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

Abstract

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 bacterium Rhodobacter 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 characte¡ by their g-and hyperfine-tensor components, their anisotropic T, relaxation as well as by the dipolar interaction between P~~sQ~-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 of R. 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 Ps which a|low to determine the full geometrical structure of the pairs even in frozen-solution RCs.

High-frequency EPR applications of open nonradiative resonators

“…At W-band and higher frequencies, the state-of-the-art in terms of minimum detectable number of spins and shortest p-pulse length is given by the cylindrical metallic cavity operating in the TE 011 mode [3][4][5][6][7][8][9][10][11][12][13]. The basic structure of the cavity is represented by a cylindrical volume delimited by metallic walls and plungers [14].…”

Pulsed EPR spectroscopy

“…This microwave pulse is then moved in time with respect to the pulses applied to the ''observer electron spin,'' and a modulation of the ESE signal can be observed. The modulation frequency is essentially determined by the magnetic coupling between the two unpaired electrons, which in turn give information about the distance and relative orientations of the spin distributions in both centers (Schnegg et al 2007). When protein dynamics influence the electron-electron distance, the distance will not be uniquely defined, but the ELDOR spectrum rather shows a distribution of distances.…”

High-field EPR

“…Their ENDOR lines appear in different frequency ranges and, providing that the nuclear Larmor frequencies are separated at the chosen Zeeman field value B 0 , the different nuclei can be immediately identified. In the case of an accidental overlap of ENDOR lines from the different nuclei at X-band (9.5 GHz, 0.34 T) the lines can be separated when working at higher Zeeman fields and mw frequencies, for instance at 3.4 T, 95 GHz (Burghaus et al 1988) or even at 12.9 T, 360 GHz (Schnegg et al 2007). This disentangling of ENDOR lines by different field/frequency settings for the EPR condition is depicted in Fig.…”

“…Afterwards, a few other laboratories have developed the instrumentation for millimeter and sub-millimeter highfield EPR spectrometers. Such spectrometers have been described for cw irradiation at 95 GHz (k % 3 mm) (Haindl et al 1985;Weber et al 1989;Burghaus et al 1992;Wang et al 1994), around 150 GHz (k % 2 mm) (Grinberg et al 1976;Becerra et al 1995;Tatsukawa et al 1995), at 122 and 244 GHz (Reijerse et al 2007), at 250 GHz (k % 1 mm) (Lynch et al 1988;Earle and Freed 1999), and in the sub-mm region (Muller et al 1989;Barra et al 1990;Tarasov and Shakurov 1991), even reaching 360 GHz/14 T EPR Grishin et al 2004;Schnegg et al 2007). Only a few additional EPR spectrometers operating at frequencies above 200 GHz appeared in the literature until the end of the 1990s (Reijerse et al 1998;Smith et al 1998;Moll et al 1999;Rohrer et al 1999).…”