Reaction centers were found to bind two ubiquinones, both of which could be removed by o-phenanthroline and the detergent lauryldimethylamine oxide. One ubiquinone was more easily removed tan the other. The lowtemperature light-induced optical and electron paramagnetic resonance (EPR) changes were eliminated and restored upon removal and readdition of ubiquinone and were quantitatively correlated with the amount of tightly bound ubiquinone. We, therefore, conclude that this ubiquinone plays an obligatory role in the primary photochemistry. The easily removed ubiquinone is thought to be the secondary electron acceptor. The low-temperature charge recombination kinetics, as well as the optical and EPR spectra, were the same for untreated reaction centers and for those reconstituted with ubiquinone. This indicates that extraction and reconstitution were accomplished without altering the conformation of the active site. Reaction centers reconstituted with other quinones also showed restored photochemical activity, although they exhibited changes in their low-temperature recombination kinetics and light-induced ( The presence of stoichiometric amounts of iron in RC's (7) and the observation of a broad electron paramagnetic resonance (EPR) signal (7-9) led to the hypothesis that iron was the primary acceptor. This hypothesis was questioned by Loach and Hall, who reported full photochemical activity in iron-depleted preparations (10). They observed a new, narrow, light-induced EPR signal that was shown to be due to a ubiquinone (UQ) radical (11, 12
Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.
The photocycle of bacterial photosynthetic reaction centers (RCs) involves electron transfer between
two quinone molecules, QA and QB. The semiquinone biradical QA
-•QB
-• forms an intermediate state in this
process. We trapped the biradical at low temperature (77 K) and investigated its EPR spectra at three microwave
frequencies, 9.6, 35, and 94 GHz, at temperatures between 1.5 and 100 K. The spectra were described with a
spin Hamiltonian that contained, in addition to the Zeeman terms, dipolar and exchange interactions, and were
fitted using the simulated annealing method (Kirkpatrick et al. Science
1983, 220, 671). From the parameters
derived from the fit, information about the spatial and electronic structure was obtained. The relative position
and orientation of the two quinones, determined from the EPR spectra, compared well with those obtained
from X-ray diffraction of RCs in the QAQB
-• state (Stowell et al. Science
1997, 276, 812). The values of the
dipolar coupling and of the exchange interaction obtained from the fits were E
d/h = (10.3 ± 0.1) MHz and
J
o/h = (−60 ± 20) MHz, respectively. The value of J
o was used to estimate a maximum electron-transfer rate,
k
ET, (QA
-•QB
-• → QAQB
=) of ∼109 s-1. This agrees within an order of magnitude with the value derived from
kinetics experiments (Graige et al. Biochemistry
1999, 38, 11465).
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