Martin R. Fuchs

Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin

The three macromolecular crystallography beamlines BL14.1, BL14.2 and BL14.3 at the BESSY II storage ring at the Helmholtz-Zentrum Berlin are described.

A high-field/high-frequency heterodyne induction-mode electron paramagnetic resonance spectrometer operating at 360 GHz

We present design details of and first measurements with a novel continuous wave (cw) high-field/high-frequency electron paramagnetic resonance spectrometer operating at a microwave frequency of 360 GHz and a magnetic field of up to 14 T. The spectrometer design incorporates a heterodyne mixer detection scheme with a quasi-optical transmission line and a bimodal induction mode Fabry–Perot cavity. First cw experiments on polycrystalline 1,1-diphenyl-2-picryl-hydrazyl and bisdiphenylene-β-phenylallyl benzolate in polystyrene at room temperature and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl in frozen solution at 190 K demonstrate the high Zeeman resolution achievable and allow an estimate of the present detection sensitivity of 4×109 spins/G at a detection bandwidth of 1 Hz.

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 380 GHz) is presented, designed for use as a source in a 360 GHz 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 360 GHz have pulse lengths from 100 ns–10 μs and a pulse power of (22±5) mW. The output in a broader frequency band between 320 and 380 GHz ranges from 20 up to 60 mW. Within a 10 μs 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.

A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source

The new version MS2 of the in situ on-axis micro-spectrophotometer at the macromolecular crystallography beamline X10SA of the Swiss Light Source supports the concurrent acquisition of Raman, resonance Raman, fluorescence and UV/Vis absorption spectra along with diffraction data.

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.

Transmissive Imaging X‐Ray Beam Position Monitors (XBPM) for Protein Crystallography (PX) Beamlines

We present the development of transmissive imaging X‐ray Beam Position Monitors (XBPM) for use in Protein Crystallography (PX) beamlines and beamlines operating at photon energies around and above 10 keV and fluxes of 1010 photons/s or higher. Results from different phosphors, substrates and coating techniques are compared with respect to transmission, layer quality and image resolution. The screen absorption is below 10 % for selected phosphors at photon energies above 10 keV. This enables continuous monitoring of both beam shape and intensity during experimental data collection at a few micron resolution. The screens have been designed for monochromatic beams, with a maximum beam diameter of 2 mm (extendible to 25 mm). The presented approach not only promises a generic solution for use in the automation of PX beamlines, but will also be extendible to other state‐of‐the‐art crystal monochromator beamlines.

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.

Transient optical absorption and time-resolved resonance Raman experiments on covalently linked porphyrin–quinone systems

Light-induced triplet electron transfer (ET) and subsequent triplet radical-pair (RP) recombination in two covalently linked porphyrin–quinone systems in highly viscous ethanol has been investigated by both transient optical absorption and time-resolved resonance Raman spectroscopy with a time resolution of 10 ns. The temperature dependence of the rates is measured between 155 and 200 K and compared with predictions of solvent-controlled adiabatic electron-transfer theory. It is shown that the triplet ET in the normal region (exergonicity ΔG0 λs) depends on the dynamics of the exchange interaction J, on the triplet–singlet mixing of the radical pair states and on the singlet recombination rate. An intermolecular ET process leading to a disproportionation reaction of the quinone moieties is also observed.

NSLS-II biomedical beamlines for micro-crystallography, FMX, and for highly automated crystallography, AMX: New opportunities for advanced data collection

We present the final design of the x-ray optics and experimental stations of two macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source-II. The microfocusing FMX beamline will deliver a flux of ∼5×1012 ph/s at 1 A into a 1 – 20 µm spot, its flux density surpassing current MX beamlines by up to two orders of magnitude. It covers an energy range from 5 – 30 keV. The highly automated AMX beamline is optimized for high throughput, with beam sizes from 4 – 100 µm, an energy range of 5 – 18 keV and a flux at 1 A of ∼1013 ph/s. A focus in designing the beamlines lay on achieving high beam stability, for example by implementing a horizontal bounce double crystal monochromator at FMX. A combination of compound refractive lenses and bimorph mirror optics at FMX supports rapid beam size changes. Central components of the in-house developed experimental stations are horizontal axis goniometers with a target sphere of confusion of 100 nm, piezo-slits for dynamic beam size changes during...