A Personal Account

In this minireview, we report on our year-long EPR work, such as electron–nuclear double resonance (ENDOR), pulse electron double resonance (PELDOR) and ELDOR-detected NMR (EDNMR) at X-band and W-band microwave frequencies and magnetic fields. This report is dedicated to James S. Hyde and honors his pioneering contributions to the measurement of spin interactions in large (bio)molecules. From these interactions, detailed information is revealed on structure and dynamics of macromolecules embedded in liquid- solution or solid-state environments. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultra-fast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy and its multi-frequency extensions to new horizons concerning sensitivity of detection, selectivity of molecular interactions and time resolution. Among the most important advances is the upgrading of EPR to high magnetic fields, very much in analogy to what happened in NMR. The ongoing progress in EPR spectroscopy is exemplified by reviewing various multi-frequency electron–nuclear double-resonance experiments on organic radicals, light- generated donor–acceptor radical pairs in photosynthesis, and site- specifically nitroxide spin-labeled bacteriorhodopsin, the light-driven proton pump, as well as EDNMR and ENDOR on nitroxides. Signal and resolution enhancements are particularly spectacular for ENDOR, EDNMR and PELDOR on frozen-solution samples at high Zeeman fields. They provide orientation selection for disordered samples approaching single-crystal resolution at canonical g-tensor orientations—even for molecules with small g-anisotropies. Dramatic improvements of EPR detection sensitivity could be achieved, even for short-lived paramagnetic reaction intermediates. Thus, unique structural and dynamic information is revealed that can hardly be obtained by other analytical techniques. Micromolar concentrations of sample molecules have become sufficient to characterize stable and transient reaction intermediates of complex molecular systems—offering exciting applications for physicists, chemists, biochemists and molecular biologists

EPR, ENDOR, and TRIPLE resonance studies of modified bacteriochlorophyll cation radicals

A series of substituted bacteriochlorophyll molecules, all used in reconstitution experiments of reaction centers of Rhodobacter sphaeroides (Struck et al. Biochim. Biophys. Acta 1991, 1060, 262-270), were characterized by EPR, electron-nuclear double (ENDOR), and electron-nuclear-nuclear triple (TRIPLE) resonance spectroscopy in their monomeric radical cation states. Effects of different substituents at position 3 in the porphyrin macrocycle were considered, especially for two «crosslinks» between plant and bacterial chlorophylls. These are 3-vinylbacteriochlorophyll where the «bacteria» acetyl group at position 3 was substituted by vinyl and 3-acetylchlorophyll where the «plant» vinyl group was substituted by acety

Biomolecular EPR Meets NMR at High Magnetic Fields

In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth. View Full-Tex

The basic properties of the electronic structure of the oxygen-evolving complex of photosystem II are not perturbed by Ca 2+ removal

Ca2+ is an integral component of the Mn4O5Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S2′ state of the Ca2+-depleted cluster from spinach is examined by X- and Q-band EPR and 55Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca2+ removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S2′ state in spinach PS II is very similar to the native S2 state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca2+ a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between YZ and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel 55Mn2+ signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca2+-depleted PS II. Mn2+ titration, monitored by 55Mn ENDOR, revealed a specific Mn2+ binding site with a submicromolar KD. Ca2+ titration of Mn2+-loaded, Ca2+-depleted PS II demonstrated that the site is reversibly made accessible to Mn2+ by Ca2+ depletion and reconstitution. Mn2+ is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn4O5Ca cluster during photoassembly and/or D1 repair

water exchange in bacterial photosynthetic reaction centers embedded in a trehalose glass studied using multiresonance EPR

Using isotope labeled water (D2O and H217O) and pulsed W-band (94 GHz) high- field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, the biologically important question of detection and quantification of local water in proteins is addressed. A bacterial reaction center (bRC) from Rhodobacter sphaeroides R26 embedded into a trehalose glass matrix is used as a model system. The bRC hosts the two native radical cofactor ions Image ID:c7cp03942e-t1.gif (primary electron donor) and Image ID:c7cp03942e-t2.gif (primary electron acceptor) as well as an artificial nitroxide spin label site-specifically attached to the surface of the H-protein domain. The three paramagnetic reporter groups have distinctly different local environments. They serve as local probes to detect water molecules via magnetic interactions (electron–nuclear hyperfine and quadrupole) with either deuterons or 17O nuclei. bRCs were equilibrated in an atmosphere of different relative humidities allowing us to control precisely the hydration levels of the protein. We show that by using oxygen-17 labeled water quantitative conclusions can be made in contrast to using D2O which suffers from proton–deuterium exchange processes in the protein. From the experiments we also conclude that dry trehalose operates as an anhydrobiotic protein stabilizer in line with the “anchorage hypothesis” of bio-protection. It predicts selective changes in the first solvation shell of the protein upon trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding network. Changes in hydrogen-bonding patterns usually have an impact on the global function of a biological system

Water oxidation in photosystem II

Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55–60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.Open access funding provided by Max Planck Society. Financial support of this work by the Max Planck Society and MANGAN (03EK3545) funded by the Bundesministeriums für Bildung und Forschung is gratefully acknowledged. N.C. acknowledges the support of the Australian Research Council (FT140100834

Water oxidation in photosystem II

Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55-60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.Open access funding provided by Max Planck Society. Financial support of this work by the Max Planck Society and MANGAN (03EK3545) funded by the Bundesministeriums für Bildung und Forschung is gratefully acknowledged. N.C. acknowledges the support of the Australian Research Council (FT140100834)