Analysis of the optical absorbance spectra of D1-D2-cytochrome b-559 complexes by absorbance-detected magnetic resonance : Structural properties of P680

Abstract

Absorbance-detected magnetic resonance (ADMR) spectroscopy was applied to D1-D2-cyt b-559 complexes isolated by different isolation methods. The triplet of the primary donor ( 3P680) does not show a homogeneous response to the microwaves; the maximum of the triplet-induced long-wavelength bleaching varied from 678 to 685.5 nm, whereas the | D|-value changed from 0.0293 to 0.0281 cm −1 with increasing monitoring wavelength within the bleaching band. The | E|-value was 0.0043 cm −1 for all detection wavelengths. Triplet-minus-singlet (T — S) difference spectra monitored with ADMR did not show any indication for a dimeric structure of P680. Linear dichroic (LD) ADMR applied to P680 yielded angles between the optical Q y transition moments and the triplet x-, y- and z- axes of 49° ± 2°, 32° ± 4° and 65° ± 3°, respectively. From LD-ADMR applied to chlorophyll a (Chl a) in vitro corresponding angles of 55° ± 6° and 43° ± 7° for the triplet x- and y-axes, respectively, were obtained. With the value of 30° ± 3° for the angle between the plane spanned by the triplet x- and y-axes and the membrane plane obtained by Van Mieghem et al. (Van Mieghem, F.J.E., Satoh, K. and Rutherford, A.W. (1991) Biochim. Biophys. Acta 1058, 379–385), it follows that the Q y optical-transition moment of P680 makes an angle with the membrane plane of 36° ± 8°. This angle is quite different from the angles calculated from the crystal structure for the dimeric or monomeric bacteriochlorophyll Q y transition moments of bacterial reaction centers (RCs). From the close correspondence between the relative orientation of the triplet axes and the Q y transition moment for both 3P680 and 3Chl in vitro as determined with LD-ADMR and the shape of the T — S spectra, which reflect a monomeric Chl a triplet state, it is concluded that P680 has little, or no interaction with other pigments from the D1-D2-cyt b-559 complex. Our observations contrast strikingly with our previous ADMR experiments on RCs of purple bacteria (Lous E.J. and Hoff, A.J. (1988) Proc. Natl. Acad. Sci. USA 84, 6147–6151) and strongly suggest that the analogy between the D1-D2-cyt b-559 complex and the RC of purple bacteria observed for the biochemical components of the complex does not extend to the conformation of the pigments. An additional triplet ascribed to a pheophytin (Pheo) was observed, having | D| = 0.0338 cm − and | E| = 0.0021 cm −1. The bleaching due to the Pheo-triplet is maximal at 680.0 ± 0.2 nm; at least one Pheo a present in the complex has its absorption maximum at this wavelength. Double-resonance experiments showed that the formation of 3Pheo depends on the population of 3P680. Simulation of the T — S spectra of 3P680 with Gaussian bands revealed three types of P680, two of which could be due to freezing in slightly different variations of a basically similar conformation. One type differs considerably from the other two with respect to the width of the bleaching, the shape of the T — S spectrum and its influence on Pheo a-triplet formation and is likely to have a conformation different from the other two. The T — S spectrum of degraded samples, composed of a mixture of intact and degraded RCs characterised by an absorption maximum below 674 nm, does not differ from that of fresh samples, which, at room temperature, have an absorption maximum at 676 nm. Together with the observation that the triplet yield of degraded samples is much lower, this indicates that for degraded RCs the triplet formation is completely inhibited.