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(1)AKADEMIA GÓRNICZO - HUTNICZA IM. STANISŁAWA STASZICA W KRAKOWIE. Wydział Fizyki i Informatyki Stosowanej. Praca doktorska mgr inż. Agnieszka Hałas. Dynamics of nanostructural organization and activity of photosynthetic systems of type II Promotor: Prof. dr hab. Kvĕtoslava Burda Współpraca: Prof. Pierre Sebban, Prof. Seiji Akimoto. Kraków, 2013.

(2) This PhD thesis was written under the supervision of prof. dr hab. Kvĕtoslava Burda from the University of Science and Technology Faculty of Physics and Applied Computer Science, AGH, Kraków, Poland. The sample preparation described in this dissertation was carried out under the supervision of prof. Pierre Sebban and under the guidance of dr Valérie Dérrien at the University of Paris XI, Orsay, France. The fluorescence measurements were performed in the laboratory of prof. Seiji Akimoto at the Molecular Photoscience Research Center, Kobe University, Japan.. Synchrotron measurements were performed under the supervision of dr Aleksandr Chumakov at ESRF, Grenoble, France..

(3) Acknowledgements I would like to express my acknowledgement to the Krakow Interdisciplinary PhDProject in Nanoscience and Advanced Nanostructures. The project was financed by the European Union Innovative Economy Program with the Foundation for Polish Science. Thank you to prof. Bartłomiej Szafran, the coordinator of this project. I would like to thank prof. dr hab. Kvĕtoslava Burda for her guidance, support and encouragement during my PhD work. I am grateful for her kindness and understanding and for the fact that while doing my PhD, I was able to count on her not only during my work in Poland but also in France and Japan. Her engagement over five years was invaluable. I would also like to thank her for a friendly working environment, the hours she dedicated to helping me understand and solve problems, for presenting me with challenges and helping me to believe in myself. I express my thanks to prof. Pierre Sebban and his team in Paris for the possibility of working in his laboratory and for having access to bacterial mutants produced in his laboratory. Thank you also for the discussions. I especially express my thanks to dr Valérie Dérrien for her patience in explaining and teaching me the methods of growing bacteria, isolating bacterial reaction centers and for having shared her experience with me. I am also grateful to prof. Seiji Akimoto from the University of Kobe in Japan for inviting me to work in his laboratory and the opportunity to carry out experiments using his equipment. Thank you to dr Makio Yokono for a invaluable help during experiments. I would like to express my thanks to the group of prof. Korecki, especially to Krzysztof Matlak for his help during Mössbauer measurements and to dr Tomasz Ślęzak for his help during synchrotron measurements and the nuclear forward scattering data analysis. I am also grateful to dr Aleksandr Chumakov for the synchrotron measurements in ESRF in Grenoble in France and to dr Marcin Zając to his help for understanding the measurements and the help in data analysis. Finally, thank you to my friends and family for their patience, understanding and support..

(4) To my sister Basia.

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(6) Abbreviations: ................................................................................................................... 3 Aim.................................................................................................................................... 4 1. THEORY................................................................................................................................ 5 1.1 Introduction ................................................................................................................. 5 1.1.1 The beginning of photosynthetic organisms ...................................................... 5 1.1.2 The process of photosynthesis............................................................................ 6 1.1.3 Photosynthetic bacteria ...................................................................................... 7 1.2 Photosynthesis in bacterial reaction centers of type II.............................................. 10 1.2.1 Bacterial reaction center complex .................................................................... 10 1.2.2 Bacterial reaction center core of type II ........................................................... 11 1.2.3 Light reactions.................................................................................................. 14 1.2.4 The non-heme iron in the quinone-iron complex............................................. 16 1.2.5 Copper ions action on photosystems of type II ................................................ 19 2. MATERIALS ....................................................................................................................... 20 2.1 Growth of bacteria..................................................................................................... 20 2.2 Purification of bacterial reaction centers................................................................... 21 2.3 Mutants description ................................................................................................... 22 2.4 The treatment of bacterial reaction centers with copper (II) ions ............................. 25 3. METHODS........................................................................................................................... 26 3.1 Mössbauer spectroscopy ........................................................................................... 26 3.1.1 Hyperfine parameters ....................................................................................... 28 3.1.2 Experimental setup........................................................................................... 32 3.2 Synchrotron radiation................................................................................................ 34 3.2.1 NFS: Nuclear forward scattering...................................................................... 35 3.2.2 NIS: Nuclear inelastic scattering...................................................................... 37 3.2.3 Experimental setup........................................................................................... 39 3.3 Fluorescence decay analysis...................................................................................... 40 4. RESULTS............................................................................................................................. 42 4.1 Bacterial reaction centers purity................................................................................ 42 4.2 Hyperfine interactions of non-heme iron .................................................................. 43 4.2.1 Mössbauer measurements ................................................................................ 43 4.2.2 Nuclear forward scattering measurements ....................................................... 47 4.3 Dynamical proprieties of non-heme iron binding sites ............................................. 52 4.3.1 Temperature dependent Mössbauer measurements.......................................... 52 1.

(7) 4.3.2 Nuclear inelastic scattering measurements ...................................................... 55 Discussion ....................................................................................................................... 59 4.4 Measurement of WT treated with copper.................................................................. 63 4.4.1 Mössbauer measurements ................................................................................ 63 4.4.2 Nuclear forward scattering measurements ....................................................... 64 Discussion ....................................................................................................................... 66 4.5 Fluorescence measurements ...................................................................................... 67 4.5.1 Steady state measurements............................................................................... 67 4.5.2 Decay time measurements................................................................................ 70 Discussion ....................................................................................................................... 71 5. Conclusions .......................................................................................................................... 75 Future aims ...................................................................................................................... 78 References ....................................................................................................................... 79. 2.

(8) Abbreviations: Asp: aspartic acid ADP: adenosine 5' – (trihydrogen diphosphate) ATP: adenosine 5' – (tetrahydrogen triphosphate) BRC: bacterial reaction center BPheo: bacteriopheophytin BChla: bacteriochlorophyll a Cyt: cytochrome EPR: electron paramagnetic resonance spectroscopy ET: electron transfer Glu: glutamic acid HF: hyperfine field LDAO: lauryldimethylamine N- oxide Leu: leucine LHC: light–harvesting complexes NADP: nicitin–amide–adenosine diphosphate NADPH: reduced form of NADP NHFe: non-heme iron NFS: nuclear forward scattering NIS: nuclear inelastic scattering (P870): special pair of bacteriochlorophylls Phe: phenylalanine PSI: photosystem I PSII: photosystem II QA, QB: ubiquinones molecules [QA – Fe – QB] complex: quinone-iron complex Rb. Sphaeroides: Rhodobacter sphaeroides TRIS: tris (hydroxymethyl) aminomethane. 3.

(9) Aim. The aim of this work was to study the influence of the non–heme iron (NHFe) in the iron-quinone complex (QA – Fe – QB complex) located on the acceptor side of the photosynthetic bacterial reaction centers of type II. NHFe is a very preserved component of the photosynthetic reaction centers of type II. It was already present in primitive photosynthetic bacterial organisms ~ 4.2 – 3.8 billion years ago. However, its role still remains unclear, especially in the stabilization of the QA and QB binding sites and the primary electron transfer (ET) between QA and QB quinone acceptors activated by temperature. In this study wild type (WT) and three mutated BRCs of Rb. sphaeroides were investigated: (i) two mutants, with point mutations changing the hydrogen network near the QA – Fe – QB complex and (ii) one mutant with modifications of residues from the second coordination sphere of NHFe at the most rigid part of the BRC protein core. In addition, WT BRCs treated with copper ions were investigated because Cu2+ is known to act as a protonophore, i.e. it may modify the hydrogen bonds in the NHFe vicinity. Bacterial reaction centers isolated from purple bacteria, grown in a medium enriched with iron isotope 57Fe, provided an opportunity to use unique techniques such as Mössbauer spectroscopy, nuclear resonant inelastic (NIS) and forward resonant X-ray scattering (FNS) of synchrotron radiation. The goal of this project is to understand the relationship between the spin and valence state of NHFe (Mössbauer spectroscopy and NFS) and the local flexibility of BRC core proteins (Mössbauer temperature dependent experiments and NIS). This knowledge will be combined with the photosynthetic activity of previously studied systems, especially with the efficiency of the energy transfer in BRCs studied by use of the ultra fast fluorescence spectroscopy resolved in time.. 4.

(10) 1. THEORY 1.1 Introduction 1.1.1 The beginning of photosynthetic organisms. The earliest fossils of life on Earth found in microfossils and stromatolites are estimated to be about 4.2 – 3.8 billon years old [1]. Stromatolites – “laminated organo-sedimentary structures formed by the trapping, binding or precipitation of minerals by microorganisms” [2] as well as a large number of. microfossils. photosynthetic. have. organisms. been [3].. assigned The. to. Earth’s. atmosphere at that time was toxic, mainly consisting of carbon dioxide (CO2), water and nitrogen with ammonia (NH3), methane (CH4) and carbon monoxide (CO). The surface of the Earth was covered with volcanoes, so the temperature in early oceans was very high (80 – 110o C). The earliest stromatolites probably descended from prokaryotic anaerobic extremophiles, which could live at. Fig. 1. Fossil stromatolites observed at the Dawn of Life trail, Pilbara Western Australia, dated to 3.46 G [1]. temperatures of up to 120° C [4] and depths below 3 km [5], on the oceanic floor or close to hydrothermal vents [6]. They absorbed near-infrared rather than visible light, reduced the carbon dioxide of the early atmosphere and produced sulfur or sulfate compounds (sulfur bacteria). Oxygenic photosynthesis probably began with cyanobacteria. Recently, it has been suggested [7] that the first photosynthetic organisms which used oxygen occurred on Earth about 1 Ga earlier than previously thought, i.e. about 3.46 Ga ago [1]. This data is consistent with isotopic and paleontological observations. The biospheric enrichment in oxygen allowed more complex life-forms to evolve. Different photosynthetic species appeared, such as algae and higher plants. The oxygen present in the atmosphere permitted the development of heterotrophic organisms, using oxygen molecules as a substrate in their respiratory process. 5.

(11) which provided them with energy. In order to build their biomass, they needed organic compounds produced during photosynthesis.. 1.1.2 The process of photosynthesis Photosynthesis is a two phase process. During the light phase, sunlight energy is transformed into a chemical free energy and later during the dark phase (called the CalvinBensen cycle), carbon dioxide assimilation and synthesis of carbohydrates occur. In the light reactions, light energy excites photosynthetic pigments to higher energy levels and this energy drives charge separation which results in the electron transfer and proton gradient formation necessary for the production of nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP). In the dark reactions high energy phosphoric anhydride bonds of ATP and the reducing power of NADPH is used to fix and reduce CO2. During the Calvin cycle, carbohydrates (monosaccharide such as fructose and glucose and polysaccharides) are produced. Carbohydrates are stable structures which can be stored by photosynthetic organisms. A general equation of photosynthesis is given by the simplified formulae:. 2n CO2 + 2n DH2 + light. 2 (CH2O)n + 2n DO. (1). where n is a positive integer.. For oxygenic photosynthesis, using water as a source of electrons, this equation is:. 6 CO2 + 12 H2O → C6 H12 O6 + 6 O2 + 6 H2O. a. (2). b. Fig. 2. a) Phytoplancton microscopic cells [8] b) Marine brown algea, Kelp [8]. 6.

(12) Oxygen is released as a waste product of oxygenic photosynthesis, when water serves as a donor of electrons and protons in the initial step of the photosynthetic process. The total photosynthetic activity in the biosphere consumes 0.1 % of the total sunlight energy reaching the surface of the Earth. It is estimated that 200 billion tons of biomass are produced by photosynthetic organisms and 10 % of the total atmospheric CO2 is fixed per year. The photosynthetic process results in the production of carbohydrates, lipids and proteins which are the main source of food for all living creatures [9]. It is estimated that marine organisms produce between 70 % and 80 % percent of the oxygen in the atmosphere. Nearly all marine “plants” are single celled photosynthetic algae [8].. 1.1.3 Photosynthetic bacteria The capacity of driving photosynthesis is often attributed to plants but most algae and some bacteria are also photoautotroph, which means that they can feed themselves by driving photosynthesis. A number of photosynthetic bacteria are known to exist. They can be differentiated according to their reaction centers activated by light.. Fig. 3. Distribution of photosynthetic reaction centre complexes among photosynthetic organism [10].. Bacterial reaction centers (BRC) occur in two types, called type I and type II [11, 12]. Anoxygenic phototrophs possess just one type, either type I or II, while all oxygenic photoautotrophs (cyanobacteria), such as higher plants and algae, possess both types. The. 7.

(13) primary distinguishing feature of the two types of BRCs are primary and secondary electron acceptors. In BRC of type I the iron-sulfur complex (FeS) serves as an electron acceptor whereas in type II quinone molecules serve the same function. For example, sulfur bacteria contain BRCs of type I and non sulfur bacteria, BRCs of type II. In bacterial cells, photosynthesis takes place in the membranes of their inner organelles, called chromatophores. In higher plants and algae, the light and dark reactions of photosynthesis occur in the thylakoid membranes of the chloroplasts.. Sulfur bacteria Another group of photosynthetic bacteria are anoxygenic bacteria. Green and purple sulfur bacteria use carbon dioxide (CO2) and an inorganic sulfur compound, for example hydrogen sulfide (H2S), as the electron donor to produce carbohydrates. In contrast to higher plants, algae and cyanobacteria, the sulfur anaerobic photosynthetic bacteria (Fig. 3) have just one photosystem similar to PS-I. These bacteria probably represent the most ancient photosynthetic microbe.. a. b. c. Fig. 4. a) Green sulfur bacteria – hot spring at Yosemite National Park (USA) [w1] b) Halophilic purple sulfur bacteria [w2] c) Cyanobacteria filamentous colonies [w3]. Non sulfur bacteria Green and purple non-sulfur bacteria (Fig. 4) can grow as: (i). photoautotrophs, (these use light as a source of energy and carbon dioxide to produce. carbohydrates during the photosynthetic process; (ii). photoheterotrophs (these use light as a source of energy, but they cannot use carbon. dioxide as a carbon source. In this case, organic compounds as succinate or malate serve as electron donors);. 8.

(14) (iii). chemoheterotrophs (these do not use light but use oxygenic respiration and obtain. energy from the oxidation of organic compounds). These organisms can switch from one mode to another depending on growth conditions, especially under the partial pressure of oxygen, on the availability of a carbon source (CO2 for autotrophic growth and organic compounds for heterotrophic growth) and of light (needed for phototrophic growth).. Cyanobacteria Cyanobacteria are oxygenic bacteria (Fig. 3), they are the predominant photosynthetic organism in anaerobic fresh and marine water. Like higher plants, they have two photosystems (PS-I and PS-II), and the waste product of photosynthesis is oxygen (Eq. 2). It is believed that cyanobacteria converted the early reduced Earth’s atmosphere into an oxidized one 2.4 – 3.5 billion years ago. The presence of O2 in the atmosphere had an influence on the evolution of living organisms on Earth by stimulating biodiversity and leading to the development of heterotrophic organisms. At the same time, the increased concentration of oxygen probably caused the extinction of some oxygen-intolerant organisms [3, 13, 14, 15].. Subject of studies Within the frame of this work, Rhodobacter sphaeroides (Rb. sphaeroides) was studied (Fig. 5). It is one of the most often studied species of purple nonsulfur bacteria. It is a gram-negative bacterium of the Proteobacteria group, which can be found in stagnant water and deep lakes. The cell dimension is about 15 µm length and 7 µm width. This bacterium is a model system for studying structural and functional role of photosystems of type II. Moreover, it has genetic accessibility, its production. is. incredibly. efficient. and. photosynthetic apparatus is relatively simple.. its. Fig. 5. Purple non-sulfur bacteria: Rhodobacter sphaeroides [w4]. 9.

(15) 1.2 Photosynthesis in bacterial reaction centers of type II 1.2.1 Bacterial reaction center complex The bacterial photosynthetic system is a membrane bound protein-lipid-pigment complex. It is composed of the core reaction center (BRC), light-harvesting complexes (LHC), the cytochrome (cyt.) bc1 complex and ATP-Synthase (Fig. 6). The inner antenna (the Periplasmic side. a. b. c. Cytoplasmic side. Fig. 6. a) Chromatophores visible in the cytoplasm of bacterial cells [w5]. b) Structural model of a chromatophore vesicle from Rb. sphaeroides containing LHC2 antenna (green), LHC1-RC dimers (red, blue), cyt. bc1 (purple) and ATP Synthase (orange). Adapted from [w6]. c) Light energy absorbed by BChls (green squares) transferred (arrows) to BRC where it is used to drive an electron transfer. Adapted from [w6]. core antenna) is called LHC1 and is closely associated with BRC. It is present in all species [16, 17] and it is synthesized in a fixed stoichiometric ratio one LHC1 per one BRC. The aim of the peripheral antenna LHC2 is to increase the cross-section of the photosynthetic unit. Its abundance increases under low light intensities [18]. The structures of LHC1 and LHC2 are similar. They are oligomers of low molecular weight, hydrophobic apoproteins (called α and β) that non-covalently bind bacteriochlorophylls a (BChls a) and carotenoids. In purple non-sulfur bacteria, bacteriochlorophylls (BChls) are the main pigments and they are related to chlorophylls occurring in plants, algae, and cyanobacteria. BChls use light at longer wavelengths, in the near infrared (IR) region, and lower wavelengths in the ultraviolet (UV) region, both of which are not absorbed by plants or cyanobacteria. The position of the absorption maximum depends on the type of bacteriochlorophyll and its protein. 10.

(16) environment.In Rb. sphaeroides the main light harvesting pigment is bacteriochlorophyll a, (BChla), (Fig. 7 a).. a. BChl a C55H74N4O6Mg. Qx band band. Qy band. b. Fig. 7. a) A porphyrin-like molecule: bacteriochlorophyll a (BChla) with an asymmetric conjugated double bond system. This results in two characteristics absorption bands, Qx and Qy (two transition dipoles) [19]. b) Absorption spectra of chlorophylls a and b and bacteriochlorophylls a and b. BChl a have three principal absorbance bands (Fig. 7 b), the Soret band in the blue/near - UV region (~ 300 - 400 nm), the Qx band in the yellow/orange region (~ 600 nm), and the Qy band in the near-infrared. Adapted from [20].. 1.2.2 Bacterial reaction center core of type II Knowledge of the structure of BRC comes from the crystallization of bacterial reaction centers and permits one to get an insight into the structure of the protein. The newest structure of the photosynthetic reaction center of wild type bacterium Rhodobacter sphaeroides at 2.65 Å resolution were determined in 2010 [21] (PDB code: 3I4D). The core. 11.

(17) of bacterial reaction center of type II of Rb. sphaeroides is composed of three polypeptides L, M and H of molecular weight 31 kDa, 34 kDa and 28 kDa respectively. The H subunit is located on the cytoplasmic side, whereas the L and M subunits are bound in the membrane. Organic and inorganic cofactors participating in light driven reactions are bound to this core proteins: four bacteriochlorophylls (BChl), two bacteriopheophytins (BPheo), two ubiquinones (QA and QB), carotenoids and a non-heme iron (NHFe) (see Fig. 8 a and 8 b) [22, 23, 24]. They are arranged along two branches, A and B, with near C2 symmetry [25], (see Fig. 8 b).. a. b. b. L M. L BPheo. H. BPheo. BChl BChl. H. Fig. 8. a) Bacterial reaction center. Three polypeptides L, M and H are represented by different colors (H subunit - thin blue line, M subunit – blue cylinders representing α-helices and L– yellow cylinders representing α-helices). Redox active cofactors are also indicated. Adapted from [w7]. b) Arrangement of BRC cofactors. The non-heme iron (NHFe) atom is marked by an orange sphere, ubiquinones* (QA and QB) are blue, bacteriopheophytins (BPheo)** are pink, bacteriochlorophyll (BChl) monomers are green, the (BChla) dimer (P870) consisting of PA and PB are yellow. The co-factors are arranged in two membrane-spanning branches around an axis of two-fold pseudo-symmetry. P870 is located close to the C2 symmetry axis. QA ubiquinone is located 25 Å away from the special pair. Only one branch is active which is called A – active branch [w8]. This Figure was constructed using PDB file 3I4D. * Ubiquinones are also known as coenzymes UQ10 **BPheo contains two H+ ions bound inside the tetrapyrole ring instead of Mg2+ ion as it is in BChl.. 12.

(18) BChl and BPheo, light active cofactors bound in the BRC, have a characteristic absorption spectrum, which is shown in Fig. 9.. Buffer solution: TRIS HCl pH8 ; 0,1% LDAO, 40mM Imidazole Reaction center WT. Absorption [a.u.]. 1,0. Qy band. 0,8. Qx band. BChl. 0,6. BPheo. 0,4. BPheo. Dimers. BChl. 0,2. 85K 0,0. 300 400 500 600 700 800 900 1000. Wavelength [nm]. Fig. 9. Absorption spectrum measured at 85 K of the bacterial reaction center isolated from Rb. sphaeroides. The band at 537 nm corresponds to the Qx transition of bacteriopheophytin (BPheo), at 590 nm to the Qx transition of the accessory bacteriochlorophyll a (BChla), at 780 nm to the Qy transition of BPheo, at 800 nm to the Qy transition of the accessory bacteriochlorophylls and finally at 865 nm to the low-energy band of the Qy transition of the bacteriochlorophyll dimer (P870). In the 450 - 550 nm region contribution of carotenoids the absorption spectrum are visible. Example spectrum obtained for WT BRCs.. The primary ubiquinone acceptor at the QA binding site is connected via the non-heme iron atom with a secondary ubiquinone acceptor at the QB site. The QA – Fe – QB complex is called the quinone-iron complex. The ubiquinone at the QA site is tightly bound, contrary to the one at the QB site. The QA ubiquinone, bound to the M subunit, is in a hydrophobic area whereas the QB ubiquinone, bound to the L subunit, is surrounded by charged and acid residues. Contrary to QA, this ubiquinone is exchangeable. QB is bound at the level of the lipid headgroups at the cytoplasmic side of the membrane and has no direct contact with the aqueous environment. Protons are delivered from the cytoplasm to QB by one or more pathways composed of interdependent hydrogen-bond networks involving titratable residues and water molecules [26 – 32]. The ubiquinone molecule is presented in Fig. 10.. 13.

(19) Fig. 10. Ubiquinone UQ10, which is natural quionone acceptor occurring in BRC of Rb. sphaeroides. 10. 1.2.3 Light reactions Light reaction begins when energy is captured by pigment molecules in the light harvesting antenna of the photosystem, producing a singlet excited electronic state of the absorbing pigment (see Fig. 11).. Fig. 11. Energy-kinetic diagram for reaction centers from purple photosynthetic bacteria. The path of linear electron flow within BRC is shown by lines [34].. The excitation reaches an energy trap called a special pair (P870). The excited special pair of chlorophylls (P870)* is a powerful reductant able to donate an electron in less than 100 ps. Charge separation, with a quantum yield near unity [33] then initiates a series of electron transfer reactions that are coupled to the translocation of protons across the membrane, generating an electrochemical proton gradient (proton-motive force) that is used for the synthesis of ATP (see Fig. 12).. 14.

(20) Fig. 12. The quinone, electron and proton transfer within BRC and cytochrome bc1 [w8]. The first step of an electron transfer occurs between the excited dimer P870* and BChla. Then electron (e-) is transferred to BPheo within 2 – 3 ps and subsequently, to the primary quinone acceptor bound at the QA site. This step lasts 200 ps and a reduced ubiquinone (semiquinone QA●-) is formed [27, 35, 36]. The electron flow from QA to a secondary quinone acceptor located at the QB site takes about 100 – 1000 µs depending on the reduced state of the QB quinone, which can accept two electrons. After one electron reduction it forms a semiquinone (QB●-) [37, 38]. These fast electron transfers result in a charge separation. During a few tens of microseconds following light excitation, the photooxidized primary electron donor (P870●+), located near the periplasmic face of the membrane, is reduced by a water-soluble cytochrome intermediate, oxydoreductase, called cytochrome c2. A second light-induced charge separation causes a double reduction in the QB ubiquinone on the acceptor side of BRC. On the donor side the second cytochrome becomes oxidized as the dimer (P870●+) becomes reduced. This linear forward electron flow occurs in the case of a non damaged BRC. Under stress conditions, the contribution of back reactions increases. The doubly reduced QB●2- picks up two protons from the cytoplasmic space, forming an ubichinol (QH2). QH2 quits its binding pocket, diffusing through the chromatophore membrane to the cytochrome bc1 complex. [39, 40]. QH2 dissociates, delivering electrons and protons to the bc1 complex [36, 41]. The oxidation of the monomers of QH2 at the Qo and Qi site results in the reduction of two cyt. c2. Electrons from the cytochrome bc1 – complex, transferred via cyt. c2 reduce the NADP+ to NADPH and protons are pumped on the. 15.

(21) periplasmic side of the membrane. This produces an electrochemical proton gradient (pH gradient) across the cellular membrane and represents stored free energy, used to drive ATP synthesis [42 – 44]. On the donor side of BRC, electrons are extracted from an organic donor. In the case of Rb. sphaeroides this donor is malate (HO2CCH2CHOHCO2H). After the reduction of NADP+ to NADPH and the generation of ATP from ADP by phosphorylation, “dark reaction” can began.. 1.2.4 The non-heme iron in the quinone-iron complex Non-heme iron in the QA – Fe – QB complex is a very important and mysterious component of the photosynthetic reaction centers of type II. It has been preserved for 3.5 billion years from primitive photosynthetic bacterial organisms to the photosynthetic apparatus of higher plants. The molecular mechanisms of the electron transfer between QA and QB quinones have been the subject of challenging studies but the role of NHFe in the stabilization of the QA and QB binding sites and in the primary electron transfer (ET) is not yet understood, especially its role in the photosynthetic charge separation process. The temperature activation of electron transfer between QA and QB quinones remains unclear [45, 46, 47]. Indeed, electron transfer is only activated above 160 K, when fast collective motions of core proteins are activated [48 – 51]. The non-heme iron is always observed in a ferrous state (Fe2+) and a change in its valence state has never been detected in any system of type II [52], which excludes the direct participation of NHFe in electron transfer within the ironquinone complex. However, it seems that a spin state of NHFe could play an important role in the stabilization of the iron-quinone complex. NHFe usually occurs in a high spin ferrous state [53] but its mixed spin states (low and high spin state) were observed in BRCs of Rb. sphaeroides and of Rhodospirillum rubrum [54, 55] and an exclusively low spin state in photosystem II of algae PSI− mutant [56, 57]. It was observed that the NHFe spin state plays a crucial role in the flexibility of the BRC core proteins [54, 55, 58]. The iron high spin state is a signature of a weak ligand field, which means increased flexibility of the protein matrix in its vicinity, whereas the low spin state indicates a high ligand field, and therefore a diminished flexibility in its surrounding (an increased rigidity).. 16.

(22) Fig. 13. The quinone iron complex and the non-heme iron ligands. The X-ray studies show that in BRCs isolated from Rb.sphaeroides, the non-heme iron (NHFe) is hexacoordinated and its ligands form a distorted-octahedral environment [59, 60]. It is coordinated by four nitrogen atoms of the imidazole ring - the functional group of histidine residues. Two histidines belong to the L subunit (L190 and L230) and two other to the M subunit (M219 and M266). Two bonds are provided by two oxygen atoms from the M234 glutamine amino acid. The average ligand-Fe distances are 2. 14 ± 0. 2 Å [61].. It has been proved that the function of a protein depends not only on its structure by also on its flexibility [62 – 65]. Theoretical calculations have shown that the flexibility of the quinone-iron complex [QA – Fe – QB] has an influence on the efficiency of the ET and proton kinetics on the acceptor side of BRCs [66]. Thus the iron atom can be a local sensor of the rigidity of the NHFe direct bonds as well as of the flexibility of the protein matrix in its vicinity. In order to gain insight into the molecular mechanism governing the spin stabilization of NHFe and the local flexibility in its neighborhood, bacterial reaction centers with point mutations were studied.. 17.

(23) Mutated bacterial reaction centers Two acidic residues situated near the QB acidic cluster, on the L protein: L212Glu and L213Asp, located within 5 – 6 Å of QB, were shown to be important in the delivery of protons to the QB quinone [67 – 70]. The removal of hydrogen bonds formed by L212 and L213 residues in BRC of Rb. sphaeroides, resulted in a reduction of 1000 times in the proton transfer to QB [71, 72]. In the case of Rb. capsulatus, BRCs carrying those two mutations called “AA”, the native pathways for the protons transfer to QB, which can be reduced by only one electron, are interrupted [70, 73]. In addition, the semiquinone QB●- state is extremely stable. At pH 8.0 its lifetime is about 12 s, i.e. 10 – times longer than that detected for the wild type (WT) BRCs, 1.2 s [73]. Since the photocycle cannot be completed, the AA strain of Rb. capsulatus is incapable of growing under photosynthetic conditions. X-ray diffraction studies of the AA mutant in Rb. sphaeroides BRC show that the replacement of L212Glu and L213Asp by alanines has unexpected structural consequences. The AA structure revealed side chain rearrangements and showed movement of the main chain segments that are contiguous with the mutation sites. The alanine substitutions cause an expansion of the cavity rather than its collapse. In addition, QB was found mainly in the binding site that is proximal to the iron-ligand complex (and closer to QA) as opposed to its more occupied distal binding site (further from QA) in the structure of the wild-type reaction center [73]. The mutant AATyr, in addition to the two previously described point mutations, carries a third point mutation near the QA binding site. The M249Ala amino acid was exchanged for a tyrosine. In Rb. sphaeroides this additional mutation partially restores the kinetics of proton uptake by QB●-. This kinetic is still delayed but it is two times faster than in the case of the double mutant AA, which allows the triple mutant to grow under photosynthetic conditions. Applying neutron inelastic scattering in dynamical studies of BRCs isolated from the double mutant AA and the triple mutant AATyr showed that their protein cores are more flexible than wild type BRCs [74].. 18.

(24) 1.2.5 Copper ions action on photosystems of type II Copper is known to be an essential microelement for photosynthetic systems [75, 76] and a cofactor in various biological systems. For example, it is an active center in plastocyanin discovered by Katoh [77], which is a mediator between the b6f complex (analogous to bc1 in BRC) and PSI. At stoichiometric concentrations, close to 1 Cu2+/ BRC, a stimulatory effect of copper on oxygen evolution due to its action on the donor side of PSII was observed [78]. However, at higher concentrations it was found that among a variety of heavy metals, Cu2+ has the highest toxicity in photosystems of type II [79, 80]. It was especially shown that it affects the acceptor side of reaction centers [80]. It was suggested that in such systems, copper bounds in the vinicity of the QA – Fe – QB complex, affecting interactions between both quinone acceptors and NHFe [81 – 83] as well as the Pheo – QA – Fe region [82]. It is believed that Cu2+ ions replace NHFe from its binding side by its substitution [84 – 86]. However, Mössbauer experiments have shown that copper at concentrations < 1000 Cu2+/ BRC causes transition of the high spin ferrous state of the non-heme iron into a low spin one (diamagnetic) in PSII and in BRCs of type II [56]. Copper action on photosystems of type II can be very complex because it is known to be a protonophore. Copper cations Cu2+ may modify protonation and deprotonation mechanisms activated in photosynthetic systems. For example, copper ions were shown to impair the photosynthetic electron transport between pheophytin and QA in photosynthetic centers of bacteria and higher plants [87]. In BRCs isolated from Rb. sphaeroides and other species of purple bacteria, the electrochromic response of the bacteriopheophytin cofactors associated with QA-QB. QAQB- electron. transfer is slowed down in the presence of Cu2+. Moreover, Cu(II) binding sites are located on various sites of the acceptor side of a photosystem of type II [88 – 91]. Additionally, a copper binding side is located on the H protein of the BRC [92]. It is still unclear how Cu2+ influences the NHFe binding site and the proton and electron transport on the acceptor side of photosynthetic RCs of type II, that is why, one of the aims of this work was to investigate the influence of copper on NHFe properties in BRCs of high purity.. 19.

(25) 2. MATERIALS 2.1 Growth of bacteria The purple bacteria, Rb. sphaeroides cultures were spread on plates filled with the growth medium and agar. Selected colonies were grown in darkness on a gyratory shaker (100 rmp) at 30° C in 50 mL flask. The malate-yeast medium (Tab. 1) was supplemented with antibiotics: kanamycin (20 µg/mL) and tetracycline (1.25 µg/ mL). Tab. 1. Malate-yeast medium enriched with iron 57Fe. GROWTH MEDIUM (pH 8.8). 1L. Base solution. 20 mL. Metal solution. 1 mL. Phosphate buffer. 20 mL. Ammonium malate with KOH. 30 mL. N-Z Amine A. 1g. Yeast extract. 1g. BASE SOLUTION. 1L. Nitrilotriacetic acid. 10 g. MgSO4 7H20. 14.45 g. CaCl2 H20. 3.33 g. (NH4) 6MO7 O244 H20 Ammonium heptamolybdate. 9.25 mg. Nicotinic acid. 50 mg. Thiamine. 25 mg. Biotin. 0.5 mg. 20.

(26) METAL SOLUTION. 100 ml. EDTA. 0.25 g. ZnSO4. 1.095 g. CuSO4. 25.8 mg. NaB407. 17.7 mg. MnSO4. 0.154 g. Co(NO3)2. 24.8 mg. 57. FeSO4. 0.7 g. PHOSPHATE BUFFER 1M pH 7 0,6 L Mono K (PO4 H2 K) + 1 L Di K (PO4 HK2 3H2O). The growth medium was deprived of natural iron and enriched in the iron isotope, 57Fe. After 48 hours, the bacteria were transferred to 2 L flask, filled with 1.2 L of medium. This procedure was repeated the next 48 hours. Then after 3 days, the cells were harvested by centrifugation at 5000 g, 10 min. The average amount of bacteria collected from eleven flasks was about 50 g.. 2.2 Purification of bacterial reaction centers Cells from the His-tagged strain of Rb. sphaeroides (see chapter 2.4) were disrupted by sonication in a 10 mM Tris HCl (pH 8.0) and 100 mM NaCl buffer. The resulting solution was centrifuged 10 min at 10000 g. Membrane solubilization was done by adding lauryldimethylamine N-oxide (LDAO; Fluka) to a final concentration of 0.8 % in dark conditions with the presence of 8 mM imidazole. After ultracentrifugation (40 000 rpm for 75 min), BRCs were incubated in 40 ml of Ni-Superflow pre-equilibrated with a 10 mM Tris HCl pH 8.0 and 0.1 % LDAO buffer. The solubilized BRCs were purified on a nickel affinity. 21.

(27) column and eluted with a buffer containing 10 mM Tris HCl pH 8.0, 0.1 % LDAO, 40 mM imidazole. The bacterial reaction centers were concentrated using a micro-concentrator (Vivaspin, MWCO 30 kDa).. The concentration of isolated BRCs was evaluated from absorbance measurements at 802 nm, using the following formula according to [91]:. C=. Abs802 nm ⋅ 100 [µM ] 288. (3). where Abs802 is the absorption at 802 nm.. The yield of obtained bacterial reaction centers are presented in Tab. 2.. Tab. 2. The yield of isolated bacterial reaction centers from subsequent cultures of bacteria. Sample. WT*. AA**. AATyr**. dFer**. Total. 108. 60. 84. 48. 300. Bacterial cell mass [g]. 273.3. 161.7. 261. 40. 736. Obtained BRCs [nmol]. 1806. 1722. 1486. 600. 5614. Volume of growth medium enriched with 57Fe [L]. *WT-wild type , ** different mutations explained in section 2.4.. 2.3 Mutants description To construct His-tagged BRCs, a sequence coding for 7 histidines was added at the 3′ terminus of the M subunit gene. This C-terminal extension facilitates a rapid and efficient recovery of purified BRCs using immobilized metal affinity chromatography [92]. The plasmid used was PRK404, carrying pufQBALMX [93]. Site-specific mutants of the Rb.. sphaeroides BRC were constructed via standard protocols [94], in the Laboratoire de Chimie Physique in Orsay, France and Argonne National Laboratory, USA [95].. 22.

(28) Mutations on the outer side of the iron-quinone complex. - Two single mutants L212Glu/Ala and L213Asp/Ala, later referred to as L212 and L213 respectively, have a single, point mutation near the QB binding site. The glutamic acid and aspartic acid respectively were replaced by neutral, non functional and small alanines. These acidic residues are located about 5 - 6 Å from the head of the quinone bound at the QB site. The structure of the L212 mutant was resolved by X-ray diffraction with a resolution of 3.10 Å [96].. - The double mutant later referred to as (AA) have two previously described mutations. (L212Glu/L213Asp. Ala/Ala), (Fig. 14). Its structure was obtained with a resolution of. 3.10 Å using X-ray crystallography [97].. QB Ala. NHFe. QA. Ala. L213Asp L212Glu. Fig. 14. The „AA” mutant. L213Asp and L212Glu amino acids was replaced by alanines. Structure obtained from 1K6N.. - The triple mutant (L212Glu/L213Asp. Ala/Ala) + (M249Ala. AATyr have a double mutation (L212Glu/L213Asp. Tyr), later referred to as. Ala/Ala) near the QB binding site and. additionally a mutation near the head of the quinone bound at the QA site (M249Ala. Tyr).. The neutral aliphatic alanine on the M protein was exchanged for an aromatic and polar. 23.

(29) tyrosine. The M249Ala distance from the QB binding site is about 18 Å. The crystal structure of AATyr was resolved by X-ray diffraction (Fig. 15) [98].. QB. NHFe. QA M249Ala. Ala. Ala. Tyr. L213Asp L212Glu. Fig. 15. The „AATyr” mutant containing L213Asp and L212Glu amino acids was replaced by alanines near the QB binding site and additionally M249Ala exchanged by tyrosines near the QA binding site.. Mutations on the outer side of the iron-quinone complex. - These mutants have point mutations near the NHFe region on the inner side of BRCs. Phenylalanine or leucine were exchanged for neutral alanines (L187Phe Ala and M216Leu. Ala). L187Leu and M216Phe are situated close to each other , with histidines as. the direct ligands of the non-heme iron. L187Leu and M216Phe are located about 7 Å and 9 Å from the non-heme iron respectively. The mutations attempt to "break" some van der Waals contacts, replacing them with short alanines instead, and/or to develop different bonds. Theoretical calculations of Brownian dynamics have shown that L187Leu and M216Phe residues are in the most rigid region of the L/M protein complex [99].. - The double mutant later referred to as dFer contains both mutations described above: L187Phe/M216Leu. Ala/Ala (Fig.16).. 24.

(30) Ala M216Leu L187Phe. QB. Ala NHFe. QA. Fig. 16. The „dFer” mutant has two point mutations in the inner part of BRC, near the non-heme iron ligands (in the second coordination sphere. Leucine and phenylalanine were replaced by neutral alanines (L187Phe/M216Leu Ala/Ala).. 2.4 The treatment of bacterial reaction centers with copper (II) ions Isolated wild type BRCs (0.3 mM) were incubated for 15 min at room temperature with a 800-fold molar excess of CuCl2 under illumination with white light and continuous stirring. Then, the unbound Cu2+ ions were removed by rinsing the sample several times with the suspension buffer (free of Cu2+). In the first step EDTA was added in order to remove the nonspecifically bound copper ions. Each time, the sample containing BRCs – Cu was stirred for 10 min in the Tris buffer at ambient temperature and then collected by ultracentrifugation. This procedure was repeated 4 times before the sample was stored at –80o C.. 25.

(31) 3. METHODS 3.1 Mössbauer spectroscopy Mössbauer spectroscopy is the recoil-free resonant emission and absorption of γ-ray by a nucleus. This technique probes transitions between the ground and excited states of the nucleus. From the momentum and energy conservation, the recoil energy ER of a system with a mass m, due to the emission or absorption of a photon which has an energy equal to Eγ is equal to:. ER =. Eγ 1 2 p2 p 2c2 mv = = = 2 2 2m 2mc 2mc 2. (4). where c is the velocity of light. For example, for an isolated iron atom, recoil energy is equal to 2 ⋅10 −3 eV which is six orders of magnitude higher than the natural linewidth ( Γ ) for the iron isotope 57Fe, being the most popular probing atom used in Mössbauer spectroscopy.. Γnat =. h. τN. ≈ 4,67 ⋅ 10 −9 eV. (5). Rudolf Ludwig Mössbauer showed that when a nucleus is bound in a solid lattice, there is a probability that the recoil energy can be transferred to the lattice vibrations. If the recoil energy is smaller than the lattice vibrational quanta, then the lattice as a whole absorbs the recoil. The recoil energy becomes then negligible. For this discovery, he received the Nobel prize in physics in 1961 [100]. It is then necessary to have an atom bound in a lattice. The mass of a single atom becomes the mass of the whole matrix (Eq. 4), which is extremely big in comparison to the mass of a free atom. In this way the recoil energy becomes negligible and the Mössbauer effect can be observed. To get a better overlapping of the resonant absorption, a Doppler effect of the first order is applied. The relative velocity between source and absorber is approximately a few mm/s in this case. The probability of the recoil free emission or absorption is determined by the Lamb-Mössbauer factor (f). Recoil effect can arise in the solid if the energy of the gamma transition is enough high to excite lattice phonons. There is a. 26.

(32) finite probability for what is called the zero-phonon processes, in which no change of oscillatory transition takes place. The fraction of zero-phonon transitions is indicated by Lamb-Mössbauer factor (LMF), f. [101].. (. f (T ) = exp − k 2 < x 2 >. ). (6). where k is the wave vector equal to:. k=. 2π Eγ hc. =. 1 A -1 0,137. (7). for the Mössbauer transition of 14.4 keV in 57Fe and <x2> is the mean square amplitude of the oscillations of the resonant nucleus in the direction of the gamma rays propagation.. Using the Debye model the recoilless fraction, can be expressed by [102]:.   3E R f (T ) = exp −  2k Bθ D . where. x=.     2 θTD    x   T   1 + 4  θ  0∫ e x − 1dx     D      . (8). hν k BT , T is the absolute temperature, kB is the Boltzmann constant and θD is a. characteristic Debye temperature defined as: θ D =. hω D kB. (9). For a given resonant nuclear transition energy, the recoil-free fraction increases with the Debye temperature. This means that the Lamb-Mössbauer factor rapidly decreases above the characteristic temperature. The Lamb-Mössbauer factor f can be simplified for temperatures higher than Debye temperature:.  6E T  f = exp − R 2  , T > θ D  k Bθ D . (10). and for temperature lower than Debye temperature:. 27.

(33)  3E R   , T < θ D f = exp −  2k Bθ D . (11). From these equations, it is possible to discover information about the mean square displacement of the probing atom. According to the Debye model, at low temperatures (T < θD) the mean square displacement of the Mössbauer atom is a constant (Eq. 11) and at high temperatures (T > θD) should be proportional to the temperature T (Eq. 10). However in biological samples, deviation from linear behavior is observed. This is a result of anharmonic vibrations due to the lattice additional vibrations of a protein matrix [103]. These lattice vibrations are distinguished into fast and slow collective motions. Fast collective motions are related to the flexibility of the protein matrix in the vicinity of the probing atom. Slow collective motions are a result of the diffusion of a whole protein and they are observed only at high temperatures, i.e. close to 0o C. The total mean square displacement of the probe is then expressed:. < x 2 >= xv + x fc + xsc. (12). where. vs: vibrational modes vfc: fast collective motions vsc: slow collective motions (diffusional modes) The temperature dependent mean square displacement provides information regarding the local dynamical proprieties of the probing atom.. 3.1.1 Hyperfine parameters 3.1.1.1 Isomer shift The isomer shift is related to the monopole electrostatic interaction coming from a Coulomb interaction between a nuclear charge and valence electrons but theses only have a finite probability of being present in the nucleus. The radius of a nucleus is different depending on whether it is in a ground or excited state. In addition, because the surrounding of the probing. 28.

(34) atom is different at the source and absorber, the density of electrons in the absorber:. ∑ φ (0). 2. A. is different from their density at the source: ∑ S φ (0 ) . 2. For a given nucleus, isomer shift is then a measure of the difference between the electron density at the source and absorber nucleus:. (. )(. ). Ze 2 2 2 IS = E A − E S = φ A (0) − φ S (0) Re2 − R g2 = K {∑ A φ (0 )2 − ∑S φ (0)2 } 10ε 0 ε r. (13). where K is the constant characterizing a nucleus. The s-electron density inside the. 57. Fe nucleus is affected by the screening effects of. d‐electrons, and by covalency and bond formation, i.e. by the chemical bonding of the iron atom. Valence electron configurations for Fe2+ and Fe3+ are 4s23d6 and 4s23d5 respectively. The isomer shift is always a relative value and it is quoted to a known absorber. For example 57Fe Mössbauer spectra are usually quoted relative to alpha-iron. If the absorber and source are the same, no isomer shift will be observed and the center of the absorption line will be at the velocity of 0 mm/s (Fig. 20).. 3.1.1.2 Quadrupole splitting Quadrupole splitting comes from the interaction between an electric quadrupole moment of a nucleus, eQ, and a gradient of an electric field (Eq. 14). Any nucleus with a spin quantum number. I>. 1 2. has a non-spherical charge distribution and this is characterized by the. quadrupole term eQ. The quadrupole splitting is influenced by the configuration of valence electrons as well as the arrangement and type of ligands. In a chemically bonded atom, the electrostatic charge distribution is usually asymmetric and therefore the interaction between the nuclear quadrupole moment and the electric field gradient results in energy level splitting. For example the excited nuclear state of iron. 57. Fe. with a nuclear spin I=3/2 splits into two sublevels:. I=. 3 3 , ± 2 2. and I =. 3 1 , ± 2 2. whereas the ground level I =. 1 1 , ± 2 2. remains unsplit. (see Fig. 17).. 29.

(35) Fig. 17. The quadrupole splitting in 57Fe [w9]. Selection rules allow only transitions for I ze − I zg = 0, ±1 and thus the spectrum comprises two lines (Fig. 17). The energy separation QS between the two lines is equal to:. ∆ = QS =. e 2 qQ 2. (14). In this case quadrupole splitting (QS) is a difference between positions of two lines forming a doublet. Because eQ is a nuclear constant for a given Mössbauer probe, the quadrupole splitting is a function of eq, so it is a function of the chemical environment. The electric field gradient is a negative second derivative of the potential at the nucleus of the whole surrounding electric charge, in which the valence electrons of the iron atom and surrounding ions contribute. If one ignores the effects of spin-orbit coupling, one can make a general prognosis about the expected quadrupole interaction. In the high spin (HS) ferrous case, in addition to a spherically symmetric subshell, a single electron in the xy state is present. This valence electron provides an asymmetric charge distribution and will give rise to a large quadrupole interaction. In the low spin (LS) ferrous case the electrons completely fill the lower triplet, the charge distribution has cubic symmetry, and no quadrupole interaction with the nucleus will result from it. However, one may expect a small contribution from the remote charges of the ligands and the more distant atoms. QS can then give information about the charge symmetry around the nucleus and it is very sensitive to electron configuration, to the type and orientation of ligands of the probing atom [104].. 30.

(36) 3.1.1.3 Magnetic hyperfine splitting It is caused by a dipole magnetic interaction of a nucleus with a magnetic field. The source of magnetic field can be internal and/or external. The total magnetic field can be expressed by a sum of these two magnetic fields: Btot = Bint ernal + Bexternal. (15). where the internal magnetic field has three components: Bint ernal = Bcontact + Borbital + Bdipolar. (16). The three terms originate from the partially filled electron shells of the probing atom. Bcontact is due to the spin on those electrons. polarizing the spin density at the nucleus, Borbital is due to the orbital moment on those. electrons and Bdipolar is the dipolar field due to Fig. 18. The magnetic splitting of nuclear energy levels in 57Fe [w9]. the spin of those electrons. The nuclear level with a spin I ≥ ½ can be split into 2I+1 levels, characterized by the quantum number ml . The. splitting of the energy levels is called the nuclear Zeeman effect and is analogous to the Zeeman effect in atomic physics. In the case of. 57. Fe, the nuclear level with I = 1/2 splits into two sublevels and the. level with I = 3/2 into four sublevels. Transitions between the excited state and ground state can only occur when ml changes by 0 or ± 1, according to the selection rules. Therefore a sextet is observed in the Mössbauer spectrum as illustrated in Fig. 18. The line spacing is proportional to Btot . The separations between the levels are given: ∆ 0 = g 0 µ N H = µ0 H / I 0. (17). ∆1 = g1µ N H = µ1 H / I1. (18). 31.

(37) where µ N is the nuclear magneton and g0 and g1 are called the g-factors for the I=1/2 and I=3/2 levels, respectively. H is a magnetic field.. 3.1.2 Experimental setup In biology the most frequently used Mössbauer isotope is. 57. Fe, as the iron atom. naturally occurs in many physiologically important proteins, for example in erythrocytes, ferritins and cytochromes or in more complex systems like photosynthetic reaction centers of higher plants, algae and bacteria. In this work the non-heme iron present in bacterial reaction centers was investigated. The source of. 57. Fe* is a radioactive isotope. 57. Co. It decays via a. 57. spontaneous electron capture producing the excited state of Fe (Fig. 19), (Eq. 19).. 57 27. 7 I= 2. Co 837 keV t1/2=240 days. Electron capture 99.84 %. I=. 5 2. 136 keV 15 %. I=. 85 %. 3 2. 14.4 keV. t1/2=1.4 .10-7 s. 14.4keV 1 I= 2. 14.4 keV. 57 26. Fe. Fig. 19. The γ-decay scheme of 57Co showing the 14.41 keV Mossbauer transition. 57 Co decays by electron capture and initially populates the 136 keV nuclear level of 57Fe with nuclear spin quantum number I = 5/2. This excited state decays after about 10 ns and populates, with 85% probability the intermediate excited level by emitting 122 keV gamma quanta and with 15% probability the ground state of 57Fe by emitting 136 keV gamma quanta. The deactivation of the intermediate excited state of 57Fe ( I=3/2 ) deactivates to the ground by emitting gamma quanta of 14.4 keV energy, the most suitable radiation for the Mössbauer spectroscopy.. 32.

(38) 57 27. 57 57 Co→ 2757 Co ∗ + −10 e − → 26 Fe ∗ → 26 Fe + γ. In our measurements 50 mCi. 57. (19). Co in a rhodium matrix was a source of 14.4 keV radiation,. usually used in Mössbauer spectroscopy. The spectral line shape of the emitted gamma ray can be described by the Lorentzian curve:. I (E ) ≈. Γ2. 4(E transition − E γ ) + Γ 2 2. (20). where Γ is the linewidth and Eγ the resonance energy.. In Fig. 20, a typical scheme of Mössbauer experiment is presented. Our samples of isolated bacterial reaction centers were frozen and put in a homemade cryostat. A proportional counter was used as a detector. Measurements were performed through a wide range of temperatures from 80 K to 260 K.. Fig. 20. An experimental scheme of Mössbauer experiment [105].. The temperature was stabilized within 0,1 K. Software “Mosiek” written by Wacław Musiał was applied for collecting experimental data. The recorded spectra were fitted using a Recoil program [106]. For Mössbauer experiments, 900 nmol, 800 nmol, 1400 nmol and 400 nmol of WT, AA and AATyr and dFer high purity BRCs were used, respectively.. 33.

(39) Mössbauer spectroscopy applied in studies of the proprieties of the non-heme iron in BRCs isolated from His-tagged Rb. sphaeroides provides information about the valence and spin state of the iron atom as well as the type and arrangement of its ligands. The temperature dependent measurements allowed one to investigate the dynamical properties of NHFe and protein local motions on the acceptor side of BRCs.. 3.2 Synchrotron radiation Synchrotron radiation is an electromagnetic radiation which is emitted by charged particles moving with velocity close to the speed of light in vacuum. Radially accelerated particles emit X-ray radiation called synchrotron radiation, tangent to the orbit. This kind of radiation was first observed in 1947 from electrons orbiting in a synchro-cyclotron [107, 108].. Fig. 21. ESFR storage ring [w10]. At the European Synchrotron Radiation Facility (ESRF) in Grenoble, electrons are first accelerated in a linear accelerator (Linac) where they are packed in “bunches” and then accelerated to energies of 200 keV. They are then injected to the booster synchrotron. The booster synchrotron has a circumference of 300 m. After several tours, electrons reach an energy of 6.04 GeV. These high-energy electrons are then injected into a large storage ring of 844.4 m circumference where they circulate in an ultra high vacuum environment, for many hours. During circulation electrons experience radiation losses. New electrons are regularly re-injected into the ring to compensate the energy loss. Nuclear resonant forward scattering (NFS) and nuclear inelastic scattering (NIS) of synchrotron radiation are new techniques, developed in 90’s and shown to be powerful methods in investigation of biological complexes containing Mössbauer nuclei. The use of. 34.

(40) synchrotron radiation overcomes some of the limitations of the conventional Mössbauer technique and provides additional information unobtainable by other methods [109] (see sections 3.2.1 and 3.2.2).. 3.2.1 NFS: Nuclear forward scattering Nuclear forward scattering corresponds to an extended Mössbauer spectroscopy into the time-domain, as it uses the time structure of synchrotron radiation to excite nuclear transitions (for 57Fe it is 14.4 keV). Nuclear forward scattering uses short flashes (100 ps) of synchrotron light to detect the 57Fe gamma-decay of a lifetime τ57Fe = 141 ns transition. For an unsplit nuclear level, the decay of the gamma ray is a simple exponential decay, whereas when the nuclear levels are split, it leads to an interference between the respective energy levels. If two degenerated levels have the same energy and are excited simultaneously, they will have the same decay constant. The probability of decay at time t for two energy levels is given by: P (t ) = ψ 1 + ψ 2. P (t ) =. 2.  itω   itω   λt   λt  λ exp −  exp − 1  + λ exp −  exp − 2  2   2  2  2  . (21). 2. (22).  ∆ωt  P (t ) = 4λ exp(− λt ) ⋅ cos 2    2 . (23). where ∆ω = ω1 − ω 2 is the frequency difference, λ – the wavelength.. This equation exhibits an exponential decay modulated with periodical oscillations. Because the energy differences between the transitions are of µeV for. 57. Fe, the corresponding. oscillation periods in the time-spectra are of the order of ns. This periodical oscillations called ‘‘quantum beats’’ and they are characteristic for the energy difference between the interfering levels. The nuclear transition in 57Fe in the presence of the magnetic field can be split up into six lines which may interfere with each other according to selection rules.. 35.

(41) a. Without the magnetic field.. b. Time. With the presence of the magnetic field.. Time. Fig. 22. Time behaviour of the reemitted photons in forward direction. The time decay shows characteristic modulations, which are caused by multiple scattering in thick samples (dynamical beats) and hyperfine interactions (quantum beats). a) The nuclear forward scattering (NFS) spectra without the magnetic field. The energetically broad synchrotron radiation pulse excites the nucleus, the reemitted radiation in forward direction contains only γ -quanta of one energy. In this case, the excitation of a single nucleus results in a simple exponential decay (dotted line). In a real experiment, for a sample with a finite thickness, coherent multiple scattering occurs. The forward scattered intensity exhibits a characteristic time modulation, the so-called dynamical beats (full line) [110]. b) NFS spectra with the presence of magnetic field. The split excited nuclear levels are coherently excited. The forward reemitted radiation contains γ - quanta with different energies, which can interfere and lead to the quantum beats (dotted line). In a thick sample (full line) the quantum beat pattern is superimposed by dynamical beats. The corresponding envelope is shown by dotted lines. NFS experiment is sensitive to the nuclear environment so that time-differential spectra provide precise values of hyperfine frequencies and relative isomer shifts. In addition, it is an elastic and coherent scattering process. It means that it takes place without energy transfer to electronic or vibronic states and is delocalized over many nuclei. NFS allows a direct determination of the Lamb-Mössbauer factor from:. f LM =. t eff d ⋅ n ⋅σ 0. (24). where d is the thickness of the sample, n, the number of Mössbauer nuclei per unit volume. 36.

(42) and σ0, the nuclear absorption cross-section at resonance, t eff is the thickness of the sample [111, 112]. In addition, NFS which can be regarded as Mössbauer spectroscopy in the time domain, overcomes some limitations of conventional Mössbauer spectroscopy: 1. The high brilliance and the extremely collimated beam of synchrotron radiation leads to a large flux of photons. The consequence is a very small size of the sample (0.1 – 1mm2) that makes it possible to measure extremely small samples [109]. The required sample crosssection for the NFS experiment is 100 times smaller that in the case of Mössbauer spectroscopy. Additionally the data acquisition time in the NFS experiment [109] is at least one order of magnitude faster. This is especially important of the case of biological samples in which the concentration of the probing atoms very low. 2. Furthermore, in NFS method, many sources of distortions are absent compared to conventional Mössbauer spectroscopy. Indeed the time spectra are almost free of background noise. They are insensitive to mechanical vibrations and have a precise and stable time scale [113]. 3. Due to the linear polarization of synchrotron radiation, NFS is especially sensitive to the direction of magnetic hyperfine fields [110]. However, data analysis of NFS spectra is complex, as only visually controlled simulations are possible [109], using the Hamiltonian:. ^ 2. → ~ → 1 E ^2 ^2 H = D[ S z − S ( S + 1) + ( S x − S y ] + β e S ⋅ g ⋅ B ext 3 D. (25). where D is zero-field splitting, E/D the rhombicity and they describe the influence of the ligand field via electronic interaction, g describes the influence Zeeman interaction, →. S represents the effective spin operator, and β e is the Bohr magneton [114, 115]. In our case. we applied the SYNFOS program for evaluation of the obtained experimental data [118]. In Mössbauer spectroscopy data are fitted using least-squares methods. Fits are performed according to the Lorentzian function [see chapter 3.1.2].. 3.2.2 NIS: Nuclear inelastic scattering NIS extends the energy range of conventional Mössbauer spectroscopy (range 10−9 to 10−7 eV) to the range of molecular vibrations (10−3 to 10−1 eV). Since NIS is sensitive only to. 37.

(43) the mean-square displacement of Mössbauer nuclei it can be used as site-selective vibrational spectroscopy [109]. Conventional Mössbauer spectroscopy therefore gives only limited information (comprised in the Lamb-Mössbauer factor) on the dynamics of a given Mössbauer nucleus (see section 3.1), while NIS spectra provide detailed spectral information on its vibrational modes. The measured NIS spectra can be directly interpreted in terms of molecular. vibrations. since. no. electronic. properties. such. as. polarizability. or. hyperpolarizability influence the intensity of the spectrum. At the time when recoilless nuclear resonant scattering was discovered by Mössbauer it was postulated that the recoil fraction of scattering is determined by lattice dynamics. Conventional Mössbauer spectroscopy did not allow to measure phonon spectra. However, the continuous spectrum of synchrotron radiation, its high intensity and pulsed character make the synchrotron radiation extremely favorable to nuclear inelastic scattering (NIS). When the energy of incident X ray coincides exactly with the energy of the nuclear transition, a peak of elastic nuclear absorption occurs. Nuclear absorption may also proceed inelastically, with the creation or annihilation of lattice vibrations. This process causes an inelastic band in the energy spectra of nuclear absorption to form around the central elastic peak [117]. From the energy spectrum it is possible to calculate the density of phonon states (DOS) of atoms that possess a nuclear resonant level. The density of vibrational states DOS, is calculated from the inelastic part of the normalized NIS spectra after subtraction of the elastic contribution using the instrumental function measured in parallel and a procedure based on Lipkin's sum rule [118 – 120]. Data processing was performed using a double-Fourier transformation routine described in [121]. The normalized probability of inelastic nuclear absorption W(E) can be decomposed into multiphonon terms [122, 117]: ∞   W ( E ) = f LM  δ (E ) + ∑ S n (E ) n =1  . (26). where δ is the Dirac function, δ(E) is a zero-phonon term, describing the elastic part of the absorption, and S n (E ) represents the inelastic absorption accompanied by the creation or annihilation of n phonons. The one phonon term is given by:. 38.

(44) S1 ( E ) =. ER ⋅ g ( E ). (27).   E    E ⋅ 1 − exp −  k T  B  . and the n-term is the harmonic approximation by:. Sn (E) =. ∞. ( ) (. ). 1 S1 E ' S n−1 E − E ' dE ' ∫ n −∞. where E R =. (28). h 2k 2 is the recoil energy of a free nucleus, m is the mass of the atom and k is the 2m. wave vector of the X-ray quantum. The g(E) function is a normalized partial DOS, which assumes an averaging over all crystallographic directions:. g ( E ) = V0. 1 (2π ) 3. →. ∑ ∫ d qδ ≥  E − hω j. j. →  ( q ) . (29). where V0 is the volume of the unit cell, the index j enumerates the branches of the dispersion →. →. relation hω j ( q ) , q is the phonon momentum, and the integral is taken in the first Brillouin zone. The detailed theory on NIS has been described elsewhere [123].. 3.2.3 Experimental setup A typical experimental set-up of NIS and NFS is presented in Fig. 24. Nuclear inelastic and forward scattering spectroscopy of synchrotron radiation measurements were performed at the nuclear resonance beamline ID18 of ESRF, in Grenoble (France). The storage ring was run in a 16-bunch mode, providing pulses of radiation energy every 176 ns. The average ring current was about 90 mA. The energy resolution was 0.5 meV. The energy of radiation was tuned in a range from –20 meV to 80 meV around the transition energy of 57. Fe (14.413 keV). The obtain spectra were collected over 24 hours.. 39.

(45) Fig. 23. Synchrotron radiation beam is produced by an undulator inserted into the storage ring. The beam is monochromatized in two stages. In the first stage, the bandwidth is reduced down to a few eV by a high-heat-load monochromator. Then, the bandwidth is reduced down to a few meV by a high-resolution monochromator [117]. A narrow energy bandpass is achieved with high-order reflection, which provides large energy–angle dispersion and has small angular acceptance. After the high-resolution monochromator, the beam passes through an ionization chamber, which monitors the flux of incident radiation, irradiates a sample and excites the resonant nuclei. Scattered radiation is counted by two avalanche photodiode (APD) detectors. The first detector is located close to the sample and counts the quanta scattered in a large solid angle. The second detector is located far away from the sample and counts the quanta scattered by the nuclei in the forward direction. The first detector monitors the nuclear inelastic scattering (NIS), and the second detector, the nuclear forward scattering (NFS) [117]. Since this is an elastic process, the data from the second detector also provide, at one and the same time the instrumental function of the spectrometer. The instrumental function of the nuclear inelastic spectrometer does not vary with energy because of the small relative energy transfer ( < 10-5)[117]. In order to distinguish the products of nuclear interaction from alternative channels of electronic absorption or scattering, the readings of both detectors are gated in time, and data is taken only between the pulses of synchrotron radiation.. 3.3 Fluorescence decay analysis Fluorescence lifetimes were determined by the simulation using a non-linear leastsquare method based on the Marquardt algorithm [124, 125]. As a profile of the excitation light, scattered light from an identical sample in the same geometry for the measurements was monitored through one or more appropriate filter(s) to avoid damage to the photomultiplier. The experimental set-up is presented in Fig. 24. Each decay curve was simulated by a multiexponential function:. n. f (t ) = ∑ An exp( − n =1. t. τn. ). (30). 40.

(46) where τ n is a time constant. As criteria of the best fit, χ2 and the Durbin-Watson parameter [127] were used. Typical examples of the decay curves are shown in Fig. 25.. Fig. 24. Fluorescence measurement experimental setup [126].. 100000. Experimental data Instrumental function. Fluorescence a.u.. 10000 1000 100 10 1. 0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Time (ns) Fig. 25. Fluorescence decay curve monitored at 400 nm. The fluorescence spectrum was obtained by integrating the decay curve throughout the time region (2.44 ps per channel). Black squares indicate experimental data and red circles, the instrumental function. Own measurement.. 41.

(47) 4. RESULTS 4.1 Bacterial reaction centers purity The protein content of isolated His-tagged bacterial reaction centers was checked using electrophoresis. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) alysis showed the presence of only three polypeptides: L, M and H, which compose the BRC protein core (Fig. 26 a). No cytochrome contamination was detected. It means that only nonheme iron is present in prepared BRCs and the heme iron present in cytochromes have been removed during the purification process on the high affinity nickel column. Wild type and mutated BRCs protein and pigment composition were additionally monitored by absorption spectroscopy in the wavelengh range from 280 nm to 1000 nm (Fig. 26 b).. Marker 150kDa 100kDa Buffer solution: 10mM TRIS HCl, 0.1% LDAO BRC WT. 80kDa 60kDa 40kDa M L. 30kDa. H. 20kDa. Absorption [a.u.]. 1,0 0,8 0,6 0,4. BPheo ooo BPheo BChl. BChl Dimers. 0,2 0,0. 300 400 500 600 700 800 900 1000. Wavelength [nm]. 10kDa a. BChl. b. Fig. 26. a) Example electrophoretic analysis (SDS-PAGE) of the polypeptide composition of BRCs isolated from Rb. sphaeroides (left and right lines show two different concentrations of the sample, about 20 µM and 15 µM of BRCs, respectively). The middle line is a protein mass marker. The electrophoresis was done according to Laemmli U. K. [128] using a 13 % gel. b) Example absorption spectrum of His-tagged BRCs of Rb. sphaeroides.. 42.

(48) A pure His-tagged reaction center must fullfill the following equation:. Absorption Absorption. 280 nm. ≤ 1 .4. (31). 802 nm. A higher value of this ratio comes form too strong an absorption peak at 280 nm, which is a signature of elevated concentration of non specific proteins. The obtained absorption spectrum (Fig. 26 b) confirms that the His-tagged BRCs have no contaminations by ligh harvesting complexes and cytochromes. No difference in absorption spectrum have been observed between wild type and mutated bacterial reaction centers (data not shown).. 4.2 Hyperfine interactions of non-heme iron 4.2.1 Mössbauer measurements Mössbauer spectroscopy was applied in order to compare the NHFe physico-chemical properties of wild type and mutated BRCs isolated from His-tagged Rb. Sphaeroides. Mössbauer spectra measured at 85 K are presented in Fig. 27. The spectrum of WT BRCs were fitted using a single symmetric doublet of two Lorentzian lines. In the case of AA mutant, two doublets were used and for the triple mutant AATyr and the double mutant dFer, a superposition of five symmetric doublets were necessary to get a fit of a good quality. The line widths of all components in BRCs are about 0.18 ± 0.01 mm/s, which indicate a high degree of homogeneity of the iron binding sites. The obtained hyperfine parameters are collected in Tab. 3. All isomer shifts are given vs. metallic α – Fe at room temperature. In wild type BRCs, a single iron binding site is observed with an isomer shift (IS) of about 1.06 mm/s and quadrupole splitting (QS) of about 2.12 mm/s. These hyperfine parameters are characteristic of NHFe in a high spin (HS) Fe2+ state [54, 58, 129, 130]. The spectrum of the double mutant AA was fitted with two Lorentzian doublets. The first component of 93 % amplitude corresponds to the high spin state of the iron atom Fe2+ with IS = 1.08 mm/s and QS = 2.16 mm/s The second component of only about 7 % contribution has a much lower isomer shift of about 0.36 mm/s and a quadrupole splitting of about 1.17 mm/s has hyperfine parameters similar to those observed for a reduced low spin (LS) state of the heme iron in cytochromes [54, 129, 131] but in our case this component is related to NHFe.. 43.