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Towards an Understanding of the Excitonic Structure of the CP47 Antenna Protein Complex of Photosystem II Revealed via Circularly Polarized Luminescence Mahboobe Jassas, Tonu Reinot, Adam Kell, and Ryszard J Jankowiak J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b00362 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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The Journal of Physical Chemistry

Towards an Understanding of the Excitonic Structure of the CP47 Antenna Protein Complex of Photosystem II Revealed via Circularly Polarized Luminescence Mahboobe Jassas,1,% Tonu Reinot,1,% Adam Kell,1 and Ryszard Jankowiak1,2,*

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Department of Chemistry, Kansas State University, Manhattan, KS 66506, USA 2

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Department of Physics, Kansas State University, Manhattan, KS 66506, USA

Contributed equally to this work

*Corresponding Author: Ryszard Jankowiak, Department of Chemistry, Kansas State University, Manhattan, KS, USA; Tel: 785-532-6785; E-mail: [email protected]

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ABSTRACT: Identification of the lowest energy pigments in the photosynthetic CP47 antenna protein complex of Photosystem II (PSII) is essential for understanding its excitonic structure, as well as excitation energy pathways in the PSII core complex. Unfortunately, there is no consensus concerning the nature of the low-energy state(s), nor chlorophyll (Chl) site energies in this important photosynthetic antenna. Although we raised concerns regarding the estimations of Chl site energies obtained from modeling studies of various types of CP47 optical spectra [Reinot, T; et al., Anal. Chem. Insights 2016, 11, 35–48] recent new assignments imposed by the shape of the circularly polarized luminescence (CPL) spectrum [Hall, J.; et al., Biochim. Biophys. Acta 2016, 1857, 1580–1593] necessitate our comments. We demonstrate that other combinations of low-energy Chls provide equally good or improved simultaneous fits of various optical spectra (absorption, emission, CPL, circular dichroism, and nonresonant hole-burned spectra), but more importantly, we expose the heterogeneous nature of the recently studied complexes and argue that the published composite nature of the CPL (contributed to by CPL685, CPL691, and CPL695) does not represent an intact CP47 protein. A positive CPL695 is extracted for the intact protein, which when simultaneously fitted with multiple other optical spectra, provides new information on the excitonic structure of intact and destabilized CP47 complexes and their lowest energy state(s).

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INTRODUCTION CP47 Complexes. Light absorbing chromophores in various photosynthetic complexes initiate a series of processes which drive many chemical reactions that support life on Earth.1 CP47 is an important photosynthetic antenna complex, being part of the Photosystem II core complex (PSII-cc) with its primary function thought to be light energy absorption and transport of excitation energy to the reaction center. The CP47 complex is a challenging protein to study, as it is more difficult to separate from the PSII-cc than, for example, the accompanying CP43 antenna.2,3 In addition, it is not known to what extent the absence and/or destabilization of the PsbH protein affects the site energies of chlorophylls (Chls) and the resulting emission spectra. It is also not clear whether all (or only some) Chl 29 have a H-bond between their 13ꞌ-keto and threonine (Thr5) of the PsbH protein. This is probably why different optical spectra have been reported over the years, which has led to disagreement about which spectra represent intact CP47 complexes and should be used in modeling studies.4-6 As a result, there is still no agreement as to which Chl(s) contribute to the lowest energy exciton state(s) and what their corresponding site energies are.7-11 While inter-pigment coupling matrix elements can be calculated from available X-ray structures,12,13 the site energies are typically extracted from simultaneous fits of various experimental data.7-9 Quantum chemical approaches can also be used,14-17 but so far the calculated site energies cannot describe experimental data. As a result, the calculated values have to be largely modified by additional shifting to lower- and/or higher-energies, via fitting algorithms.7-11 Nevertheless, it is anticipated that future higher-resolution structural data and more reliable experimental data will further aid in discarding unrealistic site energy sets obtained from the fitting algorithms, in particular when optical spectra of intact samples are analyzed (vide infra). The difficulties in finding Chl a site energies in the CP47 complex are not 3



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surprising, as this protein can be easily destabilized. For example, as mentioned above, CP47 complexes may possess an intact PsbH subunit, which could be also absent or destabilized. In the latter case, the H-bond(s) between one of the CP47 Chls and the amino acid residues of PsbH and/or the H-bond(s) between Chls and water molecules (serving as axial ligands) could be broken or weakened.6 Thus, the key problem concerning the uncertainties in pigment site energies is sample integrity and stability of the complex after isolation. Further Challenges Facing Determination of Chl a Site Energies in CP47. Furthermore, interrogation of CP47 complexes using a large excitation fluence at lowtemperatures may also lead to modification of optical spectra; i.e., the latter can change the shapes of absorption, and, in particular, the shapes of emission and circularly polarized luminescence (CPL) spectra due to reversible hole-burning (HB) and/or irreversible photodamage phenomena.4,5 While the effect of HB can be eliminated by sample annealing, one needs to be mindful of photodamage that is irreversible.4,5 Another important obstacle in providing a unified description of the structure-function relationship in CP47 (or any other photosynthetic complex) is that different shapes of the phonon spectral density, Jph(ω), are used to describe its electronic structure.5,8,10,11 In addition, there is insufficient experimental information on site-dependent inhomogeneities and site-dependent Jph(ω). Therefore, we argued recently that a single solution of pigment site energies based on modeling studies of typically measured optical spectra alone may not exist, and a larger number of experimental constraints is needed to narrow the possible choices; for example, the resulting Hamiltonian needs to be able to describe all frequency- and time-domain data. However, most importantly, the consensus (not available as of yet) must exist as to which spectra constitute an intact/stable complex and should be used in modeling studies.11 To provide more insight we analyze and model multiple spectra 4


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obtained for both destabilized10 (at least in our view; vide infra) and intact4 CP47 complexes to identify the most likely low-energy Chls in intact samples. CPL Spectra. We begin with a discussion of the recently published theoretical fits of the low-temperature CPL and circular dichroism (CD) spectra obtained at liquid helium temperatures for CP47, which, as argued by the authors of ref 10, offer a clear identification of the lowest energy exciton state of intact CP47. While we agree that the CPL spectrum has the advantage in that it is selective for the lowest energy state, the key questions are: i) does the interrogated lowest energy state(s) represent the intact, destabilized, or a mixture of intact and destabilized complexes, especially under experimental conditions used in ref 10; ii) which emission spectrum (whose reported maxima vary in the literature from 689-695 nm,4,5,18-20 with a variable contribution of the so-called 685 nm, 691 nm, and 695 nm emissions4) originates from the lowest energy state of intact/stable CP47 and/or intact PSII-cc; and iii) how reliable is the identification of the low-energy Chls based on the fits of the CPL and CD spectra reported in ref 10, that is, is there a unique solution to account for the lowest energy pigment, i.e., Chl 11, as suggested in ref 10? What is the Assertion of This Work? We suggest that in contrast to the conclusions of ref 10, the CP47 complex studied by Hall et al. was likely structurally modified during or after the isolation procedure (i.e., the sample was heterogeneous), and for that reason its optical spectra cannot be considered to represent intact/stable complex, and as a result should not be directly used in modeling studies. The complex shape of the CPL curve is in perfect agreement with the variable 685 nm, 691 nm, and 695 nm contributions observed over the years in the CP47 emission spectra.4 Although in the main part of this work we focus on intact CP47 complexes (again, in our opinion), for the sake of argument we will also fit spectra reported by Hall et al.10 5



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using our algorithm; see Supporting Information (SI). However, by doing this, we will demonstrate that in contrast to their fit different sets of low-energy Chls provide improved or equally good fits of their optical spectra. In summary, we raise concerns about the recently proposed interpretation and innovative implications suggested for new understanding of the excitonic structure of isolated CP47 complex and the entire PSII-cc, and proposed regulation of energy transfer, as described in ref 10. An alternative view is proposed, emphasizing the new interpretation of the recently published CPL spectrum. Finally, the implications of our new assignments are also briefly discussed.

METHODS To test the key assertion of ref 10 we first model their optical spectra, focusing, as an example, on the newly measured CPL spectra. Our modeling approach is described in ref 11; in brief, the disorder is introduced into the diagonal matrix elements (i.e., ) by a Monte-Carlo approach with normal distributions centered at (n labeling various pigments, i.e., n = 1-16) and with fwhm representing Γinh, which can be site-dependent or independent. Eigen decomposition of the interaction matrix provides eigen-coefficients ( ) and eigenvalues ( ). Phonon and vibrational Huang-Rhys (S) factors are used as free or fixed parameters and are optimized simultaneously against the experimental spectra. We use experimentally determined phonon spectral density Jph(ω)11 and vibrational spectral density Jvib(ω).8 Intramolecular vibrational modes (Jvib) are dynamically localized21 and only contribute to absorption, HB, linear dichroism (LD) and fluorescence spectra.22

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For easy comparison, the Chls within the CP47 complex are divided into the same five excitonic domains of coupled Chls as in ref 10 with Vc = 30 cm-1. In this case, the Poisson-TrEsp coupling matrix elements (based on the 1.9 Å crystal structure of PSII from cyanobacteria, PDB ID: 3ARC12) are used in all modeling studies. Isolated Chls are placed in separate domains. We also use somewhat larger TrEsp Vnm values with Vc = 30 cm-1 which leads to three domains. We hasten to add that a division into well-defined domains is not straightforward and often impossible. However, two criteria for defining domains have been proposed,23 both relating to circumstances which can cause localization of the excited state. That is, pigment m is considered in a domain if the following is true for the coupling constant of pigments m and n, where n is already assigned to a particular domain, i.e., 1) |Vnm| is larger than the coupling cutoff Vc (which is on the order of the reorganization energy); and 2) |Vnm| > |(En – Em)|/6 (with E representing each pigment’s site energy). These criteria are simple approximations for the consideration of dynamic localization and localization due to large energetic separation. The latter is critical when inhomogeneous broadening is large or weakly coupled Chls have very different site energies. Thus, three cases (A, B, and C) could be considered. In Case A, the complex is divided into five domains as in ref 10 using Poisson-TrEsp Vnm and the domain cutoff of Vc = 30 cm-1 with excitons delocalized over an entire domain. In Case B we also put all intradomain Vnm values smaller than Vc = 30 cm-1 to zero. Finally, Case C models spectra with TrEsp Vnm assuming that both intra- and interdomain Vnm < Vc are set to zero. The results obtained for all cases are compared below. The intermediate case, which would explicitly consider disorder within the second criterion (|Vnm| > |(En – Em)|/6) (taking into account the inhomogeneous broadening for all Chls in the Monte Carlo simulations), is beyond the scope of this work, as, in the first

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approximation, we anticipate that the resulting fits should lie somewhere between the above mentioned limiting cases. In simulations we used a non-Markovian reduced density matrix theory24 with a NelderMead Simplex algorithm for parameter optimization.25 It is assumed that the phonon spectral density (weighted phonon profile) can be described by a continuous function, which is chosen to be a lognormal distribution,26 =

√

(1)

where ! is the cutoff frequency, " is the standard deviation, and ≤ 0 = 0. The real and imaginary parts of the Fourier transfer of the energy gap correlation function can be described in terms of the spectral density as % &' = ( )*1 + - . + - − −0 < 6 78 9

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% 12 = 4 5

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