Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas reinhardtii

Abstract

It is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction center (RC) preparations is pheophytin a (Pheo a) within the D1 protein (Pheo(D1)), while Pheo(D2) (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the past two decades placed the Q(y)-states of Pheo(D1) and Pheo(D2) bands near 678-684 and 668-672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986 - 998; Cox et al. J. Phys. Chem. B 2009, 113, 12364 - 12374] of the electronic structure of the PSII RC reversed the assignment of the active and inactive Pheos, suggesting that the mean site energy of Pheo(D1) is near 672 nm, whereas Pheo(D2) (~677.5 nm) and Chl(D1) (~680 nm) have the lowest energies (i.e., the Pheo(D2)-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Q(y) absorption maxima at 676-680 nm [Germano et al. Biochemistry 2001, 40, 11472 - 11482; Germano et al. Biophys. J. 2004, 86, 1664 - 1672]. To provide more insight into the site energies of both Pheo(D1) and Pheo(D2) (including the corresponding Q(x) transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch Pheo(D1) is genetically replaced with chlorophyll a (Chl a). We show that the Q(x)-/Q(y)-region site energies of Pheo(D1) and Pheo(D2) are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803 - 8814]. The latter values should be used to model excitonic structure and excitation energy transfer dynamics of the PSII RCs.

Critical assessment of the emission spectra of various photosystem II core complexes

“…We proposed recently (Acharya et al 2012a) that the NPHB process in isolated RCs of PSII can also take place Photosynth Res during the stage when the continuous wave laser is on, and the primary electron donor is in the long-lived 3 Chl D1 triplet (Renger and Schlodder 2010), although due to a broad distribution of CT rates the slower charge separation kinetics may also contribute to persistent hole formation (Riley et al 2004). Thus it is feasible that the persistent nonphotochemical holes burned in isolated RCs could correspond to a bleach of the briefly decoupled Pheo D1 (during the long-lived triplet and/or charge-separated states if Q A is present), which could explain why the shallow persistent holes burned in isolated PSII RCs (within the zero-phonon hole action spectrum) revealed an excited state lifetime close to the Pheo radiative lifetime of a few ns (Zazubovich et al 2003) even though charge separation occurs on a ps time scale (Acharya et al 2012b;Chauvet et al 2015;Kwa et al 1994).…”

“…3 in Supporting Information), we predict that the origin band of the Pheo D1 emission (in PSII-cc and PSII-m) should be much narrower (as observed) than the corresponding emission band observed in isolated RCs. This is because in isolated RCs, in the absence of the long wavelength CT state(s) and in most cases with Q A lost, the charges may also recombine to the excited state of the Chl exciton leading to additional emission and resulting in broadening of the total fluorescence band (Acharya et al 2012a, b). In fact, it has been shown that emission (4-77 K) of isolated PSII RC from spinach has signature modes of both Chl and Pheo, although the Pheo contribution is at most 10 % (Konermann et al 1997;Kwa et al 1994;Peterman et al 1998).…”

“…Pheo D1 -, which after fast recombination forms a long-lived 3 Chl D1 (*2 ms) (van Kan et al 1990). Assuming that Pheo D1 contributes to exciton states near 680-684 nm in isolated RCs (Acharya et al 2012a;Chauvet et al 2015) and/or near 685 nm in PSII-cc/PSII-m (Masters et al 2001), Pheo D1 could be temporarily decoupled during the long-lived triplet. That is, excitation of this temporarily decoupled pigment could constitute an energy trap for EET from states excited by CW laser at *665 nm.…”

“…However, many critical aspects of PSII have remained controversial for years. This includes the nature of the charge-separating assembly (Acharya et al 2012a, b;Jankowiak et al 2011Jankowiak et al , 2012Lewis and Ogilvie 2012;van der Weij-der Wit et al 2011), the assignment of pigments contributing to the lowest energy states and site energies of cofactors in the reaction center (RC). Further open questions include the site energies of the antenna chlorophylls (Chls) coordinated by the protein subunits CP43 (Müh et al 2012;Raszewski and Renger 2008;Reppert et al 2008;Shibata et al 2013) and CP47 (Raszewski and Renger 2008;Reppert et al 2010;Shibata et al 2013), as well as details of the excitation energy transfer (EET) pathways that connect them, and the shape and origin of PSII fluorescence emission (Andrizhiyevskaya et al 2005;Brecht et al 2014;Hughes et al 2007;Krausz et al 2005;Masters et al 2001;Mathis 1996;Shibata et al 2013;van Dorssen et al 1987;Wang et al 2002).…”

Pseudo-Rephasing and Pseudo-Free-Induction-Decay Mechanism in Two-Color Three-Pulse Photon Echo of a Binary System

“…In this paper, we propose an alternative mechanism based on pseudo-rephasing and pseudo-free-induction-decay (FID) in a binary system and understand the negative peak shift as the result of macroscopic interference 1,16 . We evaluate the effect of the electronic energy gap and electronic coupling constants asymmetry between the two components of the binary system [17][18][19][20][21] . A specific example of such a system is the photosynthetic reaction center, which in all known cases posses an approximate C 2 symmetry.…”