Red Antenna States of Photosystem I from Synechocystis PCC 6803 † Marc Brecht,* Volker Radics, Jana B. Nieder, Hauke Studier, and Robert Bittl Fachbereich Physik, Freie UniVersita¨t Berlin, Arnimalle 14, 14195 Berlin, Germany ReceiVed January 22, 2008; ReVised Manuscript ReceiVed March 20, 2008 ABSTRACT: Single-molecule spectroscopy at low temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the red-shifted chlorophyll a (Chla) molecules responsible for the fluorescence from photosystem I (PSI). Emission spectra of single PSI complexes from the cyanobacterium Synechocystis PCC 6803 show zero-phonon lines (ZPLs) as well as broad intensity distributions without ZPLs. ZPLs are found most frequently on the blue side of the broad intensity distributions. The abundance of ZPLs decreases almost linearly at longer wavelengths. The distribution of ZPLs indicates the existence of at least two pools with maxima at 699 and 710 nm. The pool with the maximum at 710 nm is assigned to chlorophylls absorbing around 706 nm (C706), whereas the pool with the maximum at 699 nm (F699) can be assigned to chlorophylls absorbing at 692, 695, or 699 nm. The broad distributions dominating the red side of the spectra are made up of a low number of emitters assigned to the red-most pool C714. The properties of F699 show close relation to those of F698 in Synechococcus PCC 7002 and C708 in Thermosynechococcus elongatus. Furthermore, a high similarity is found between the C714 pool in Synechocystis PCC 6803 and C708 in Synechococcus PCC 7002 as well as C719 in T. elongatus. Two transmembrane pigment-protein complexes, photo- system I (PSI) 1 and photosystem II (PSII), are essential for the light-driven oxygenic photosynthesis. This study focuses on PSI which transports, after photoexcitation, electrons from reduced plastocyanin or cytochrome c 6 on the luminal side to ferredoxin on the stromal side of the photosynthetic membrane (1). The first step of the light reaction is the absorption of a photon in the antenna system of PSI with subsequent energy transfer to the reaction center. At this point the captured excitation energy is used to induce a trans- membrane charge separation starting from a pigment with main absorption at 700 nm (P700) (1-3). The absorption spectra of PSI from green plants, algae, and cyanobacteria show the existence of chlorophyll a (Chla) molecules absorbing at lower energy than the usual Q y absorption at 680 nm of Chla in solution and, in particular, at lower excitation energy than the primary donor P700. These red- shifted Chlas are often called the “red pools” or the “red- most” chlorophylls [for a review, see Gobets et al. (4) and Karapetyan et al. (5)]. Due to their spectral properties, Chla dimers have been discussed as candidates for the red-pool Chlas (see, e.g., refs 6-8). If the excitation energy is localized within this “red-pool” Chla, the stored energy is no longer sufficient to directly excite P700 to P700*. Additional activation energy, e.g., thermal energy of the phonon bath, is necessary to excite P700 and start the charge separation process. The question concerning the origin of the red shift and the physiological role of the red pool is puzzling (9-11). Even though the PSI complex from cyanobacteria differs in organization and polypeptide content to higher plants (12) and algae, the composition of the core structure of PSI from cyanobacteria and plants shows high similarity (13). Therefore, PSI from cyanobacteria is used to investigate key properties of all types of PSI. The well- resolved structural model for the cyanobacterium Thermo- synechococcus elongatus (T. elongatus, previously Synecho- coccus elongatus) from X-ray crystallography (14-16) is used as a reference structure for PSI in general. For PSI from T. elongatus four red pools with different spectroscopic properties were found. They were called C708, C715, C719, and C735 according to their proposed mean absorption wavelength. Two of those pools (C708 and C719) were identified by absorption (17), one by hole-burning spectros- copy (C715) (18), and one (C735) (19) by single-molecule spectroscopy (SMS). The red absorption in PSI from Synechocystis PCC 6803 at low temperature consists of a single band centered at 708 nm (20). With hole-burning spectroscopy it was shown that the red side of this absorption band shows much stronger electron-phonon coupling than the blue side, suggesting that there are (at least) two states underlying the absorption (18, 21-23). These two states were named C706 and C714, due to their mean absorption wavelength at 706 and 714 nm, respectively. Both show large inhomogeneous broadening and remarkable spectral overlap. Stark hole-burning spectra on PSI (24) suggest a large heterogeneity of the induced dipole moment changes in the close vicinity of the respon- sible chromophores (18) or large variations of the inherent dipole moment change of the Chla species (22). The low- temperature fluorescence spectrum of Synechocystis PCC † This work was supported by Volkswagenstiftung in the framework of the program: Physics, chemistry and biology with single molecules (I/78361). * To whom correspondence should be addressed: e-mail, brecht@ hydrogenase.de; phone, ++49-30-838-56047; fax, ++49-30-838- 56046. 1 Abbreviations: PSI, photosystem I; Chla, chlorophyll a; SMS, single-molecule spectroscopy; ZPL, zero-phonon line; PW, phonon wing. Biochemistry 2008, 47, 5536–5543 5536 10.1021/bi800121t CCC: $40.75  2008 American Chemical Society Published on Web 04/23/2008