On the Conflicting Estimations of Pigment Site Energies in Photosynthetic Complexes: A Case Study of the CP47 Complex

Citation: Reinot, T., Chen, J. H., Kell, A., Jassas, M., Robben, K. C., Zazubovich, V., & Jankowiak, R. (2016). On the Conflicting Estimations of Pigment Site Energies in Photosynthetic Complexes: A Case Study of the CP47 Complex. Analytical Chemistry Insights, 11, 35-48. doi:10.4137/aci.s32151We focus on problems with elucidation of site energies (E-0(n)) for photosynthetic complexes (PSCs) in order to raise some genuine concern regarding the conflicting estimations propagating in the literature. As an example, we provide a stern assessment of the site energies extracted from fits to optical spectra of the widely studied CP47 antenna complex of photosystem II from spinach, though many general comments apply to other PSCs as well. Correct values of E-0(n) for chlorophyll (Chl) a in CP47 are essential for understanding its excitonic structure, population dynamics, and excitation energy pathway(s). To demonstrate this, we present a case study where simultaneous fits of multiple spectra (absorption, emission, circular dichroism, and nonresonant hole-burned spectra) show that several sets of parameters can fit the spectra very well. Importantly, we show that variable emission maxima (690-695 nm) and sample-dependent bleaching in nonresonant hole-burning spectra reported in literature could be explained, assuming that many previously studied CP47 samples were a mixture of intact and destabilized proteins. It appears that the destabilized subpopulation of CP47 complexes could feature a weakened hydrogen bond between the 13(1)-keto group of Chl29 and the PsbH protein subunit, though other possibilities cannot be entirely excluded, as discussed in this work. Possible implications of our findings are briefly discussed

Excited-state structure and energy-transfer dynamics in various photosynthetic antenna complexes: hole-burning and modeling studies

Doctor of PhilosophyDepartment of ChemistryRyszard J. JankowiakNatural photosynthesis has been an inspiration for solving humankind’s urgent demand for replacing fossil energy sources with renewable forms of energy. Knowledge of the molecular mechanisms that lies behind photosynthetic processes is essential for designing novel devices capable of producing solar fuel. Great efforts are being made to understand the first steps of photosynthesis, in particular light-harvesting and excitation energy transfer (EET). In this work, to overcome the static disorder in protein complexes and provide insight into both inhomogeneous and homogeneous line broadening, as well as the excitonic structure and dynamics in various photosynthetic proteins, we use site-selective frequency-domain hole burning (HB) spectroscopy. Complexes studied in detail include: i) wild type (WT) CP29 and CP47 antenna complexes of Photosystem II (PSII), and ii) chlorosome-baseplate proteins of two different green bacteria families (Cb. tepidum and Cfx. aurantiacus). Experimental and modeling results obtained for these complexes shed more light on their excitonic structure and EET dynamics. Simultaneous modeling of various types of optical spectra is based on a non-Markovian reduced density matrix approach. For example, we demonstrate that improved simultaneous fits of absorption, emission, circularly polarized luminescence, circular dichroism, and nonresonant hole-burned spectra, provide new information on the excitonic structure of intact and destabilized CP47 complexes and their lowest energy state(s). Regarding the reconstituted wild-type CP29 protein antenna we show that, depending on the laser excitation frequency, reconstituted complexes display two (independent) low-E states (i.e., the A and B traps) with different HB and emission spectra. We argue that with two subpopulations identified, only the major one corresponds to the native folding of CP29, whereas the minor conformation occurs only in reconstituted complexes. The lowest energy state of the major subpopulation is mostly delocalized over the a611, a612, a615 Chl trimer, and that of the minor one is localized on Chl a604. Studies of the Cb. tepidum and Cfx. aurantiacus baseplates reveal that in both complexes excitation energy is transferred to a localized low-energy trap state near 818 nm with similar rates, most likely via exciton hopping. These data are consistent with the model in which baseplate CsmA proteins are arranged as dimers containing two Bchl a molecules sandwiched between the hydrophobic protein regions

On light-induced photoconversion of B800 bacteriochlorophylls in the LH2 antenna of the purple sulfur bacterium Allochromatium vinosum

The B800-850 LH2 antenna from the photosynthetic purple sulfur bacterium Allochromatium vinosum exhibits an unusual spectral splitting of the B800 absorption band; i.e., two bands are well-resolved at 5 K with maxima at 805 nm (B800R) and 792 nm (B800B). To provide more insight into the nature of the B800 bacteriochlorophyll (BChl) a molecules, high-resolution hole-burning (HB) spectroscopy is employed. Both white light illumination and selective laser excitations into B800R or B800B lead to B800R → B800B phototransformation. Selective excitation into B800B leads to uncorrelated excitation energy transfer (EET) to B800R and subsequent B800R → B800B phototransformation. The B800B → B800R EET time is 0.9 ± 0.1 ps. Excitation at 808.4 nm (into the low-energy side of B800R) shows that the lower limit of B800R → B850 EET is about 2 ps, as the B800R → B800B phototransformation process could contribute to the corresponding zero-phonon hole width. The phototransformation of B800R leads to a ∼ 200 cm–1 average blue-shift of transition energies, i.e., B800R changes into B800B. We argue that it is unlikely that B800-B850 excitonic interactions give rise to a splitting of the B800 band. We propose that the latter is caused by different protein conformations that can lead to both strong or weak hydrogen bond(s) between B800 pigments and the protein scaffolding. Temperature-dependent absorption spectra of B800, which revealed a well-defined isosbestic point, support a two-site model, likely with strongly and weakly hydrogen-bonded B800 BChls. Thus, BChls contributing to B800R and B800B could differ in the position of the proton in the BChl carbonyl-protein hydrogen bond, i.e., proton dynamics along the hydrogen bond may well be the major mechanism of this phototransformation. However, the effective tunneling mass is likely larger than the proton mass

Dichotomous Disorder versus Excitonic Splitting of the B800 Band of Allochromatium vinosum

The LH2 antenna complex of the purple bacterium Allochromatium vinosum has a distinct double peak structure of the 800 nm band (B800). Several hypotheses were proposed to explain its origin. Recent 77 K two-dimensional electronic spectroscopy data suggested that excitonic coupling of dimerized bacteriochlorophylls (BChls) within the B800 ring is largely responsible for the B800 split [M. Schröter et al., <i>J. Phys. Chem. Lett.</i> <b>2018</b>, <i>9</i>, 1340]. Here we argue that the excitonic interactions between BChls in the B800 ring, though present, are weak and cannot explain the B800 band split. This conclusion is based on hole-burning data and modeling studies using an exciton model with dichotomous protein conformation disorder. Therefore, we uphold our earlier interpretation, first reported by Kell et al. [<i>J. Phys. Chem. B</i> <b>2017</b>, <i>121</i>, 9999], that the two B800 sub-bands are due to different site-energies (most likely due to weakly and strongly hydrogen-bonded B800 BChls)

Toward an Understanding of the Excitonic Structure of the CP47 Antenna Protein Complex of Photosystem II Revealed via Circularly Polarized Luminescence

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., <i>Anal. Chem. Insights</i> <b>2016</b>, 11, 35–48] recent new assignments imposed by the shape of the circularly polarized luminescence (CPL) spectrum [Hall, J.; et al., <i>Biochim. Biophys. Acta</i> <b>2016</b>, 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)

Band Structure of the Rhodobacter sphaeroides Photosynthetic Reaction Center from Low-Temperature Absorption and Hole-Burned Spectra

Persistent/transient spectral hole burning (HB) and computer simulations are used to provide new insight into the excitonic structure and excitation energy transfer of the widely studied bacterial reaction center (bRC) of Rhodobacter (Rb.) sphaeroides. We focus on site energies of its cofactors and electrochromic shifts induced in the chemically oxidized (<i>P</i>⁺) and charge-separated (<i>P</i>⁺<i>Q</i>_<i>M</i>^–) states. Theoretical models lead to two alternative interpretations of the <i>H</i>-band. On the basis of our experimental and simulation data, we suggest that the bleach near 813–825 nm in transient HB spectra in the <i>P</i>⁺<i>Q</i>_<i>M</i>^– state, often assigned to the upper exciton component of the special pair, is mostly due to different electrochromic shifts of the <i>B</i>_{<i>L</i>/<i>M</i>} cofactors. From the exciton compositions in the charge-neutral (CN) bRC, the weak fourth excitonic band near 780 nm can be denoted <i>P</i>_<i>Y</i>+, that is, the upper excitonic band of the special pair, which in the CN bRC behaves as a delocalized state over <i>P</i>_<i>M</i> and <i>P</i>_<i>L</i> pigments that weakly mixes with accessory BChls. Thus, the shoulder in the absorption of Rb. sphaeroides near 813–815 nm does not contain the <i>P</i>_<i>Y</i>+ exciton band

Conformational Complexity in the LH2 Antenna of the Purple Sulfur Bacterium <i>Allochromatium vinosum</i> Revealed by Hole-Burning Spectroscopy

This work discusses the protein conformational complexity of the B800–850 LH2 complexes from the purple sulfur bacterium <i>Allochromatium vinosum</i>, focusing on the spectral characteristics of the B850 chromophores. Low-temperature B850 absorption and the split B800 band shift blue and red, respectively, at elevated temperatures, revealing isosbestic points. The latter indicates the presence of two (unresolved) conformations of B850 bacteriochlorophylls (BChls), referred to as conformations 1 and 2, and two conformations of B800 BChls, denoted as B800_R and B800_B. The energy differences between average site energies of conformations 1 and 2, and B800_R and B800_B are similar (∼200 cm^–1), suggesting weak and strong hydrogen bonds linking two major subpopulations of BChls and the protein scaffolding. Although conformations 1 and 2 of the B850 chromophores, and B800_R and B800_B, exist in the ground state, selective excitation leads to 1 → 2 and B800_R → B800_B phototransformations. Different static inhomogeneous broadening is revealed for the lowest energy exciton states of B850 (fwhm ∼195 cm^–1) and B800_R (fwhm ∼140 cm^–1). To describe the 5 K absorption spectrum and the above-mentioned conformations, we employ an exciton model with dichotomous protein conformation disorder. We show that both experimental data and the modeling study support a two-site model with strongly and weakly hydrogen-bonded B850 and B800 BChls, which under illumination undergo conformational changes, most likely caused by proton dynamics

Structure-Based Exciton Hamiltonian and Dynamics for the Reconstituted Wild-type CP29 Protein Antenna Complex of the Photosystem II

We provide an analysis of the pigment composition of reconstituted wild type CP29 complexes. The obtained stoichiometry of 9 ± 0.6 Chls <i>a</i> and 3 ± 0.6 Chls <i>b</i> per complex, with some possible heterogeneity in the carotenoid binding, is in agreement with 9 Chls <i>a</i> and 3.5 Chls <i>b</i> revealed by the modeling of low-temperature optical spectra. We find that ∼50% of Chl <i>b</i>614 is lost during the reconstitution/purification procedure, whereas Chls <i>a</i> are almost fully retained. The excitonic structure and the nature of the low-energy (low-E) state(s) are addressed via simulations (using Redfield theory) of 5 K absorption and fluorescence/nonresonant hole-burned (NRHB) spectra obtained at different excitation/burning conditions. We show that, depending on laser excitation frequency, reconstituted complexes display two (independent) low-E states (i.e., the A and B traps) with different NRHB and emission spectra. The red-shifted state A near 682.4 nm is assigned to a minor (∼10%) subpopulation (sub. II) that most likely originates from an imperfect local folding occurring during protein reconstitution. Its lowest energy state A (localized on Chl <i>a</i>604) is easily burned with λ_B = 488.0 nm and has a red-shifted fluorescence origin band near 683.7 nm that is not observed in native (isolated) complexes. Prolonged burning by 488.0 nm light reveals a second low-E trap at 680.2 nm (state B) with a fluorescence origin band at ∼681 nm, which is also observed when using a direct low-fluence excitation near 650 nm. The latter state is mostly delocalized over the <i>a</i>611, <i>a</i>612, <i>a</i>615 Chl trimer and corresponds to the lowest energy state of the major (∼90%) subpopulation (sub. I) that exhibits a lower hole-burning quantum yield. Thus, we suggest that major sub. I correspond to the native folding of CP29, whereas the red shift of the Chl <i>a</i>604 site energy observed in the minor sub. II occurs only in reconstituted complexes