Photosynthesis Research (v.102, #2-3)

Introduction to imaging methods in photosynthesis by Egbert J. Boekema (107-109).

Optical microscopy in photosynthesis by Richard Cisek; Leigh Spencer; Nicole Prent; Donatas Zigmantas; George S. Espie; Virginijus Barzda (111-141).
Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.
Keywords: Nonlinear microscopy; Second harmonic generation; Third harmonic generation; Chlamydomonas ; Chloroplasts; Cyanobacteria

Fluorescence lifetime-resolved imaging by Yi-Chun Chen; Robert M. Clegg (143-155).
This is a short account of fluorescence lifetime-resolved imaging, in order to acquaint readers who are not experts with the basic methods for measuring lifetime-resolved signals throughout an image. We present the early FLI (fluorescence lifetime imaging) history, review shortly the instrumentation and experimental design, discuss briefly the fundamentals of the measured fluorescence response, and introduce the basic measurement methodologies. We also emphasize the complex nature of the fluorescence response in FLI signals, and introduce certain analysis methods that are appropriate and informative for complex fluorescence decays. The advantages of model independent analyses are discussed and examples given.
Keywords: Fluorescence lifetime-resolved imaging; Microscopy; Model independent analysis; Fluorescence lifetime-resolved imaging microscopy; Fluorescence lifetime-resolved imaging; Spectral FLIM; Time-domain; Frequency domain

Fluorescence lifetime imaging microscopy (FLIM) is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope. We present a scanningless implementation of FLIM based on a time- and space-correlated single photon counting (TSCSPC) method employing a position-sensitive quadrant anode detector and wide-field illumination. The standard time-correlated photon counting approach leads to picosecond temporal resolution, making it possible to resolve complex fluorescence decays. This allows parallel acquisition of time-resolved images of biological samples under minimally invasive low-excitation conditions (<10mW/cm2). In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided. Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope, where focusing of the excitation beam into a tight spot is required. Therefore, wide-field FLIM permits to study Photosystem II (PS II) in a way so far not possible with a laser scanning microscope. The potential of the wide-field TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.
Keywords: Fluorescence lifetime imaging; FLIM; Time-correlated single photon counting; QA detector; Photosynthesis; Chlorophyll; Acaryochloris marina

Imaging of multi-color fluorescence emission from leaf tissues by Zuzana Benediktyová; Ladislav Nedbal (169-175).
Multi-color fluorescence emission from leaf tissues is presented as a powerful reporter on plant biochemistry and physiology that can be applied both at macro- and micro-scales. The blue–green fluorescence emission is typically excited by ultraviolet (UV) excitation. However, this approach cannot be applied in investigating intact leaf interior because the UV photons are largely absorbed in the epidermis of the leaf surface. This methodological barrier is eliminated by replacing the UV photon excitation by excitation with two infra-red photons of the same total energy. We demonstrate this approach by using two-photon excitation for microscopy of Arabidopsis thaliana leaves infected by pathogenic bacterium Pseudomonas syringae. The leaf structures are visualized by red chlorophyll fluorescence emission reconstructed in 3-D images while the bacteria are detected by the green emission of engineered fluorescence protein.
Keywords: Chlorophyll fluorescence; Blue–green fluorescence; Pyridine nucleotide; Two-photon microscopy

Exploring photosynthesis by electron tomography by Martin F. Hohmann-Marriott; Robert W. Roberson (177-188).

Single particle electron microscopy by Egbert J. Boekema; Mihaela Folea; Roman Kouřil (189-196).
Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structures at low, medium, and high resolution. Since electron micrographs of biological objects are very noisy, improvement of the signal-to-noise ratio by image processing is an integral part of EM, and this is performed by averaging large numbers of individual projections. Averaging procedures can be divided into crystallographic and non-crystallographic methods. The crystallographic averaging method, based on two-dimensional (2D) crystals of (membrane) proteins, yielded in solving atomic protein structures in the last century. More recently, single particle analysis could be extended to solve atomic structures as well. It is a suitable method for large proteins, viruses, and proteins that are difficult to crystallize. Because it is also a fast method to reveal the low-to-medium resolution structures, the impact of its application is growing rapidly. Technical aspects, results, and possibilities are presented.
Keywords: Electron microscopy; Single particle analysis; Photosynthesis

Photosynthesis both in the past and present provides the vast majority of the energy used on the planet. The purple photosynthetic bacteria are a group of organisms that are able to perform photosynthesis using a particularly simple system that has been much studied. The main molecular constituents required for photosynthesis in these organisms are a small number of transmembrane pigment–protein complexes. These are able to function together with a high quantum efficiency (about 95%) to convert light energy into chemical potential energy. While the structure of the various proteins have been solved for several years, direct studies of the supramolecular assembly of these complexes in native membranes needed maturity of the atomic force microscope (AFM). Here, we review the novel findings and the direct conclusions that could be drawn from high-resolution AFM analysis of photosynthetic membranes. These conclusions rely on the possibility that the AFM brings of obtaining molecular resolution images of large membrane areas and thereby bridging the resolution gap between atomic structures and cellular ultrastructure.
Keywords: AFM; Photosynthetic apparatus; PSU; ICM; LH2; LH1; RC; Supramolecular assembly; Membrane structure

MRI of intact plants by Henk Van As; Tom Scheenen; Frank J. Vergeldt (213-222).
Nuclear magnetic resonance imaging (MRI) is a non-destructive and non-invasive technique that can be used to acquire two- or even three-dimensional images of intact plants. The information within the images can be manipulated and used to study the dynamics of plant water relations and water transport in the stem, e.g., as a function of environmental (stress) conditions. Non-spatially resolved portable NMR is becoming available to study leaf water content and distribution of water in different (sub-cellular) compartments. These parameters directly relate to stomatal water conductance, CO2 uptake, and photosynthesis. MRI applied on plants is not a straight forward extension of the methods discussed for (bio)medical MRI. This educational review explains the basic physical principles of plant MRI, with a focus on the spatial resolution, factors that determine the spatial resolution, and its unique information for applications in plant water relations that directly relate to plant photosynthetic activity.
Keywords: Xylem; Phloem; Flow conducting area; Hydraulic conductance; Water content; Storage pools; Dynamic behavior

Protein crystallization by Mei Li; Wen-rui Chang (223-229).

Structures of proteins and cofactors: X-ray crystallography by J. P. Allen; C. Seng; C. Larson (231-240).
Protein crystallography is the predominately used technique for the determination of the three-dimensional structures of proteins and other macromolecules. In this article, the methodology utilized for the measurement and analysis of the diffraction data from crystals is briefly reviewed. As examples of both the usefulness and difficulties of this technique, the determination of the structures of several photosynthetic pigment–protein complexes is described, namely, the reaction center from purple bacteria, photosystem I and photosystem II from cyanobacteria, the light-harvesting complex II from purple bacteria, and the FMO protein from green bacteria.
Keywords: Protein crystallography; X-ray diffraction; Reaction centers; Photosystem I; Photosystem II; Light-harvesting complexes

X-ray absorption spectroscopy by Junko Yano; Vittal K. Yachandra (241-254).
This review gives a brief description of the theory and application of X-ray absorption spectroscopy, both X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS), especially, pertaining to photosynthesis. The advantages and limitations of the methods are discussed. Recent advances in extended EXAFS and polarized EXAFS using oriented membranes and single crystals are explained. Developments in theory in understanding the XANES spectra are described. The application of X-ray absorption spectroscopy to the study of the Mn4Ca cluster in Photosystem II is presented.
Keywords: Photosystem II; Water oxidation; Oxygen evolution; Manganese cluster; X-ray spectroscopy; EXAFS; XANES; X-ray dichroism

X-ray emission spectroscopy by Uwe Bergmann; Pieter Glatzel (255-266).
We describe the chemical information that can be obtained by means of hard X-ray emission spectroscopy (XES). XES is presented as a technique that is complementary to X-ray absorption spectroscopy (XAS) and that provides valuable information with respect to the electronic structure (local charge- and spin-density) as well as the ligand environment of a 3d transition metal. We address non-resonant and resonant XES and present results that were recorded on Mn model systems and the Mn4Ca-cluster in the oxygen evolving complex of photosystem II. A brief description of the instrumentation is given with an outlook toward future developments.
Keywords: X-ray spectroscopy; X-ray emission; Synchrotron radiation; Electronic structure; Photosystem II; Manganese; Oxygen evolving complex

Advances in X-ray light sources and detectors have created opportunities for advancing our understanding of structure and structural dynamics for supramolecular assemblies in solution by combining X-ray scattering measurement with coordinate-based modeling methods. In this review the foundations for X-ray scattering are discussed and illustrated with selected examples demonstrating the ability to correlate solution X-ray scattering measurements to molecular structure, conformation, and dynamics. These approaches are anticipated to have a broad range of applications in natural and artificial photosynthesis by offering possibilities for structure resolution for dynamic supramolecular assemblies in solution that can not be fully addressed with crystallographic techniques, and for resolving fundamental mechanisms for solar energy conversion by mapping out structure in light-excited reaction states.
Keywords: X-ray scattering; Photosynthesis; Artificial photosynthesis; Solution structure; Supramolecular chemistry; Molecular dynamics; Structure-function

Protein dynamics investigated by neutron scattering by Jörg Pieper; Gernot Renger (281-293).
This contribution describes incoherent quasielastic neutron scattering (QENS) as a suitable tool for investigations of protein dynamics with special emphasis on applications in photosynthesis research. QENS characterizes protein dynamics via the measurement of energy and momentum exchange between sample system and incident low-energy neutrons (1 meV < E < 20 meV). This method is especially sensitive for picosecond motions of hydrogen atoms because it makes use of the exceptionally large incoherent neutron scattering cross section of protons and their almost homogeneous distribution in proteins. After a short introduction into the basic principles of neutron scattering, a more detailed description of QENS will be presented including a short overview on instrumentation and theory. Recent QENS results will be discussed for the antenna complex LHC II and PS II membrane fragments. It is shown that diffusive protein dynamics is indispensable for enabling $$ { ext{Q}}_{ ext{A}}^{ - ullet } $$ reoxidation by QB at temperatures above 240 K, which explains the strong dependence of this electron transfer step on temperature and hydration level of the sample. Finally, a new laser-QENS pump-probe technique will be introduced which permits in situ monitoring of protein dynamics correlated with a change of the functional state of the sample, i.e. a direct observation of structure-dynamics-function relationships in real time.
Keywords: Neutron scattering; Protein dynamics; QENS; Photosystem II; Electron–phonon coupling; Electron transfer; Time-resolved QENS

Freeze-quench 57Fe-Mössbauer spectroscopy: trapping reactive intermediates by Carsten Krebs; J. Martin Bollinger Jr. (295-304).
57Fe-Mössbauer spectroscopy is a method that probes transitions between the nuclear ground state (I = 1/2) and the first nuclear excited state (I = 3/2). This technique provides detailed information about the chemical environment and electronic structure of iron. Therefore, it has played an important role in studies of the numerous iron-containing proteins and enzymes. In conjunction with the freeze-quench method, 57Fe-Mössbauer spectroscopy allows for monitoring changes of the iron site(s) during a biochemical reaction. This approach is particularly powerful for detection and characterization of reactive intermediates. Comparison of experimentally determined Mössbauer parameters to those predicted by density functional theory for hypothetical model structures can then provide detailed insight into the structures of reactive intermediates. We have recently used this methodology to study the reactions of various mononuclear non-heme-iron enzymes by trapping and characterizing several Fe(IV)-oxo reaction intermediates. In this article, we summarize these findings and demonstrate the potential of the method.
Keywords: Mössbauer spectroscopy; Iron; Intermediate; Oxygen activation; C–H activation

Electron paramagnetic resonance (EPR) and, more recently, solid-state nuclear magnetic resonance (NMR) have been employed to study photosynthetic processes, primarily related to the light-induced charge separation. Information obtained on the electronic structure, the relative orientation of the cofactors, and the changes in structure during these reactions should help to understand the efficiency of light-induced charge separation. A short introduction to the observables derived from magnetic resonance experiments is given. The relation of these observables to the electronic structure is sketched using the nitroxide group of spin labels as a simple example.
Keywords: Magnetic resonance; EPR; NMR; Photosynthesis; Nitroxides

High-field EPR by Anton Savitsky; Klaus Möbius (311-333).
Among the numerous spectroscopic techniques utilized in photosynthesis research, high-field/high-frequency EPR and its pulse extensions ESE, ENDOR, ESEEM, and PELDOR play an important role in the endeavor to understand, on the basis of structure and dynamics data, dominant factors that control specificity and efficiency of light-induced electron- and proton-transfer processes in primary photosynthesis. Short-lived transient intermediates of the photocycle can be characterized by high-field EPR techniques, and detailed structural information can be obtained even from disordered sample preparations. The chapter describes how multifrequency high-field EPR methodology, in conjunction with mutation strategies for site-specific isotope or spin labeling and with the support of modern quantum-chemical computation methods for data interpretation, is capable of providing new insights into the photosynthetic transfer processes. The information obtained is complementary to that of protein crystallography, solid-state NMR and laser spectroscopy.
Keywords: Photosynthesis; Reaction centers; Electron transfer; Radical pairs; High-field EPR; ENDOR; ESEEM; Orientation-resolved PELDOR

The light induced electron transfer in photosynthesis generates a series of sequential spin polarized radical pairs, and transient electron paramagnetic resonance (TREPR) is ideally suited to study the lifetimes and physical and electronic structures of these radical pairs. In this article, the basic principles of TREPR are outlined with emphasis on the electron spin polarization (ESP) that develops during the electron transfer process. Examples of the analysis of TREPR data are given to illustrate the information that can be obtained. Recent applications of the technique to study the functionality of reaction centers, light-induced structural changes, and protein–cofactor interactions are reviewed.
Keywords: Spin polarization; Electron transfer; Radical pairs; Photosystem I

The primary energy conversion steps of natural photosynthesis proceed via light-induced radical ion pairs as short-lived intermediates. Time-resolved electron paramagnetic resonance (EPR) experiments of photosynthetic reaction centers monitor the key charge separated state between the oxidized primary electron donor and reduced quinone acceptor, e.g., P 865 + Q A of purple photosynthetic bacteria. The time-resolved EPR spectra of P 865 + Q A are indicative of a spin-correlated radical pair that is created from the excited singlet state of P 865 in an ultra-fast photochemical reaction. Importantly, the spin-correlated radical pair nature of the charge separated state is a common feature of all photosynthetic reaction centers, which gives rise to several interesting spin phenomena such as quantum oscillations, observed at short delay times after optical excitation. In this review, we describe details of the quantum oscillation phenomenon and present a complete analysis of the data obtained from the charge separated state of purple bacteria, P 865 + Q A . The analysis and simulation of the quantum oscillations yield the three-dimensional structure of P 865 + Q A in the photosynthetic membrane on a nanosecond time scale after light-induced charge separation. Comparison with crystallographic data reveals that the position of Q A is essentially the same as in the X-ray structure. However, the head group of Q A has undergone a 60° rotation in the ring plane relative to its orientation in the crystal structure. The results are discussed within the framework of the previously suggested conformational gating mechanism for electron transfer from Q A to the secondary quinone acceptor Q B.
Keywords: Conformational gating; Electron transfer mechanism; Purple photosynthetic bacteria; Quantum oscillations; Spin-correlated radical pair; Structure of charge separated state; Time-resolved electron paramagnetic resonance; Ubiquinone A

Pulsed EPR spectroscopy by Maurice van Gastel (367-373).

Erratum to: Pulsed EPR spectroscopy by Maurice van Gastel (375-375).

Spin labeling EPR by Johann P. Klare; Heinz-Jürgen Steinhoff (377-390).
Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin–transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26.
Keywords: DEER; Nitroxide; PELDOR; Polarity; Photosynthetic reaction center; Sensory rhodopsin; Spin label accessibility; Spin label mobility

Electron-nuclear double resonance by Leonid Kulik; Wolfgang Lubitz (391-401).
The application of electron-nuclear double resonance (ENDOR) spectroscopy for the investigation of photosynthetic systems is reviewed. The basic principles of continuous wave and pulse ENDOR are presented. Selected examples of the application of the ENDOR technique for studying stable and transient paramagnetic species, including cofactor radical ions, radical pairs, triplet states, and the oxygen-evolving complex in plant Photosystem II (PSII) are discussed. Limitations and perspectives of ENDOR spectroscopy are outlined.
Keywords: Continuous wave ENDOR; Pulse ENDOR; Photosynthesis; Primary donor radical cation; Quinone acceptor radical anion; Carotenoid triplet state; Radical pair; Manganese cluster; Oxygen-evolving complex; Photosystem II; Photosystem I

Optically Detected Magnetic Resonance (ODMR) is a double resonance technique which combines optical measurements (fluorescence, phosphorescence, absorption) with electron spin resonance spectroscopy. After the first triplet-state ODMR experiments in zero magnetic field reported in 1968 by Schmidt and van der Waals, the number of double resonance studies on excited triplet states grew rapidly. Photosynthesis has proven to be a fruitful field of application due to the intrinsic possibility of forming photo-induced pigment triplet states in many sites of the photosynthetic apparatus. The basic principles of this technique are described and examples of application in Photosynthesis are reported.
Keywords: ODMR; Triplet state; FDMR; ADMR; T–S; Chlorophyll; Carotenoid

In the last two decades, Magic Angle Spinning (MAS) NMR has created its own niche in studies involving photosynthetic membrane protein complexes, owing to its ability to provide structural and functional information at atomic resolution of membrane proteins when in the membrane, in the natural environment. The light-harvesting two (LH2) transmembrane complex from Rhodopseudomonas acidophila is used to illustrate the procedure of the technique applicable in photosynthesis research. One- and two-dimensional solid-state NMR experiments involving 13C- and 15N-labeled LH2 complexes allow to make a sequence-specific assignment of NMR signals, which forms the basis for resolving structural details and the assessment of charge transfer, electronic delocalization effects, and functional strain in the ground state.
Keywords: Magic angle spinning NMR; LH2 complex; Electronic structure; Membrane protein; Isotope labeling

The solid-state photo-CIDNP effect by Jörg Matysik; Anna Diller; Esha Roy; A. Alia (427-435).
The solid-state photo-CIDNP effect is the occurrence of a non-Boltzmann nuclear spin polarization in rigid samples upon illumination. For solid-state NMR, which can detect this enhanced nuclear polarization as a strong modification of signal intensity, the effect allows for new classes of experiments. Currently, the photo- and spin-chemical machinery of various RCs is studied by photo-CIDNP MAS NMR in detail. Until now, the effect has only been observed at high magnetic fields with 13C and 15N MAS NMR and in natural photosynthetic RC preparations in which blocking of the acceptor leads to cyclic electron transfer. In terms of irreversible thermodynamics, the high-order spin structure of the initial radical pair can be considered as a transient order phenomenon emerging under non-equilibrium conditions and as a first manifestation of order in the photosynthetic process. The solid-state photo-CIDNP effect appears to be an intrinsic property of natural RCs. The conditions of its occurrence seem to be conserved in evolution. The effect may be based on the same fundamental principles as the highly optimized electron transfer. Hence, the effect may allow for guiding artificial photosynthesis.
Keywords: Electron transfer; Spin polarization; Radical pair; Bacterial RC; Solid-state NMR

Theory and molecular modeling play an increasingly important role in complementing the experimental findings and supporting the interpretation of the data. Owing to the increase in computational power combined with the development of more efficient methods, computer simulations and modeling have emerged as primary ingredients of modern scientific inquiry. Here, we introduce the methods that in our view bring the largest promises in photosynthesis research, indicate how they have already contributed, and can in the near future assume a significant role in this field. Particular emphasis is given to density functional theory and its combination with molecular dynamics simulations. We point out the need for a multi-scale approach in facing the challenging task of describing processes which cover several orders of magnitude both in the time scale and in the size of the systems of interest.
Keywords: DFT; Molecular dynamics; QMMM; Multi-scale simulations; Free energy calculations

Density functional theory by Maylis Orio; Dimitrios A. Pantazis; Frank Neese (443-453).
Density functional theory (DFT) finds increasing use in applications related to biological systems. Advancements in methodology and implementations have reached a point where predicted properties of reasonable to high quality can be obtained. Thus, DFT studies can complement experimental investigations, or even venture with some confidence into experimentally unexplored territory. In the present contribution, we provide an overview of the properties that can be calculated with DFT, such as geometries, energies, reaction mechanisms, and spectroscopic properties. A wide range of spectroscopic parameters is nowadays accessible with DFT, including quantities related to infrared and optical spectra, X-ray absorption and Mössbauer, as well as all of the magnetic properties connected with electron paramagnetic resonance spectroscopy except relaxation times. We highlight each of these fields of application with selected examples from the recent literature and comment on the capabilities and limitations of current methods.
Keywords: Density functional theory; Spectroscopic properties; Photosystem; Oxygen evolving complex

The MoD-QM/MM methodology for structural refinement of photosystem II and other biological macromolecules by Eduardo M. Sproviero; Michael B. Newcomer; José A. Gascón; Enrique R. Batista; Gary W. Brudvig; Victor S. Batista (455-470).
Quantum mechanics/molecular mechanics (QM/MM) hybrid methods are currently the most powerful computational tools for studies of structure/function relations and structural refinement of macrobiomolecules (e.g., proteins and nucleic acids). These methods are highly efficient, since they implement quantum chemistry techniques for modeling only the small part of the system (QM layer) that undergoes chemical modifications, charge transfer, etc., under the influence of the surrounding environment. The rest of the system (MM layer) is described in terms of molecular mechanics force fields, assuming that its influence on the QM layer can be roughly decomposed in terms of electrostatic interactions and steric hindrance. Common limitations of QM/MM methods include inaccuracies in the MM force fields, when polarization effects are not explicitly considered, and the approximate treatment of electrostatic interactions at the boundaries between QM and MM layers. This article reviews recent advances in the development of computational protocols that allow for rigorous modeling of electrostatic interactions in extended systems beyond the common limitations of QM/MM hybrid methods. We focus on the moving-domain QM/MM (MoD-QM/MM) methodology that partitions the system into many molecular domains and obtains the electrostatic and structural properties of the whole system from an iterative self-consistent treatment of the constituent molecular fragments. We illustrate the MoD-QM/MM method as applied to the description of photosystem II as well as in conjunction with the application of spectroscopically constrained QM/MM optimization methods, based on high-resolution spectroscopic data (extended X-ray absorption fine structure spectra, and exchange coupling constants).
Keywords: Quantum mechanics; Molecular mechanics; Photosystem II; EXAFS; MoD-QM/MM

This mini-review summarizes our current theoretical knowledge about excitation energy transfer in pigment–protein complexes. The challenge for theory lies in the complexity of these systems and in the fact that the pigment–pigment and the pigment–protein interactions are of equal magnitude. The first part of this review contains an introduction to the theory of light harvesting and to structure-based calculations of the parameters of the theory. The second part provides a discussion of the standard Förster and Redfield theories of excitation energy transfer, which are valid in the limit of weak and strong pigment–pigment coupling, respectively. Afterward, we provide a description of recent extensions of the standard theories and discuss challenging problems to be solved in the future.
Keywords: Pigment–protein complex; Light harvesting; Förster theory; Redfield theory; Modified Redfield theory; Generalized Förster theory; Site energies; Excitonic coupling; Spectral density

Oxygen detection in biological systems by Gernot Renger; Bertram Hanssum (487-498).
This article presents a brief description of analytical tools for monitoring evolution and consumption of molecular dioxygen in biological organisms. Based on its nature as a gas and its physical and chemical properties of the ground state $$^{3}sum {_{ ext{g}} { ext{O}}_{2} } , $$ different approaches have been developed for quantitative determinations: (i) manometry, (ii) formation of titratable sediments, (iii) solid state electrodes, (iv) EPR oximetry, (v) luminescence quenching, (vi) biological sensoring, (vii) mass spectrometry and (viii) amperometry. Among these methods mass spectrometry and amperometry are of special relevance for studies on the mechanisms of photosynthetic dioxygen evolution. Mass spectrometry is described in the article of Beckman et al. in this special issue. Therefore, the major part of this contribution focuses on amperometric methods that are currently widely used. Two different types of electrodes are described: (i) Clark-type electrode and (ii) Joliot-type electrode. The complementary advantages of both systems are outlined. A more detailed description comprises the potential of the Joliot-type electrode for mechanistic studies on the reactivity of the different redox states of the water oxidizing complex (WOC).
Keywords: Oxygen detection; Photosystem II; Water oxidizing complex; Joliot-type electrode; Clark-type electrode

Determination of thermodynamic parameters of water oxidation at the photosystem II (PSII) manganese complex is a major challenge. Photothermal beam deflection (PBD) spectroscopy determines enthalpy changes (ΔH) and apparent volume changes which are coupled with electron transfer in the S-state cycle (Krivanek R, Dau H, Haumann M (2008) Biophys J 94: 1890–1903). Recent PBD results on formation of the Q A /Y Z •+ radical pair suggest a value of ΔH similar to the free energy change, ΔG, of −540 ± 40 meV previously determined by the analysis of recombination fluorescence, but presently the uncertainty range of ΔH values determined by PBD is still high (±250 meV). In the oxygen-evolving transition, S3 → S0, the enthalpy change may be close to zero. A prominent non-thermal signal is associated with both Q A /Y Z •+ formation (<1 μs) and the S3 → S0 transition (~1 ms). The observed (apparent) volume expansion (ΔV of about +40 Å3 per PSII unit) in the S3 → S0 transition seems to revert, at least partially, the contractions on lower S-transitions and may also comprise contributions from O2 and proton release. The observed volume changes show that the S3 → S0 transition is accompanied by significant nuclear movements, which likely are of importance with respect to energetics and mechanism of photosynthetic water oxidation. Detailed PBD studies on all S-transitions will contribute to the progress in PSII research by providing insights not accessible by other spectroscopic methods.
Keywords: Enthalpy change; Manganese complex; Oxygen evolution; Photoacoustic; Photosynthesis; Photosystem II; Photothermal methods

On-line mass spectrometry: membrane inlet sampling by Katrin Beckmann; Johannes Messinger; Murray Ronald Badger; Tom Wydrzynski; Warwick Hillier (511-522).
Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O2 evolution versus O2 uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H2 generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.
Keywords: Membrane-inlet mass spectrometry; Oxygenic photosynthesis; Water-splitting; Carbonic anhydrase; Water binding; Artificial photosynthesis

Analytical approaches to photobiological hydrogen production in unicellular green algae by Anja Hemschemeier; Anastasios Melis; Thomas Happe (523-540).
Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of “anaerobic oxygenic photosynthesis”, whereby photosynthetically generated oxygen is consumed by the cell’s own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular “hydrogen metabolism” process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H2 production in Chlamydomonas can be monitored and regulated.
Keywords: Anaerobiosis; Green microalgae; Hydrogen; Photosynthesis; Screening; Sulphur

Dynamic electrochemical experiments on hydrogenases by Fraser A. Armstrong (541-550).
A powerful approach for studying hydrogenases, applying a suite of dynamic electrochemical techniques known as protein film electrochemistry, is trailblazing fresh discoveries and providing a wealth of quantitative data on these complex enzymes. The information now stemming from experiments on tiny quantities of hydrogenases ranges from their kinetics and catalytic bias (a preference to operate in H2 oxidation vs. H2 production) to wide differences in the ways they react with oxygen and other inhibitors. Tolerance of hydrogenase catalysis to oxygen is essential if organisms are to be exploited for photosynthetic hydrogen production, and is crucial in enabling aerobes to use trace H2 as an energy source. Experiments described in this article may be adapted for other complex enzymes.
Keywords: Hydrogen; Hydrogenase; Photosynthesis; Electrochemistry