A. Schnegg

High-field EPR spectroscopy applied to biological systems: characterization of molecular switches for electron and ion transfer

The last decade witnessed a tremendous growth in combined efforts of biologists, chemists and physicists to understand the dominant factors determining the specificity and directionality of transmembrane transfer processes in proteins. A large variety of experimental techniques is being used including X-ray and neutron diffraction, but also time-resolved optical, infrared and magnetic resonance spectroscopy. This is done in conjunction with genetic engineering strategies to construct site-specific mutants for controlled modification of the proteins. As a general perception of these efforts, the substantial influence of weak interactions within the protein and its membrane interfaces is recognized. The weak interactions are subject to subtle changes during the reaction cycle owing to the inherent flexibility of the protein-membrane complex. Specific conformational changes accomplish molecular-switch functions for the transfer process to proceed with optimum efficiency. Characteristic examples of time varying non-bonded interactions are specific H-patterns and/or polarity effects of the microenvironment. The present perception has emerged from the coupling of newly developed spectroscopic techniques - and advanced EPR certainly deserves credit in this respect - with newly developed computational strategies to interpret the experimental data in terms of protein structure and dynamics. By now, the partners of this coupling, particularly high-field EPR spectroscopy and DFT-based quantum theory, have reached a level of sophistication that applications to large biocomplexes are within reach. In this review, a few large paradigm biosystems are surveyed which were explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three biosystems are characterized with respect to structure and dynamics: (1) Light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from the photosynthetic bacterium Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The detailed information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide highly desired detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales. The review introduces the audience to origins and basic experiments of EPR in relation to NMR, describes the underlying strategies for extending conventional EPR to high-field/high-frequency EPR, and highlights those details of molecular information that are obtained from high-field EPR in conjunction with genetic engineering and that are not accessible by classical spectroscopy. The importance of quantum-chemical interpretation of the experimental data by DFT and advanced semiempirical molecular-orbital theory is emphasized. A short description of the laboratory-built 95 GHz and 360 GHz EPR/ENDOR spectrometers at FU Berlin is also presented. The review concludes with an outlook to future opportunities and challenges of advanced bio-EPR in interdisciplinary research.

Pulsed Orotron—A new microwave source for submillimeter pulse high-field electron paramagnetic resonance spectroscopy

A vacuum-tube device for the generation of pulsed microwave radiation in the submillimeter range (up to 380u2002GHz) is presented, designed for use as a source in a 360u2002GHz high-field/high-frequency electron paramagnetic resonance (EPR) spectrometer—the pulsed Orotron. Analogous to the known continuous wave (cw) version, in the pulsed Orotron microwave radiation is generated by the interaction of a nonrelativistic electron beam with a diffraction grating (stimulated Smith–Purcell radiation) in feedback with an open Fabry–Perot resonator construction. The presented design extends the cw Orotron by a gate electrode and a high-voltage pulsing unit to control the electron beam current. The generated pulses at 360u2002GHz have pulse lengths from 100u2002ns–10u2002μs and a pulse power of (22±5)u2002mW. The output in a broader frequency band between 320 and 380u2002GHz ranges from 20 up to 60u2002mW. Within a 10u2002μs time slot, incoherent pulse trains of arbitrary duration can be generated. The pulsed Orotron has been incorporated in the qua...

High-field EPR, ENDOR and ELDOR on bacterial photosynthetic reaction centers

We report on recent 95 and 360 GHz high-field electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and pulsed electron-electron double resonance (PELDOR) studies of wild-type and mutant reaction centers (RCs) from the photosynthetic bacteriumRhodobacter sphaeroides. Taking advantage of the excellent spectral and temporal resolution of EPR at 95 and 360 GHz, the electron-transfer (ET) cofactors radical ions and spin-correlated radical pairs were characterized by theirg- and hyperfine-tensor components, their anisotropicT2 relaxation as well as by the dipolar interaction between P865•+QA•− radical pairs. The goal of these studies is to better understand the dominant factors determining the specificity and directionality of transmembrane ET processes in photosynthetic RC proteins. In particular, our multifrequency experiments elucidate the subtle cofactor-protein interactions, which are essential for fine-tuning the ET characteristics, e.g., the unidirectionality of the light-induced ET pathways along the A branch of the RC protein. By our high-field techniques, frozen-solution RCs of novel site-specific single and double mutants ofR. sphaeroides were studied to modulate the ET characteristics, e.g., even to the extent that dominant B branch ET prevails. The presented multifrequency EPR work culminates in first 360 GHz ENDOR results from organic nitroxide radicals as well as in first 95 GHz high-field PELDOR results from orientationally selected spin-polarized radical pairs P865•+QA•−, which allow to determine the full geometrical structure of the pairs even in frozen-solution RCs.

Multifrequency EPR study of the mobility of nitroxides in solid-state calixarene nanocapsules

Multifrequency continuous wave (cw) and echo-detected (ED) electron paramagnetic resonance (EPR) was employed to study the mobility of nitroxides confined in nanocapsules. The complexes p-hexanoyl calix[4]arene with 4-methoxy-2,2,6,6-tetramethylpiperidine-N-oxyl (MT) and N-(2-methylpropyl)-N-(1-diethylphosphono-2,2-dimethylpropyl)-aminoxyl (DEPN) were studied by X-, W-band and 360 GHz cw EPR at temperatures between 90 and 370 K. Thereby we were able to extract the canonical values of the hyperfine and g-tensors of the encapsulated radicals as well as information on restricted orientational dynamics of the caged spin probes. Comparing cw and ED-EPR data obtained on MT@C6OH we found that between 90 and 200 K the caged nitroxide undergoes isotropic small-angle fluctuations (librations), whereas at higher temperatures restricted rotations of the radical with correlation times of 0.75 x 10(-10) s and 1.2 x 10(-10) s dominate at 325 and 300 K, respectively. The activation energy of the rotational motion of encapsulated MT radicals was evaluated as E(a) = 1.0 kcal mol(-1), which is comparable to the magnitude of van der Waals interactions. Compared to MT, the reorientational motion of DEPN was found to be slower and more isotropic.

The g-tensor of the flavin cofactor in (6–4) photolyase: a 360 GHz/12.8 T electron paramagnetic resonance study

The g-tensor of the neutral radical form of the flavin adenine dinucleotide cofactor FADH• of (6–4) photolyase from Xenopus laevis has been determined by very high-magnetic-field/high-microwave-frequency electron-paramagnetic resonance (EPR) performed at 360u2009GHz/12.8u2009T. Due to the high spectral resolution the anisotropy of the g-tensor could be fully resolved in the frozen-solution continuous-wave EPR spectrum. By least square fittings of spectral simulations to experimental data, the principal values of the g-tensor have been established: gX u2009=u20092.00433(5), gY u2009=u20092.00368(5), gZ u2009=u20092.00218(7). A comparison of very high-field EPR data and proton and deuteron electron-nuclear double resonance measurements yielded precise information concerning the orientation of the g-tensor with respect to the molecular frame. This data allowed a comparison to be made between the principal values of the g-tensors of the FADH• cofactors of photolyases involved in the repair of two different DNA lesions: the cyclobutane pyrimidine dimer (CPD) and the (6–4) photoproduct. It was found that gX and gZ are similar in both enzymes, whereas the gY component is slightly larger in (6–4) photolyase. This result clearly shows the sensitivity of the g-tensor to subtle differences in the protein environment experienced by the flavin.

The primary donor cation P+ in photosynthetic reaction centers of site-directed mutants of Rhodobacter sphaeroides: g-tensor shifts revealed by high-field EPR at 360 GHz/12.8 T

Abstract The frozen solution electron paramagnetic resonance spectrum of the primary donor cation P + in reaction centers of site-directed mutants of Rhodobacter ( Rb. ) sphaeroides has been obtained at a microwave frequency ν =360 GHz and a magnetic field B 0 =12.8 T. Due to the high Zeeman resolution of the powder pattern, all three principal components of the rhombic g -tensors at T =160 K could be determined with high accuracy. We compare spectra of the site-directed mutants, in which the axial ligand histidine M202 of the primary donor is replaced by glutamic acid (HE(M202)) or leucine (HL(M202)), with those of the strain R26, whose primary donor is similar to that of the wild type and only lacks the carotenoid. For HE(M202), this is the first determination of its g -tensor with the principal components g xx =2.00335(3), g yy =2.00236(2) and g zz =2.00191(2). While in R26 the primary donor is a bacteriochlorophyll a dimer, the HL(M202) and HE(M202) mutants have previously been shown to be bacteriochlorophyll:bacteriopheophytin heterodimers. Their g -tensor anisotropy Δ g = g xx − g zz shows significant variations in opposite directions when compared with R26, with an increased anisotropy for HE(M202) and a decreased one for HL(M202). Calculations employing Density Functional Theory suggest that the observed shifts originate in different torsional angles of the acetyl group attached to the spin-carrying bacteriochlorophyll half L of the dimer.

Towards an identification of chemically different flavin radicals by means of theirg-tensor

Theg-tensors of two chemically different flavin mononucleotide (FMN) radicals, one of which is covalently bound via N(5) of its 7,8-dimethyl isoalloxazine moiety, and the other one non-covalently bound to mutant LOV domains of the blue-light receptor phototropin, LOV1 C57M and LOV2 C450A, respectively, have been determined by very high microwave frequency and high magnetic field electron paramagnetic resonance (EPR) performed at 360 GHz and 12.8 T. Due to the high spectral resolution of the frozen-solution continuous-wave EPR spectra, the anisotropy of theg-tensors could be fully resolved. By least-squares fittings of spectral simulations to expermental data, the principal values ofg have been established:gX=2.00554(5),gY=2.00391(5), andgZ=2.00247(7) for the N(5)-alkyl-chain-linked FMN radical in LOV1 C57M-675, andgX=2.00427(5),gY=2.00360(5), andgZ=2.00220(7) for the noncovalently bound FMN radical in LOV2 C450A-605. By a comparison of these values to the ones from the flavin adenine dinucleotide radicals in two photolyases, the radical in LOV2 C450A-605 could be clearly identified as a neutral FMN radical, FMNH. In contrast, LOV1 C57M-675 exhibits significantly shifted principal components ofg, the differences being caused by spin-orbit coupling of the nearby sulfur from the reactive methionine residue, and the modified chemical structure due to the covalent attachment at N(5) of the radical to the apoprotein. The results clearly show the potential of using theg-tensor as probe of the global electronic and chemical structure of protein-bound flavin radicals.