The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from 13C chemical shift data. A new experimental approach involving multispin 13C labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly 13C biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state 13C magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state 13C-13C correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many 13C resonances of the [U-13C]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of 13C shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.
A new and rapid procedure was developed for the isolation of the reaction center core (RCC)-complex from the green sulfur bacterium Prosthecochloris aestuarii. Reaction center preparations containing the Fenna Matthews Olson (FMO) protein were also obtained. The procedure involved incubation of broken cells with the detergents Triton X-100 and SB12, sucrose gradient centrifugation and hydroxyapatite chromatography. Three different pigment protein complexes were obtained: one containing (about) three FMO trimers per RCC, one with one FMO per RCC and one consisting of RCC only. The last one contained polypeptides with apparent molecular masses of 64 kDa (pscA) and 35 kDa (pscB, the FA/FB, FeS subunit), but no cytochrome. Bacteriochlorophyll a and the chlorophyll a isomer functioning as primary electron acceptor were present at a ratio of 4.8:1. The complexes were also characterized spectroscopically and in terms of photochemical activity, at room temperature as well as at cryogenic temperatures. Illumination caused oxidation of the primary donor P840, with the highest activity in the RCC complex (DeltaA840/A810 = 0.06). At room temperature in the RCC complex essentially all of the P840+ produced in a flash was re-reduced slowly in the dark (several seconds). At low temperatures (150-10 K) a triplet was formed in a fraction of the reaction centers, presumably by a reversal of the charge separation, whereas in others P840+ formed in the light was re-reduced in 40-50 ms.
A new method for portal dosimetry using CCD camera-based electronic portal imaging devices (CEPIDs) is demonstrated. Unlike previous approaches, it is not based on a priori assumptions concerning CEPID cross-talk characteristics. In this method, the nonsymmetrical and position-dependent cross-talk is determined by directly imaging a set of cross-talk kernels generated by small fields ("pencil beams") exploiting the high signal-to-noise ratio of a cooled CCD camera. Signal calibration is achieved by imaging two reference fields. Next, portal dose images (PDIs) can be derived from electronic portal dose images (EPIs), in a fast forward-calculating iterative deconvolution. To test the accuracy of these EPI-based PDIs, a comparison is made to PDIs obtained by scanning diode measurements. The method proved accurate to within 0.2+/-0.7% (1 SD), for on-axis symmetrical and asymmetrical fields with different field widths and homogeneous phantom thicknesses, off-axis Alderson thorax fields and a strongly modulated IMRT field. Hence, the proposed method allows for fast, accurate portal dosimetry. In addition, it is demonstrated that the CEPID cross-talk signal is not only induced by optical photon reflection and scatter within the CEPID structure, but also by high-energy back-scattered radiation from CEPID elements (mirror and housing) towards the fluorescent screen.
A permanent hole burning study on the Fenna-Matthews-Olson, or FMO, antenna complex of the green sulfur bacterium Prosthecochloris aestuarii was carried out at 6 K. Excitation resulted not only in relatively sharp features resonant with the burn wavelength but also in broad absorbance changes in the wavelength region of 800-820 nm. The shape of the latter changes was almost independent of the wavelength of excitation. Evidence is given that they are induced by a different mechanism than that which causes the resonant holes and that they may be due to a conformational change of the protein. The original spectrum was restored upon warming to 60 K. The effective dephasing times T2, as obtained from the homogeneous line widths, increased from about 0.5 ps at 803 nm to >/=20 ps at 830 nm and are in good agreement with recent measurements of accumulated photon-echo and time-resolved absorbance changes.
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