In the biological research the electron microscopy was at first the tool of anatomists and histologists. After some time, also biochemists and physicists started to use the electron microscope to examine cells and tissue in many different ways. The importance of electron microscopy for the biologists is emphasized by the fact that electron micrographs had appeared in most text books and research papers on cell biology and anatomy. There are commercially available two most common types of electron microscopes: the Transmission electron microscope (TEM) and the Scanning electron microscope (SEM).

In the SEM, the specimen is scanned with a focused beam of the electrons which produces "secondary" electrons as the beam hits the specimen. These secondary electrons are detected and converted into the three-dimensional image of the surface of the specimen.

Specimens in the TEM are exposed to passing electron beam, revealing detail information of the internal structure of specimens.

Transmission electron microscope (TEM)

The TEM is about 2 meters long evacuated metal column. Like a source of illumination the TEM uses a cathode, placed at the top of the column. A high voltage (accelerating voltage) from 40,000 to 100,000 volts is passed between the cathode and the anode and heats the cathode tungsten filament which emits the electrons. The positive charged anode is placed below the heated filament. Through a tiny hole in the anode some accelerated electrons are passed and form an electron beam which passes through the sample down the

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column. The speed at which the electrons are accelerated to the anode depends on the amount of accelerating voltage present. To focus, the electron beam is placed in a column of electro-magnets, like glass lenses in the light microscope. The double electro-magnet condenser lenses focus the electron beam onto the specimen clamped into the removable specimen stage. Some electrons are scattered but those which pass through the specimen are focused by the objective lens either onto a phosphorescent screen, photographic film or CCD camera to form an image. To enhance the image contrast there is an objective aperture to block out the unfocussed electrons. The contrast of the resulting image can be increased by reducing the size of this aperture. The intermediate lens is used to control the magnification on the TEM. The last TEM lens that corresponds to the ocular lens of the light microscope is a projector lens. This lens forms a real image on the fluorescent screen at the base of the microscope column.

Figure 1. Comparison of the optical system of LM and a TEM. (adopted from Hajibagheri 1999)

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Theory of electron optics

Electrons, which are normally considered as particles, have a wave-like character and show the phenomena normally associated with waves: interference and diffraction. From the comparison between the structure of electron microscope and the optical microscope is clear that transmission electron microscope uses principles originated in light microscopy, based on the wave properties of electrons. Because of the negative charge of electron we can use the magnetic forces to focus an electron beam to a point, which is a basic function of electron lenses.

An electromagnetic lens consists of a coil of copper wires inside the iron pole pieces. When the electrical current is passed through the coils, an electromagnetic field is created between the pole pieces. In this electromagnetic field the electrons are affected by the Lorentz force F:

F = -e(E + vB)

E: strength of electric field B: strength of magnetic field

e/v: charge/velocity of electrons The focusing effect of the electromagnetic lens therefore increases with the magnetic field B, which can be controlled via the current flowing through the coils. This is a useful property of the electromagnetic lenses, because of the magnification of images in the electron microscope can be adjusted quickly and easily by the simple adjustment of the current. A conventional TEM operates up to 100-120 kV, usually with several lower steps (e.g., 40, 60, 80 kV). The varied focus length is the main difference between the electromagnetic and glass lens. Otherwise the lenses behave in the same way and have the same types of aberration: • Spherical aberration is determined by the lens design and means that the magnification in the centre of the lens is different from that at the edges. • Chromatic aberration means that the magnification of the lens varies with the wavelength of the electrons in the beam. This aberration is reduced by keeping the accelerating voltage as stable as possible. • Astigmatism show a circle in the specimen like an ellipse in the image and can be corrected by using variable electromagnetic compensation coils. Described aberrations have important implications for the resolution of the electron microscope.

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Resolution of the microscope

By definition, resolving power is the ability of a lens to show two adjacent objects as discrete entities. A major role in the determination of the resolving power of the light microscope plays the property of the objective lens terms of numerical aperture (NA). The numerical aperture can be expressed:

NA = n.sin α

n: refractive index for the medium through the light passes (n air = 1.0; n water = 1.3; n oil = 1.5)

α: angle of one half of the angular aperture of the lens The resolving power of the optical system can be expressed:

R = λ/2NA

R: distance between distinguishable points λ: wavelength of the illumination source

NA: numerical aperture of the objective lens The resolving power of the light microscope is limited by two facts: • NA of the objective lens can reach max 1.5 (α cannot be greater than 90 degrees, and n cannot be greater than 1.5) • relatively long wavelength of the light used for illumination. The optimal resolving power for a light microscope with the NA=1.5 obtained with blue illumination (λ = 436 nm) is 145 nm. Very short wavelength of the electrons makes the resolving power of the TEM approximately three orders of magnitude greater than that of the light microscope. The wavelength of electrons, accelerated through a potential difference of volts (U), is equal to:

λ = 1.226/U1/2 Therefore, an electron accelerated with voltage (U) 50,000 volt has a wavelength (λ) of 0.0055 nm. The electromagnetic lenses are extremely narrow (α = 1 degree), so that the numerical aperture is reduced to hundredths and if these values are entered into the resolution formula:

R = λ/2NA

then the theoretical resolution of TEM is 0.27 nm.

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Depth of field in the microscope

In addition to the resolving power, microscopes have another limiting factor, called the depth of field. This term is used in photomicroscopy as well as in ordinary photography and means the distance between the nearest subject and the farthest subject that are in focus when the picture is being taken. In photography the depth of field can be increased by the use of a smaller aperture when focusing on a given object. The depth of field is shorter with increased NA of objective lenses and with higher the magnification, so in microscopy the depth of field is reduced. The limitation on the depth of field can be eliminated by the use of the objectives with a low NA. The depth of field of the electron microscope equipped with a low NA objective lens is several hundred times greater than that of a light microscope. As a result of this, there is no problem to have a normal thin section (80-90 nm) on one plane in a focus. This also means that the position of the image recording apparatus is not critical and can be easily placed several centimeters above or below the viewing screen. Magnification

Magnification is how much bigger a sample appears to be under the microscope than it is in real life. The useful magnification obtainable by an objective lens is dependent upon the NA (like the depth of field and the resolution of the lens). The useful magnification of any objective lens is approximately 1000 times its NA and any magnification higher than this will not resolve more detail but will only give "empty magnification". The most electron microscopes are created to provide at least 200,000x magnification, which is necessary to produce the theoretical 0.3 nm resolution. There are also microscopes with magnification up to 500,000x, though this is of little practical value to biologists. Image formation

The absorption of the specimen is in the light microscope the most important image-forming process. Absorption gives rise to amplitude contrast, or differences in intensity, to which the eye is sensitive. By using the different absorptions at different wavelengths we can get colored images.

In the electron microscope, absorption is not so important and contrast is formed by scattering. Electrons scattered do not reach the viewing screen thus the dark spots remains there. There are two types of scattering:

• elastic scattering (nuclear interactions between the electron beam and the specimen) • inelastic scattering (electron interactions where the primary electrons dislodge secondary electrons from the specimen). The inelastic scattering is the most important aspect of image formation in a TEM. Dark parts of the image correspond with regions where the scattering is strong, and bright parts with where it is weak.

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One of the factors that strongly determines the resolution of TEM is the specimen preparation method. The main problem of the examination of biological material with the electron microscope is the fact that the specimens must be exposed to the unphysiological conditions (high vacuum 10-5 to 10-8 Torr). The biological specimen must be fixed and dried so that its ultrastructure is as close to that in the living material as possible.

2. Electron microscopy and macromolecular structural studies

Electron microscopy (EM) is a powerful tool not only for determination of cell structure but also for visualization of large enough macromolecular structure. Samples of macromolecular complexes for electron microscopy are placed on an EM grid which is covered with a low absorption thin film. The biological macromolecules are mainly composed of carbon, oxygen, nitrogen, and hydrogen, which are light atoms that scatter electrons weakly and therefore produce a low contrast image. In order to improve the contrast of biological macromolecules, the negative staining technique is used.

Figure 2. A sample deposited on a carbon coated grid and surrounded by stain.

Negative staining is simple method based on staining of samples with a heavy metal salt that readily scatter electrons (Boekema 1991, Harris and Scheffler 2002). This is usually lead, tungsten, molybdenum, vanadium, or depleted uranium. The name "negative staining" is caused by the fact that microscope doesn’t visualize the object itself, but an area empty of stain surrounded by stain. The empty stain area is because the sample (protein for example) prevents the stain from sedimenting onto the carbon layer, however stain is able to penetrate between small surface projections and to delineate them.

For microscopical high resolution macromolecular structural studies the cryo-electron microscopy can also be used (Frank 2001, Henderson 2004).

Cryo-electron microscopy is the technique when the sample is viewed frozen in a

very cold liquid refrigerant (liquid nitrogen) in order to preserve and protect it during observation. Biggest advantage of cryo EM over negative staining is that there is no stain which could generate artifacts and distort the sample. The staining process requires drying, which can also damage the sample in many ways. Advantage of negative staining over cryo EM is better signal to noise ratio. Preparation of the samples is easy and not so time

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consuming. In cryo EM a cubic ice, which absorbs electrons very easily and make the frozen sample worthless, can be formed.

With both techniques (negative staining and cryo EM) the macromolecular structure at resolutions spanning from molecular to near atomic can be visualized. Electron micrographs of specimens must be recorded at very low electron beam to minimize radiation damage, resulting in low image contrast. This is the main complication of visualization of macromolecules by EM which limits the resolution to about 3 Å. During the recording of the image some degrades of image quality can occur (because of mechanical specimen drift and electron beam-induced charging). There are also two important sources of noise of micrographs of biological macromolecules: • The main is that carbon layer itself has a structure which image appears superimposed on the image of macromolecule. • The second source of noise is because of variations in thickness of the heavy metal salts used as stain. This necessitates the use of computer image processing methods to align and average thousands of individual projections of molecules to increase the signal-to-noise ratio (van Heel et al. 2000, Frank 2002). EM of macromolecules followed by averaging procedures of projections can be divided into crystallographic and non-crystallographic methods.

Electron crystallography of 2D crystals has the potential to determine structures to atomic resolution and represents an alternative to X-ray crystallography and NMR spectroscopy of proteins that are too large for solution NMR or resist forming well ordered three-dimensional (3D) crystals.

Non-crystallographic methods are based on aligning and averaging of single particles. Resolution limits in single particle analysis is about 10-25 Å and requires very large numbers of individual particles to be processed, aligned, and averaged.

The electron microscopy single particle structure determination methods have been developed over the past 30 years (Thuman-Commike 2001). There are several Software packages which are designed for single particle structure determination. The community of structural biologists commonly uses IMAGIC (van Heel et al. 1996) and SPIDER (Frank 1996). There are also other general purpose electron microscopy software packages: SUPRIM (Schroeter and Bretaudier 1996), EM (Hegerl 1996) and EMAN (Ludtke et al. 1999) that can be used for data processing steps in single particle structure determination.

Computer image processing is based on three data processing sequences. First, particle picking is performed to cut out individual particles within the micrograph followed by centering of the projections. Next, particle alignment is performed to change the rotational and translational positions of the particles in order to similar positions of all individual particles. The last step is the classification of the particles which is done in order to identify groups that represent the same views (orientation and shape) of the particles. Centered/aligned/classified particles are then ready to be averaged with Multivariate Statistical Analysis (Frank 1990).

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Figure 3. Depiction of single particle structure determination.

A: Cartoon representation of a sample micrograph. B: Nine selected particle images from the micrograph shown in A. C: Aligned particle images. D: Classified particle images.

(adopted from Thuman-Commike 2001)

3. Electron microscopy and photosynthetic pigment-protein structural studies

Introduction to photosynthesis

Photosynthesis is a process by which plants, algae and some bacteria convert light energy into chemical energy. The raw materials are carbon dioxide and water and the end-products are oxygen and carbohydrates. Driving force of this process is sunlight absorbed by photosynthetic pigments. Certain types of bacteria synthesize organic compounds using the light energy but with no production of oxygen. Such process is called anoxygenic photosynthesis. In plants and algae the photosynthesis process occurs in chloroplasts.

Chloroplast

Chloroplasts are one of the many different types of organelles in the cell. The endosymbiotic theory suggests that chloroplast origins by endocytosis or gene fusion of photosynthetic bacteria by early eukaryotic cells to form the first plant cells. This theory promotes the fact that chloroplasts have many similarities with photosynthetic bacteria including prokaryotic-type ribosomes, circular chromosome and similar proteins in the

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photosynthetic reaction centers. Morphologically, chloroplast has a flat discs shape, usually with 2 to 10 micrometer in diameter. The chloroplast is bounded by an envelope that consists of an inner and an outer phospholipid membrane with an intermembrane space. The material inside the chloroplast is called stroma and contains circular DNA, ribosomes and many other constituents and compounds (starch, oil, etc.). The main parts of chloroplasts are membrane-bound compartments called thylakoids. They are the primary site of the light-dependent reactions of photosynthesis.

Figure 4. A. Chloroplast location in eukaryotic cell. B. Schematic structure of chloroplast. C. Electron micrograph of ultrathin section of chloroplast thylakoid membrane.

Thylakoid

Thylakoids are system of membrane sacs, usually with flattened disk shape. In eukaryotic cells (except some algae) the thylakoids are arranged in stacks called grana. Grana as a single functional compartment are connected with intergrana also called stroma thylakoids or lamellae. Grana thylakoids and stroma thylakoids can be distinguished by their different protein composition. The compartment inside the thylakoid is called thylakoid lumen. During the light-dependent reaction of photosynthesis, protons are pumped across the

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thylakoid membrane into the thylakoid lumen. There are four major integral membrane protein complexes:

Photosystem I

Photosystem II Cytochrome b6f complex

ATP synthase

Imunolabelig shows that Photosystem II is located mostly in the grana thylakoids, while Photosystem I and ATP synthase are located in outer layers of grana, but mostly in the stroma thylakoids (Allen and Forsberg 2001). The cytochrome b6f complex is distributed evenly throughout thylakoid membranes.

Figure 5. Schematic diagram of membrane proteins of thylakoid membrane with electron transport chain. (adopted from Ort and Yocum 1996)

Except these integral membrane protein complexes, there are also electron carriers (plastoquinone, plastocyanin and ferredoxin) required to shuttle electrons among photosystems. Plastoquinones are lipid-soluble and therefore move within the thylakoid membrane and shuttles electrons from photosystem II to the cytochrome b6f (Crane and Henninger 1966). Plastocyanin is present in the lumen and carries electrons from the cytochrome b6f complex to photosystem I (Katoh 1960). Ferredoxin is located on stromal surface and transfers electrons from the photosystem I to ferredoxin reduktase to synthetize NADPH. The electron transport chain (shown in the figure 5.) generates a chemiosmotic potential across the membrane. Chemiosmotic potential is used by the ATP synthase to make ATP.

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All oxygenic photosynthetic organisms utilize both PSI and PSII in sequence, with the use of water as the reductant. Anoxygenic photosynthesizers utilize only a single photosystem that uses other electron donors. Photosystem II

PSII is an integral membrane pigment-protein complex made of more than 25 subunits and several cofactors (Barber et al. 1997, Hankamer et al. 2001). The positions of PSII subunits is shown in 3-D structural models of the dimeric PSII core complex from cyanobacteria (Zouni et al. 2001, Kamiya and Shen 2003, Ferreira et al. 2004, Loll et al. 2005). The heterodimer core of PSII reaction center is composed of polypeptides D1 (PsbA) and D2 (PsbD) that binds six chlorophyll a, two β-carotene, two plastoquinone molecules named Qa and Qb, two pheophytins a and an iron atom (Bricker and Frankel 2002). The reaction center is surrounded by chlorophyll a binding core antennae complexes CP43 (PsbC) and CP47 (PsbB). Closely associated with the RC are proteins PsbE and PsbF bearing hem and know as Cytochrome b559. At the lumenal site of the PSII the oxygen-evolving complex (OEC) is bound. It is composed of a cluster of four Mn2+ ions and one calcium atom and three extrinsic proteins OEE1 (PsbO), OEE2 (PsbP) and OEE3 (PsbQ) (Seidler 1996). In cyanobacteria and red algae the proteins PsbP and PsbQ are replaced by the Cytochrome c-550 (PsbV) and 12 kDa (PsbU) protein (Shen et al. 1992). Finally, there are several small proteins, from PsbH to PsbZ associated with PSII. Some of them are involved in photoprotection against the damaging effects of the reactive oxygen species generated during photosynthesis, some are required for the stability or dimerisation of the PSII complex, but the functional role of most of them is not yet clear (Barber et al. 1997, Hankamer et al. 2001).

Photosystem II uses light energy to oxidize water molecules to molecular oxygen (O2) and to form a transmembrane pH gradient. The derived excitation energy is passed to a primary donor of PSII, made of chlorophyll a molecules known as P680. P680 is excited with an energy of photon to P680*, which is sufficiently reducing to transfer the electron to the primary electron acceptor pheophytin (Knaff et al. 1977). This process of charge separation takes about 2-4 ps. From pheophytin the electron moves in hundreds of ps to the first plastoquinone molecule Qa (Klimov et al. 1977) which is tightly bound to D2 subunit of PSII. From Qa the electron is transferred in 100 µs to second plastoquinone Qb that is loosely bound to D1 subunit and after it accumulates two electrons it is removed and diffuses to Cytochrome b6f complex. The electron that reduces remaining P680+ is extracted from water 2H2O (O2 + 4 H+ + 4e-) by OEC.

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Figure 6. Schematic diagram of subunit composition end electron transport of PSII from

higher plant. (drawn by Jon Nield) Photosystem I

PSI is an integral membrane multi-subunit pigment-protein complex. The structure of the PSI complex isolated from pea and cyanobacteria has been determined and the positions of many of the subunits and electron transfer components have been located (Krauss et al. 1996, Schubert et al. 1997, Jordan et al. 2001, Ben-Shem et al. 2003). There are 3 subunits known as PsaA, PsaB (core heterodimer) and PsaC that are the enzymatic heart of the complex. In the reaction center of PSI there is a pair of chlorophyll a molecules ("the special pair") known as P700. Four additional chlorophyll molecules and two phylloquinone molecules are also present. Feature unique to photosystem I is the set of three iron-sulfur clusters (Fx, Fa, Fb) with function as early electron acceptors (Nugent 1996). One of the Fe-S clusters, called Fx is held between the PsaA and PsaB proteins, while the FA and FB iron-sulfur clusters are found in the PsaC protein. The PsaD and PsaE proteins are located on the stromal site of the complex and are involved in ferredoxin docking (Fromme et al. 2001). The PsaF protein is located on the lumenal site and is implicated in the plastocyanin binding. There are also 9 small proteins from PsaG to PsaO. Some of these proteins are only found in cyanobacteria and some only in eukaryotic organisms. The function of these proteins is not well understood.

Photosystem I uses light energy to reduce NADP+ to NADPH. Upon absorption of a photon, special pair P700 is excited to P700*, which is able to reduce another chlorophyll a molecule called A0. Primary acceptor A0 is reduced in about 2 ps. The electron then moves in 25 ps to one of the phylloquinone called A1 and from there to iron-sulfur cluster FX in about 200 ns. Next adjoining subunits, FA and FB accept the electrons in hundreds of ns. Electrons

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are then donated to a soluble protein Ferredoxin and ultimately NADP+ is reduced. The reduction of NADP+ catalyzes the enzyme FNR (ferredoxin NADP+ reductase). The remaining P700+ is reduced in most organisms by electrons from the Cytochrome b6f complex by copper binding protein Plastocyanin (Blankenship 2002).

Photosystem I can run in noncyclic and cyclic modes (Arnon et al. 1957). With the presence of NADP+ the electron transport will run in the noncyclic mode generating NADPH. When the NADPH/NADP+ ratio is high, the electron transport in PSI can run in a cyclic mode, that generates a proton gradient without producing NADPH. In the cyclic electron transport, the energized electron is passed down a chain that ultimately returns it in base state via plastocyanin to the special pair P700+.

Figure 7. Schematic diagram of subunit composition end electron transport of PSI from higher plant. (drawn by Jon Nield)

The light-harvesting system

Light harvesting is recognized as a primary process in the photosynthesis. The function of harvesting solar energy is fulfilled by a series of light-harvesting complexes (Blankenship 2002). The function of light-harvesting complexes is based on energy transfer process that involves the migration of excited states from one molecule to another. This is a pure physical process, which depends on a weak energetic coupling of the antenna pigments. In light-harvesting complexes, the pigments are bound to proteins in highly specific associations. Common antenna pigments are chlorophylls, cartotenoids and open-chain tetrapyrrole bilin pigments found in phycobilisome antenna complexes.

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Due to the fact that sunlight is a relatively dilute energy source, antenna system increases the amount of energy that can be absorbed by photosystem reaction centers. Under certain conditions, especially if the organism is exposed to some form of stress, more light energy can be absorbed than it can be productively used. If unchecked, this can lead to severe damage of photosystems. Antenna systems (as well as reaction centers) have therefore extensive and multifunctional regulation, protection and repair mechanisms (state transitions, xanthophylle cycle, etc.).

Antenna systems usually work like energetic spatial funneling mechanism, in which pigments that are on the periphery of the complex absorb at shorter wavelengths and therefore higher excitation energies that pigments at the core. The funneling of energy from the LHCs to RC is achieved through a process known as resonance energy transfer, also known as the Förster mechanism (Blankenship 2002). Pigment is excited by the light at a particular wavelength, and the consequential de-excitation of this pigment, associated with a loss of energy to heat or fluorescence, leads to the excitation of another lower energy pigment. Series of these excitations and de-excitations of pigments create an energy cascade which leads to the excitation of special pair in reaction center with longer wavelength, than was absorbed by the antenna system.

Figure 8. Schematic diagram of photosynthetic antenna and energy transport to RC.

A remarkable variety of antennae differing in a structure or even in types of pigments

are found in various photosynthetic organisms. Almost in all antenna complexes there are pigments specifically associated with proteins to the unique structures. The only known exception is the chlorosome antenna complex of green photosynthetic bacteria, in which the pigments are aggregated with another pigments without a help of proteins.

Antenna complexes can be divided into outer membrane complexes and integral membrane complexes: • In outer membrane complexes, the antenna complex is not a part of a membrane, however, it is usualy connected or anchored to the membrane with a help of other proteins.

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• Integral membrane antennae contain proteins that cross the lipid bilayer. The pigments are often deeply buried in the membrane.

Integral membrane antennae known as core antenna complex are closely associated with the RC. They have a fixed pigment stoichiometry and a well-defined physical arrangement to the reaction center complex. The other group of integral membrane antenna complexes is called accessory antennae. They are found in variable amounts in addition to the core antennae (Blankenship 2002).

LHCI and LHCII are the most important accessory integral membrane antennae of plants and many algae. The light-harvesting complexes I (LHCI) is associated with photosystem I and light-harvesting complexes II (LHCII) with PSII. In some cases LHCII can bind also to PSI (Kouril et al. 2005). The structure of LHCII complex was determined to a resolution of 2.7 Å (Liu et al. 2004) and 2.5 Å (Standfuss et al. 2005). The structural model of LHCI (Mozzo et al. 2006) based on the structure of PSI-LHCI (Ben-Shem et al. 2003) shows some similarity to structure of LHCII. Both LHC complexes consist of three transmembrane helices that coordinate several molecules of chlorophyll (9 in LHCI, 12 in LHCII) and some carotenoid molecules (3 in LHCI, 2 in LHCII) which help to hold the LHC complex together (Corbet et al. 2007). Major complex of LHCII consists of three proteins Lhcb1-3 which form the trimeric structure (Peter and Thornber 1991). Lhcb4, Lhcb5 and Lhcb6 usually form the monomeric CP29, CP26, and CP24 (Bassi and Dainese 1992). By contrast, Lhca proteins form dimers of LHCI. Lhca1+4 compose the LHCI-730 (Schmid et al. 1997), and Lhca2+3 another dimer called LHCI-680 (Bassi et al. 1985).

FCP (fucoxanthin-chlorophyll protein) is antenna complex of chromophytic (or chlorophyll c-containing) algae. The name FCP is caused by the fact that fucoxanthin is the most prominent carotenoid in majority chromophytes organisms. The chromophyte FCP peptides consist like LHC peptides of three transmembrane helices but are usually smaller (Hiller et al. 1991, Caron et al. 1987, Buchel and Garab 1997). FCP antenna complex can be in trimeric and higher oligomeric form (Buchel 2003, Beer et al. 2006, Guglielmi et al. 2005, Brakemann et al. 2006).

Phycobilisomes are large outer membrane antenna complexes found in cyanobacteria and red algae (Seidler 1994, Grossman et al. 1995, MacColl 1998). Phycobilisomes consist of two or three types of pigment-proteins known as biliproteins, along with a number of additional proteins known as linkers. The major types of biliproteins are: phycoerythrin, phycocyanin and allophycocyanin. Most of the biliproteins are arranged into six rods (phycoerythrin, phycocyanin), which attach in a fanlike arrangement to a biliprotein core (allophycocyanin) that is attached to a stromal side of the thylakoid membrane, close to PSI-II.

PCB light-harvesting antenna was found in specific clade of cyanobacteria, called Prochlorophytes. Main pigments of PCB antenna are chlorophyll a and b or their analogs (Partensky and Garczarek 2003). Proteins of PCB are encoded by pcb genes and are predicted to have six transmembrane helices.

Except mentioned light-harvesting antennae, there are also photosynthetic dinoflagellates PCP (peridinin-chlorophyll-protein) antenna complexes and some photosynthetic bacteria antenna complexes described: Chlorosome, LHI and LHII.

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EM and PSI

The first structural studies of PSI particles by electron microscopy were carried out on cyanobacterial PSI (Newman and Sherman 1978). Few years later single particle analysis showed, that cyanobacterial PSI could occur in monomeric and trimeric form (Boekema et al. 1987, Ford and Holyenburg 1988, Rogner et al. 1990). Both different forms of PSI are interchangeable and depend on growth conditions. The trimerisation of PSI was observed also in chlorophyll b containing prochlorophyte Prochlorotrix hollandica (van der Staay et al. 1993). Electron microscopy studies revealed that phycobilisomes (cyanobacterial peripheral antennae) under conditions of iron deficiency, can be replaced by an antenna ring around trimeric PSI made of 18 chlorophyll a-binding IsiA proteins (Bibby et al. 2001b, Boekema et al. 2001). Similar light harvesting antenna ring (made of PCB proteins) around trimeric PSI has been found in the prochlorophyte Prochlorococcus marinus (Bibby et al. 2001a) and Prochlorotrix hollandica (Bumba et al. 2005b). Single particle analysis followed by electron microscopy of green algae and higher plants PSI showed monomeric particles with half-moon shaped LHCI antenna on one side of the PSI core complex (Boekema et al. 2001, Germano et al. 2002, Kargul et al. 2003). Transitional state between cyanobacteria and chloroplasts of photosynthetic eukaryotes is represented by the red algae. The image analysis of red alga PSI complexes from Cyanidium caldarium shows monomeric particles similar to the PSI core or PSI–LHCI supercomplexes observed in higher plants or Chlamydomonas reinhardtii (Gardian et al. 2007).

Figure 9. Overall structure of PSI complexes obtained by EM and single particle image analysis. A: Monomeric PSI core complex from C. reinhardtii (Kargul et al. 2003). B: Monomeric PSI-LHCI complex from C. caldarium (Gardian et al. 2007). C: Trimeric PSI complex from Synechocystis 6803 (Boekema et al. 2001). D: Trimeric PSI-PCB supercomplex from P. hollandica (Bumba et al. 2005b). Supercomplex is overlaid with cyanobacterial X-ray model of the PSI and ring of cyanobacterial CP43 antenna proteins.

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EM and PSII

The first higher resolution electron microscopy structural studies of PSII complexes were made by the single particle analysis of cyanobacerial and spinach PSII (Boekema et al. 1995). These structural studies showed a dimeric organization of PSII core complexes. On the side-view projections of the PSII complexes there were small protrusions found on the lumenal sides. They were attributed to the extrinsic proteins of the oxygen-evolving complex (Kuhl et al. 1999, Bumba et al. 2004). Also higher plants PSII with its light harvesting antennae was studied by the electron microscope. LHCII was found in a form of two trimeric and four monomeric complexes situated around the dimeric PSII core complex (Boekema et al. 1995). It was proved (Boekema et al. 1998, Yakushevska et al. 2001) that trimeric LHCII can be attached to PSII in strong, moderate or loose positions. 3-D structure of higher plants PSII-LHCII supercomlex was obtained by the single particle analysis at a resolution of 24 Å (Nield et al. 2000a). The interaction of the PSII-LHCII supercomplexes has also been studied in adjacent layers of stacked chloroplast thylakoid membranes (Bumba et al. 2004). Single particle analysis of PSII isolated from phycobilisome containing red alga (C. caldarium) revealed cyanobacterial type of the PSII complex, with no LHCII (Gardian et al. 2007). A combination of His-tagged subunits and Ni-NTA Nanogold label, EM can also identify the location of the subunit within the photosystem complex (Bumba et al. 2005a).

Figure 10. Overall structure of PSII complexes obtained by EM and single particle image analysis. A: Top-view projection of dimeric PSII core complex from Synechocystis 6803 (Bumba et al. 2006). B: PSII core complex in his side-view projection, isolated from C. caldarium (Gardian et al. 2007). C: Side-view projection of pair of two PSII-LHCII supercomplexes from Pea plants (Bumba et al. 2004). D: Top-view projection of dimeric PSII-LHCII supercomplex from Spinach, overlaid with a model of PSII-LHCII supercomplex based on models derived from electron crystalography (Hankamer et al. 1999, Nield et al. 2000b).

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Outline of the thesis

The aim of this work is to describe the use of electron microscopy in the photosynthetic studies. For investigation of the structure of photosynthetic pigment-protein complexes we use the method of single particle image analysis of electron micrographs of pigment-protein complexes. For the studies of thylakoid membranes changes in cyanobacteria mutant, we use the EM method of ultra thin specimens. Chapter 2 describes the organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium. This organisation of photosynthetic pigment-protein complexes was investigated with the consideration of red algae being the intermediate evolution stage between the cyanobacteria and the green algae. Chapter 3 describes the importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes. In this chapter we used the electron microscopy as a technique supplemental to the molecular biology to describe the changes of photosynthetic apparatus in cyanobacterial mutant. Chapter 4 describes the results of the structural characterization of Photosystem I, Photosystem II and FCP complexes from chromophytic alga Xanthonema debile.

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Chapter 2

Regular paper Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts Z. Gardian, L. Bumba, A. Schrofel, M. Herbstova, J. Nebesarova and F. Vacha Published in: Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1767, Issue 6, June 2007, Pages 725-731

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Biochimica et Biophysica Acta 1767 (2007) 725–731

Copyright © 2007 Elsevier B.V. All rights reserved

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial

and higher plant concepts

Received 29 September 2006; received in revised form 19 January 2007; accepted 30 January 2007; available online 7 February 2007

Zdenko Gardian a,b, Ladislav Bumba a,c, Adam Schrofel d, Miroslava Herbstova a,e, Jana Nebesarova a,e, Frantisek Vacha a,b,e,*

a. Biological Centre, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic b. Institute of Physical Biology, University of South Bohemia, Zámek 136, 373 33 Nové Hrady, Czech Republic c. Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic d. Department of Biochemistry, Faculty of Science, Charles University, Albertov 6, 128 43 Praha 2, Czech Republic e. Faculty of Biological Sciences, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic

Abstract

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ´) extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed. Keywords: Photosynthesis; Red algae; Cyanidium caldarium; Photosystem I; Photosystem II; Electron microscopy Anotace česká: Struktura fotosystému I a II červené řasy Cyanidium caldarium byla určena elektronovou mikroskopií a obrazovou analýzou jednotlivých částic. Struktura fotosystému II je podobná struktuře známé u sinic. Fotosystém I byl nalezen v monomerní formě. Abbreviations: Chl, chlorophyll; DEAE, diethyaminoethyl; DM, n-dodecyl-β-d-maltoside; EM, electron microscopy; HL, high light; LHCI, light-harvesting complex of Photosystem I; LHCII, light-harvesting complex of Photosystem II; LL, low light; MES, 2-morpholinoethanesulfonic acid; OEC, oxygen evolving complex; PSI, Photosystem I; PSII, Photosystem II; SDS-PAGE, polyacrylamide gel electrophoresis in a presence of sodium dodecylsulfate * Corresponding author. Biological Centre, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic. Tel.: +420 387775533; fax: +420 385310356. E-mail address: [email protected] (F. Vacha).

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Chapter 3

Regular paper Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes U. Dühring, R. Sobotka, J. Komenda, E. Peter, Z. Gardian, M. Tichy, B. Grimm, A. Wilde Published in: JBC, 283, NO. 38, September 2008, Pages 25794 –25802

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HE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 38, pp. 25794 –25802, September 19, 2008

© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A

Importance of the Cyanobacterial Gun4 Protein for Chlorophyll Metabolism and Assembly of Photosynthetic Complexes* Received for publication, May 16, 2008, and in revised form, July 11, 2008 Published, JBC Papers in Press, July 14, 2008, DOI 10.1074/jbc.M803787200

Roman Sobotkaa,b,1, Ulf Dühringc,1, Josef Komendaa,b, Enrico Peterc, Zdenko Gardiand, Martin Tichya,b, Bernhard Grimmc, and Annegret Wildec,2

From the aInstitute of Physical Biology, University of South Bohemia, 37333 Nove Hrady, Czech Republic, bDepartment of Autotrophic Microorganisms, Institute of Microbiology, Opatovicky mlyn, 37971 Trebon, Czech Republic, cInstitute of Biology, Humboldt-University Berlin, Chausseestrasse 117, 10115 Berlin, Germany, dInstitute of Plant Molecular Biology, Branisovska 31, 37005 Ceske Budejovice, Czech Republic

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional

analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished

* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB429 and TPA8 (to A. W. and B. G.), by Institutional Research Concept AV0Z50200510, by Ministry of Education of the Czech Republic project MSM6007665808 (to J. K.), and by Grant Agency of the Czech Academy of Sciences project IAA500200713 (to R. S. and M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “ advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 These authors equally contributed to this work. 2 Present address and to whom correspondence should be addressed: Institute of Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Hei- nrich-Buff-Ring 26-32, 35392 Giessen, Germany. Tel.: 496419935545; Fax: 496419935549; E-mail: [email protected]. 3 The abbreviations used are: Chl, chlorophyll; PIX, protoporphyrin IX; PS, photosystem; DM, β-dodecyl maltoside; BN-PAGE, blue native polyacrylamide gel electrophoresis. HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid.

photoautotrophic growth of the resulting

gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein

complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.

Anotace česká: Naše výsledky demonstrují že Gun4 protein hraje nezbytnou roli při vázaní hořčíku při tetrapyrolovej biosyntéze a má dramaticky vliv na obsah chlorofylu v buňkách. V gun4 mutantech bol pozorovaný značný nedostatek proteinů vázajících chlorofyl, hlavně proteinů fotosystému II.

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REFERENCES 1. Papenbrock, J., and Grimm, B. (2001) Planta 213, 667-681. 2. Vavilin, D. V., and Vermaas, W. F. (2002) Physiol Plant 115, 9-24. 3. Grossman, A. R., Lohr, M., and Im, C. S. (2004) Annu Rev Genet 38, 119-173. 4. Beale, S. I. (2005) Trends Plant Sci 10, 309-312. 5. Herrin, D. L., Battey, J. F., Greer, K., and Schmidt, G. W. (1992) J Biol Chem 267, 8260-8269. 6. Plumley, G. F., and Schmidt, G. W. (1995) Plant Cell 7, 689-704. 7. Brusslan, J. A., and Peterson, M. P. (2002) Photosynth Res 71, 185-194. 8. Susek, R. E., Ausubel, F. M., and Chory, J. (1993) Cell 74, 787-799. 9. Mochizuki, N., Brusslan, J. A., Larkin, R., Nagatani, A., and Chory, J. (2001) Proc Natl Acad Sci U S A 98, 2053-2058. 10. Larkin, R. M., Alonso, J. M., Ecker, J. R., and Chory, J. (2003) Science 299, 902-906. 11. Davison, P. A., Schubert, H. L., Reid, J. D., Iorg, C. D., Heroux, A., Hill, C. P., and Hunter, C. N. (2005) Biochemistry 44, 7603-7612. 12. Verdecia, M. A., Larkin, R. M., Ferrer, J. L., Riek, R., Chory, J., and Noel, J. P. (2005) PLoS Biol 3, e151. 13. Wilde, A., Mikolajczyk, S., Alawady, A., Lokstein, H., and Grimm, B. (2004) FEBS Lett 571, 119-123. 14. Rippka, R., Desrulles, J., Waterbury, J. B., Herdman, M., and Stanier, R. Y. (1979) J Gen Microbiol 11, 419-436. 15. Tous, C., Vega-Palas, M. A., and Vioque, A. (2001) J Biol Chem 276, 29059-29066. 16. Papenbrock, J., Mock, H. P., Kruse, E., and Grimm, B. (1999) Planta 208, 264-273.

17. Komenda, J., Reisinger, V., Muller, B. C., Dobakova, M., Granvogl, B., and Eichacker, L. A. (2004) J Biol Chem 279, 48620-48629. 18. Schägger, H., and von Jagow, G. (1991) Anal Biochem 199, 223-231. 19. Komenda, J., Lupinkova, L., and Kopecky, J. (2002) Eur J Biochem 269, 610-619. 20. Jänsch, L., Kruft, V., Schmitz, U. K., and Braun, H. P. (1996) Plant J 9, 357-368. 21. Dühring, U., Irrgang, K. D., Lünser, K., Kehr, J., and Wilde, A. (2006) Biochim Biophys Acta 1757, 3-11. 22. Porra, R. J., Thompson, W. A., and Kriedmann, P. E. (1989) Biochem Biophys Acta 975, 384-394. 23. Shen, G., and Vermaas, W. F. (1994) J Biol Chem 269, 13904-13910. 24. He, Q., Brune, D., Nieman, R., and Vermaas, W. (1998) Eur J Biochem 253, 161-172. 25. Eaton-Rye, J. J., and Vermaas, W. F. (1991) Plant Mol Biol 17, 1165-1177. 26. Dobakova, M., Tichy, M., and Komenda, J. (2007) Plant Physiol 145, 1681-1691. 27. Yang, H., Inokuchi, H., and Adler, J. (1995) Proc Natl Acad Sci U S A 92, 7332-7336. 28. Sobotka, R., McLean, S., Zuberova, M., Hunter, C. N., and Tichy, M. (2008) J Bacteriol 190, 2086-2095. 29. Kada, S., Koike, H., Satoh, K., Hase, T., and Fujita, Y. (2003) Plant Mol Biol 51, 225-235. 30. Vavilin, D., and Vermaas, W. (2007) Biochim Biophys Acta 1767, 920-929. 31. He, Q., and Vermaas, W. (1998) Proc Natl Acad Sci U S A 95, 5830-5835. 32. Sobotka, R., Komenda, J., Bumba, L., and Tichy, M. (2005) J Biol Chem 280, 31595-31602.

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Chapter 4

Regular paper Structural characterization of Photosystem I, Photosystem II and FCP complexes from chromophytic alga Xanthonema debile Z. Gardian and F. Vacha To be submitted

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Structural characterization of Photosystem I, Photosystem II and FCP complexes from

chromophytic alga Xanthonema debile

Zdenko Gardian1,2, František Vácha1,2,3

1 Institute of Physical Biology, University of South Bohemia, Zámek 136, Nové Hrady 373 33, Czech Republic 2 Biology centre ASCR, Branišovská 31, České Budějovice 370 05, Czech Republic 3 Faculty of Science, University of South Bohemia, Branišovská 31, České Budějovice 370 05, Czech Republic Abstract

Photosynthetic carbon fixation by Chromophytes is one of the significant components of the carbon cycle on the Earth. Their photosynthetic apparatus is different in pigment composition from that of green plants and algae. In this work we report structural maps of PSI, PSII and light harvesting antenna complexes isolated from the soil chromophytic alga Xanthonema debile. The single particle analysis folowed by electron microscopy of negatively stained preparations reveal that overall structure of Chromophytes PSI and PSII reaction centers is similar to that known from higher plants or algae. Averaged top-view projection of light harvesting antenna complexes (FCP) shows two groups of particles. The smaller ones correspond to trimeric form of FCP. The bigger particles resemble the oligomeric form of FCP. Keywords: Photosynthesis, Chromophytes, Xanthonema debile, Photosystem I, Photosystem II, FCP, Electron Microscopy

Introduction

The Chromophytes (Heterokont or chlorophyll c-containing algae) contribute significantly to the photosynthetic carbon fixation in the oceans. Except water, some classes inahabit also soil environments. Xanthonema debile is an example of soil chromophytic alga that differs from not only green plants and algae but also other Chromophytes. There are no grana stacking and no segregation of photosystems [1]. Their photosynthetic apparatus contain Chl a, Chl c, β-Carotene, diadinoxanthin, diatoxanthin, heteroxanthin, and vaucheriaxanthinester.

Abbreviations: Chl, chlorophyll; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DEAE, diethyaminoethyl; DM, n-dodecyl-β-D-maltoside; EM, electron microscopy; FCP, fucoxanthin-chlorophyll proteins; LHCI, light-harvesting complex of Photosystem I; LHCII, light-harvesting complex of Photosystem II; PSI, Photosystem I; PSII, Photosystem II; SDS-PAGE, polyacrylamide gel electrophoresis in a presence of sodium dodecylsulfate

Fucoxanthin, which is present in other Chromophytes classes, is absent in the Xanthophyceae [2-4].

Because fucoxanthin is the most prominent carotenoid in the majority of Chromophytes, their light harvesting antenna proteins are usually called fucoxanthin-chlorophyll proteins (FCP). The chromophytic FCP proteins are usually smaller (17-24 kDa) than the light-harvesting complexes (LHC) of plants and green algae [5-7]. Several years ago, one type of FCP for both photosystems has been purified from Chromophytes [8-12], recently trimeric and higher oligomeric state of Chromophytes FCP complexes were described in Diatoms [7,13-15].

Electron microscopy followed by a single particle analysis offers a powerful means to visualise and study structure of large proteins and protein complexes. This brought a number of structural data of

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photosynthetic pigment-protein complexes during last decades. For example at the end of 80’s single particle analysis shows that cyanobacterial PSI can occur in a monomeric and a trimeric form [16-18]. The trimerisation of PSI was observed also in chlorophyll b containing prochlorophyte Prochlorotrix hollandica [19]. Electron microscopy followed by the single partical analysis of green alga, red alga and higher plats showed PSI only as a monomeric particle with half-moon shaped LHCI antenna on one side of the PSI complex [20-23]. The electron microscopy structural studies of cyanobacterial and spinach PSII [24] showed a dimeric organization of PSII core complexes. Small protrusions on the lumenal side of the PSII complexes were attributed to the extrinsic proteins of the oxygen-envolving complex [25,26]. Electron microscope was also used to study higher plants PSII with their light harvesting antennae. LHCII was found like a two trimeric and four monomeric complexes around the dimeric PSII core complex [24].

When compare the number of information about the organization of higher plant, algae or cyanobacteria photosynthetic complexes and their LHC, there are no such structural data obtained for Chromophytes’ photosynthetic apparatus. Only one reord has been published on FCP-antennae of Diatomes showing PSI-FCP complex in the EM micrograph as a monomer [27].

In this contribution we report structural maps of PSI and PSII complexes and light harvesting antennae isolated from the chromophytic alga Xanthonema debile. The aim of this work was to characterise the organisation of PSI and PSII complexes with a particular respect to the arrangement of light harvesting antennae complexes.

Materials and methods

The chromophytic alga Xanthonema debile was batch cultivated in 5 l flasks at room temperature in BBM (Bold-Basal/Bristol Medium) medium [28] and bubbled with filtered air. The light irradiance

was 100 µmol photon m-2s-1. Cells were harvested by centrifugation at 1800×g for 5 min, washed with distilled water, and resuspended in a buffer containing 10 mM HEPES (pH 7.4), 2 mM MgCl2, 2 mM MnCl2, 10 mM KCl, 1 M sorbitol and protease inhibitors (1 mM benzamidine and 1 mM phenylmethanesulfonylfluoride).

Cells were broken by a double French press cycle at 15,000 psi. The unbroken cells were removed by centrifugation for 5 min at 3000×g. The supernatant was then centrifuged for 1 h at 60,000×g to pellet thylakoid membranes. Membranes were resuspended and solubilised with 5% Digitonin at a chlorophyll concentration of 1 mg (Chl) ml-1 for 60 min.

The unsolubilised material was removed by centrifugation for 20 min at

Fig. 1. (A) SDS-PAGE analysis of pigment–protein complexes from thylakoid membranes of Xanthonema debile. Lane (1) represents PSI-PSII-FCP fraction eluted from DEAE Sepharose CL-6B anion-exchange column with 120 mM NaCl. (2) Pure PSI-PSII zone and (3) FCP zone resolved after sucrose density gradient centrifugation of PSI-PSII-FCP anion-exchange fraction. Molecular weight markers (in kDa) are indicated on left. (B) Sucrose density gradient of PSI-PSII-FCP fraction. The sample was divided into zone contained free FCP antennae and zone contained mainly PSI and PSII core complexes.

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60,000×g and the supernatant was loaded onto DEAE Sepharose CL-6B (Amersham Biosciences, Sweden) anion-exchange column equilibrated with 10 mM HEPES (pH 7.4), 2 mM MgCl2, 2 mM MnCl2, 0.03% DM. Photosynthetic complexes were eluted from the column with concentration of 120 mM NaCl and loaded onto a fresh 0–1.2 M continuous sucrose density gradient prepared by freezing and thawing the centrifuge tubes filled with a buffer containing 10 mM HEPES (pH 7.4), 2 mM MgCl2, 2 mM MnCl2, 10 mM KCl, 0.03% DM, 0.6 M sucrose. The following centrifugation was carried out at 4 °C using a P56ST swinging rotor (Sorvall) at 150,000×g for 14 h. After centrifugation the sample was separated into a zone with light-harvesting antennae and a zone with PSI and PSII core complexes. Zones resolved in the sucrose density gradient were further purified and desalted by gel filtration using Sephadex G-25 (Amersham Biosciences, Sweden).

Fig. 2. 77K fluorescence emission spectra (A) and absorption spectra (B) of sucrose density gradient zones. The FCP (solid line) and PSI-PSII (dashed line) zones were obtained by a sucrose density gradient centrifugation of PSI-PSII-FCP anion-exchange fraction. Spectra are normalised to their maxima.

Chlorophyll concentration was determined according to Ogawa and Vernon [29]. Room temperature absorption spectra were recorded with a UV300 spectrophotometer (Spectronic Unicam, Cambridge, UK). Fluorescent emission spectra were measured at a liquid nitrogen temperature using a Fluorolog-2 spectrofluorometer (Jobin Yvon, Edison, NJ, USA) with an excitation wavelength of 435 nm and a chlorophyll concentration of 10 µg (Chl) ml-1. The protein composition was determined by SDS-PAGE using a 12.5% polyacrylamide gel containing 6 M urea and stained with Coomassie Brilliant Blue or Silver Staining Kit.

Fig. 3. Electron micrographs of (A) PSI-PSII and (B) FCP complexes in their top-view projections. Samples were negatively stained with 2% uranyl acetate. The scale bar represents 50 nm

Freshly prepared photosynthetic complexes were immediately used for electron microscopy (EM). The specimen was placed on glow-discharged carbon-coated copper grids and negatively stained with 2% uranyl acetate. EM was performed with JEOL 1010 transmission electron microscope (JEOL, Japan) using 80 kV at 60,000× magnification. EM micrographs were digitized with a pixel size corresponding to 5.1 Å at the specimen level. Image analyses were carried out using Spider and Web software package [30]. The selected projections were rotationally and translationally aligned, and treated by multivariate statistical analysis in combination with classification procedure [31,32]. Classes from each of the subsets were used for refinement of alignments and subsequent classifications. For the final sum,

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the best of the class members were summed using a cross-correlation coefficient of the alignment procedure as a quality parameter. Results and discussion

PSII and PSI complexes from chromophytic algae have been previously isolated using sucrose density gradient centrifugation [33,34] or ion exchange chromatography [27,35]. In order to isolate pure photosynthetic complexes of Xanthonema debile, we combined both techniques. At first, ion exchange chromatography was used to obtain puryfied mixture of PSI, PSII and FCP complexes from digitonin solubilized thylakoid membranes, in the second step, sucrose density gradient centrifugation separated the sample into two zones of free FCP and a mixture of PSI, PSII complexes.

From DEAE Sepharose CL-6B anion-exchange column, two chlorophyll-containing fractions were eluted with a linear gradient of 0–600 mM NaCl. The first fraction can be attributed to free pigments. The second fraction eluted with 120 mM NaCl contained polypeptides of PSI, PSII

complexes and light-harvesting antennae (FCP) as indicated by SDS-PAGE (Fig. 1A, line 1). This fraction contained a 60 kDa band typical for the PsaA/B reaction center proteins of PSI, and the protein bands characteristic for the PSII, the intrinsic antennae CP47, CP43 and reaction centre proteins D2 and D1. SDS-PAGE shows also a prominent band about 20 kDa, corresponding to antenna polypeptides of FCP [5-7].

The second fraction from anion-exchange column chromatography was loaded onto sucrose density gradient. After 14 hour centrifugation at 150,000×g the sample was divided into a two zones (Fig. 1B). SDS-PAGE analysis showed that the bottom zone contained mainly PSI and PSII core complexes while the upper zone contained free FCP antennae (Fig. 1A, lanes 2 and 3).

77 K fluorescence emission spectra of sucrose density gradient zones are shown in figure 2A. The upper zone is characterized by a peak at 682 nm, a typical region of fluorescence emission of FCP antennae. The bottom zone peaks at 691 nm, corresponding

Fig. 4. Single particle analysis of top-view projection maps of X. debile photosynthetic pigment-protein complexes. (A–D) The most representative class averages obtained by classification of 6900 particles from the PSI-PSII sucrose density gradient zone. (E–H) The most representative class averages obtained by classification of 6120 particles from the FCP sucrose density gradient zone. The number of summed images is: 524 (A), 486 (B), 258 (C), 281 (D), 442 (E), 412 (F), 322 (G) and 356 (H). The scale bar represents 5 nm.

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to the emission of PSII, with a broad long wavelength shoulder of the emission of PSI. The significant long-wavelength fluorescence component found in many species is missing due to the absence of specific “red” chlorophyll molecules [36] as it was reported previously in e.g. cyanobacteria [37,38] and red algae [39].

Room temperature absorption spectra of the PSI-PSII zone and FCP zone are in figure 2B, showing higher content of carotenoids in the FCP antennae zone.

Purified photosynthetic complexes were negatively stained by 2% uranyl acetate, visualized by electron microscopy and processed by image analysis. Typical electron microscopy images of PSI-PSII and FCP zone are shown in Fig. 3. To process the particle images by single particle analysis, we have selected 6900 particles from the images of PSI-PSII zone and 6120 particles from FCP zone. The selected projections were aligned, treated with multivariate statistical analysis and classified into classes. After the classification steps, selected photosynthetic complexes were decomposed into fifteen and eighteen classes for PSI-PSII and FCP zones, respectively. The projections of PSI-PSII

zone can be divided into two groups of particles shown in figure 4A,B and figure 4C,D, respecitely.

One group (Fig. 4A,B) had an oval shape and represent monomeric PSI core compexes. PSI from Xanthonema debile did not reveal dimeric or trimeric form of PSI particles as found in cyanobacteria [40]. The size and shape of these particles are very similar to the PSI core complexes previously observed in algae [21-23] or higher plants [20]. Fig. 5A presents the most representative class average of 524 top-view projections of PSI complexes, overlaid with an X-ray structure of PsaA/B heterodimer of PSI from Synechococcus elongatus [41].

The top-view projections of the second group of particles from PSI-PSII zone show diamond-shaped particles with two-fold rotational symmetry (Fig. 4C,D). These particles resemble the PSII core complexes isolated from cyanobacteria, algae and higher plants [24,25,42-44]. Class average of 281 top-view projections of PSII complexes is shown in Fig 5B. The PSII particle is overlaid with PSII crystal structure obtained from Thermosynechococcus elongatus [45].

Fig. 5. Schematic representation of the overal structure of the PSI and PSII complexes isolated from X. debile. The most representative top-view PSI (A) and PSII (B) projection maps of negatively stained particles from PSI-PSII sucrose density gradient zone. The projections are overlaid with a cyanobacterial X-ray model of the PSI (A) and PSII (B) core complexes. The coordinates are taken from Protein Data Bank (http://www.rcsb.org/pdb). (A) code 1JB0 [41] and (B) code 2AXT [45]. The scale bar represents 5 nm.

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Fig. 6. Schematic representation of the FCP subunits organisation in FCP complexes isolated from X. debile. (A) The most representative class averages of top-view trimeric FCP projections overlaid with a model of FCP trimer [7]. (B) The most representative class averages of top-view projections of oligomeric FCP overlaid with a model of five trimeric FCP. The scale bar represents 5 nm. A comparison of amount of the PSII and PSI complexes in the electron micrographs shows that only a few PSII complexes are fixed to the EM grids compare to the PSI. However, it is hard to speculate on the number of PSI and PSII complexes in the thylakoid membrane because PSII lumenal sufrace has a much lower afinity to the support carbon film than PSI [24,25].

The most representative classes of single particle analysis of FCP complexes are in Fig. 4E-H. Other studies showed that FCP complexes can be organized in a trimeric or even higher oligomeric states [7,13-15]. Our averaged top-view projections of FCP show two groups of particles. The smaller one, with a size of about 7 nm, coresponds to the trimeric form of FCP. Fig. 6A represents class average of this trimeric FCP, made of 442 summed images and overlaid with a model of trimeric FCP [7]. The bigger particles (about 12 nm) had an oval shape and resemble the oligomeric form of FCP. It was reprted that the higher oligomers consist of six to nine monomers of FCP [7], but oligomers consisting of even seven trimers were suggested on the bases of calculations [46,47]. Our results suggest five trimers in one oligomeric FCP. The incorporation of the

five trimeric FCP antennae into the the most representative class average of 356 top-view projections of oligomeric FCP complexes is shown in Fig. 6B.

In this paper we have confirmed, that the overall structure of Chromophytes PSI and PSII reaction centers is similar to that known from higher plants or algae. We have also demonstrated the structure of trimeric and oligomeric forms of FCPs, obtained by electron microscopy and single particle image analysis. Since we were not able to obtain photosynthetic reaction centers asociated with light harvesting antenna FCP, the question how are the FCP antennae bound to PSI or PSII, or whether they are attributed to a certain photosystem at all is still open.

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Summary

Photosynthesis is directly or indirectly the most important process on the Earth. It is carried out by a wide variety of photosynthetic organisms and provides energy in a form of reduced carbohydrates that is necessary for all living organisms. The light reaction of the photosynthesis is performed by the special pigment-containing protein complexes that are associated to photosynthetic thylakoid membranes. In higher plants, algae and cyanobacteria the thylakoid membranes accommodate two different types of photosystems: Photosystem I (PSI) and Photosystem II (PSII). Main function of these Photosystems inheres in conversion of the solar energy into the chemically fixed energy. General architecture of PSII and PSI is quite similar. Both Photosystems consist of two main domains: core complex and peripheral light-harvesting antenna complex.

For better understanding of the function of PSI, PSII and excitation energy transfer from light-harvesting antenna, it is necessary to know the structure of these photosynthetic pigment-protein complexes. In this thesis the overall structure of photosynthetic pigment-protein complexes was investigated using the electron microscopy followed by the single particle image analysis. The electron microscopy was also used to investigate the changes in shape of cyanobacteria thylakiod membranes in dependence on the photosynthetically important gun4 gene deletion.

Chapter 1 gives an introduction to the main principles of electron microscopy and also an introduction to the electron microscopy techniques used in the structural studies of macromolecular complexes. This chapter also briefly describes general knowledge about the photosynthetic apparatus and summarizes information about the investigation of photosynthetic pigment-protein complexes by the electron microscopy. Chapter 2 describes the organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium, determined by electron microscopy and single particle image analysis. The overall structure of PSII was found to be similar to that known from cyanobacteria. PSI was determined as a monomeric unit with varying amounts of antenna subunit attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. Since we have observed only PSI monomers, we suggest that the trimerisation is not caused by a simple absence of PsaH subunit in PSI complex. Also, we assume that the presence of PsaG protein in PSI is not essential for binding of LHCI subunits. Chapter 3 describes the importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes. Function of this Gun4 protein in Synechocystis sp. PCC 6803 was analyzed by blue native polyacrylamide gel electrophoresis, immunoblot analyses, electron microscopy and radiolabeling of the membrane protein complexes. Our results demonstrate that Gun4 plays an essential role in the magnesium branch of tetrapyrrole biosynthesis and dramatically affects the Chl content in the cell. Chl deficiency arising from the inactivation of gun4 affects accumulation of both photosystems. The more extensive deficiency of PSII was most probably related to the insufficient

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accumulation of CP47. This Chl-binding protein appears to be the most sensitive protein to changes in the Chl availability and may, therefore, represent an important target for the mutual regulation of tetrapyrrole biosynthesis and formation of new PSII complexes. Chapter 4 describes the results of structural characterization of Photosystem I, Photosystem II and FCP (fucoxanthin-chlorophyll protein) antenna complexes from chromophytic alga Xanthonema debile. The single particle analysis of electron micrographs showed that overall structure of Chromophytes PSI and PSII reaction centres is similar to that known from higher plants or algae. The averaged top-view projection of FCP complexes shows two groups of particles. The smaller ones correspond to trimeric form of FCP. The bigger particles had an oval shape and resemble the oligomeric form of FCP.