1 The Trapping Dynamics in Photosystem I- Light Harvesting Complex

Subscriber access provided by UNIV NEW ORLEANS

Article

The Trapping Dynamics in Photosystem I- Light Harvesting Complex I of Higher Plants Is Governed by the Competition Between Excited State Diffusion From Low Energy States and Photochemical Charge Separation Egle Molotokaite, William Remelli, Anna Paola Casazza, Giuseppe Zucchelli, Dario Polli, Giulio Cerullo, and Stefano Santabarbara J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07064 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

The Trapping Dynamics in Photosystem I- Light Harvesting Complex I of Higher Plants is Governed by the Competition Between Excited State Diffusion from Low Energy States and Photochemical Charge Separation. Egle Molotokaite1, William Remelli1§, Anna Paola Casazza2, Giuseppe Zucchelli1, Dario Polli3,4, Giulio Cerullo3*, Stefano Santabarbara1* 1

Centro Studi sulla Biologia Cellulare e Molecolare delle Piante, CNR, Via Celoria 26, 20133 Milan,

2

Italy; Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via Bassini 15a, 20133 Milano, Italy; 3Istituto di Fotonica e Nanotecnologie del CNR, Dipartimento di Fisica, Politecnico di Milano, P.zza Leonardo da Vinci 32, 20133 Milano, Italy;

4

Center for Nano Science and Technology at

Polimi, Istituto Italiano di Tecnologia, Via Giovanni Pascoli, 70/3, 20133 Milano, Italy.

*To whom correspondence should be addressed.

S.S. Centro Studi sulla Biologia Cellulare e Molecolare delle Piante, CNR, Via Celoria 26, 20133 Milan, Italy. Tel: + 39 02 503 14857; e-mail: [email protected] G.C. Dipartimento di Fisica, Politecnico di Milano, P.zza Leonardo da Vinci 32, 20133 Milano, Italy; e-mai: [email protected] §

present address: Sacco S.r.l., Via A. Manzoni 29/A, 22071 Cadorago (CO), Italy

List of Abbreviations: PSI, Photosystem I; Chl, Chlorophyll; LHCI, Light Harvesting Complex I; RC, Reaction center; ER, electron transfer; RP, radical pair; TCSPC, Time-correlated Single Photon Counting; FWHM, full-width at half-maximum; IRF, Instrument Response Function; DA(D)S, Decay Associated (difference) Spectrum; SA(D)S, species associated (difference) spectrum; GSB, ground state bleaching.

1 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract. The dynamics of excited state equilibration and primary photochemical trapping have been investigated in the Photosystem I-Light Harvesting Complex I isolated from spinach, by the complementary time-resolved fluorescence and transient absorption approaches. The combined analysis of the experimental data indicates that the excited state decay is described by lifetimes in the ranges of 12-16 ps, 32-36 ps and 64-77 ps, for both detection methods, whereas faster components, having lifetimes of 550-780 fs and 4.2-5.2 ps, are resolved only by transient absorption. A unified model capable of describing both the fluorescence and the absorption dynamics has been developed. From this model it appears that the majority of excited state equilibration between the bulk of the antenna pigments and the reaction center occurs in less than 2 ps, that the primary charge separated state is populated in ~4 ps and that the charge stabilisation by electron transfer is completed in ~70 ps. Energy equilibration dynamics associated with the long wavelength absorbing/emitting forms harbored by the PSI external antenna are also characterized by a time mean lifetime of ~75 ps, thus overlapping with radical pair charge stabilization reactions. Even in the presence of a kinetic bottleneck for energy equilibration, the excited state dynamics are shown to be principally trap-limited. However, direct excitation of the low energy chlorophyll forms is predicted to lengthen significantly (~2-folds) the average trapping time.


Page 2 of 41

Page 3 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Photosystem I (PSI) is a large macromolecular, multi-subunit, cofactor-protein supercomplex that is a fundamental component of photosynthetic electron transport in oxygen evolving phototrophic organisms e.g. 1-3. In land plants, PSI is present in the thylakoids membrane in the form of a supercomplex (PSI-LHCI) consisting in two functional units, a core complex and an external light-harvesting complement, LHCI e.g. 2-5. LHCI is composed of several transmembrane Chlorophyll (Chl) a/b-carotenoid-binding complexes organized in pairs of dimers, Lhca2/3 and Lhca1/4, and located on one side of the Core complex in a crescent-like shape4,5. LHCI of land plants harbors the lowest energy Chl spectral forms of the photosystem (reviewed in refs. 6, 7), commonly referred to as “red forms”, absorbing at lower energy than the photochemical reaction center (RC). The RC, coordinated by the core complex, absorbs light at about 700 nm, whereas the several red spectral forms identified in LHCI absorb in the 710-730 nm window e.g. 8-11. The PSI core complex contains the large PsaA and PsaB subunits, which form a heterodimer that binds the vast majority of light harvesting cofactors (∼ 90 Chls a, 22 β-carotenes), those involved in the electron transfer (ET) reactions (6 Chls a, 2 phylloquinones) as well as a 4Fe-4S cluster, known as FX. Two other [4Fe-4S] clusters, FA and FB, act as terminal electron transfer acceptors and are coordinated by the PsaC subunit1,12. Most of the bound chromophores of the core complex serve as an internal antenna, transferring the energy of the absorbed photons to the RC, where photochemical charge separation takes place. The photochemical RC of PSI is composed by at least six Chl a molecules organised as three pseudo-dimers (see Figure 1 for a graphic representation). One pair, bound at the PsaA:PsaB interface, is almost perpendicular to the complex symmetry axis1,12 and is identified as the terminal electron donor, P700. The two other pairs contain the so-called accessory Chl (otherwise referred as Chl eC2 according to the structure nomenclatue12) and the Chl electron acceptor A0 (or Chl eC3). Each eC2/eC3 Chl pair, which is tilted by about 30 degrees with respect to the symmetry axis1,12, is part of one of the two ET chains that, at odds with RCs of purple bacteria and Photosystem II, are both redox active (reviewed in refs.13-16). Charge separation from the RC results in the population of the [P700+A0-] radical pair in less than 20 ps e.g. 13,14,17-20

. The electrons are then transferred to the next acceptor, A1, a phylloquinone molecule,

forming the [P700+A1-] radical pair. The A1 acceptor is oxidized with polyphasic kinetics13-16, 21 by the common acceptor to both ET chains, FX, coordinated at the PsaA:PsaB interface12. The electrons are then transferred sequentially to FA and FB, which ultimately reduces the soluble electron carrier Ferredoxin at the stromal side of the thylakoid membrane. Although the structural, biochemical, spectroscopic and functional properties of PSI have been intensively investigated, there remain some open issues concerning the details of energy 3 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 41

transfer and primary photochemical reactions. These aspects are of particular interest, considering that PSI possesses the highest photochemical quantum conversion efficiency amongst photosystems, generally estimated to be larger than 0.95 3, 7 and often larger than 0.98 e.g. 22-24. The main points of debate, that relate to the interpretation of ultrafast transient absorption and fluorescence lifetime experiments used to study these processes, are: i) the impact of excited state diffusion processes, mainly due to the presence of the red spectral chlorophyll forms, on the overall kinetics of photochemical energy conversion and ii) the mechanisms and pathways of primary charge separation. In a simplified framework, two limiting case scenarios have been discussed in the literature to interpret fluorescence lifetime e.g. 25-42 and transient absorption results e.g. 43-55. In one case, photochemical energy trapping is determined almost exclusively by the photochemical reactions themselves, whereas energy transfer and equilibration in the antenna matrix are significantly faster (trap-limited scenario e.g. 20, 35, 37, 49, 51, 53). In the other, the overall time required by the excited state energy to reach the photochemical RC is comparable, or longer, than the photochemical reactions and, therefore, limits the kinetics of trapping (transfer-to-trap limited scenario e.g. 6, 19, 31, 40, 46) . The other point of contention, not entirely disconnected from the first, relates to the mechanisms and the kinetics of primary photochemistry. Two reaction mechanism are currently discussed (Figure 1). In the first mechanism, defined as “one-step” for briefness, photochemistry takes place populating the [P700+A0-] radical pair directly from the excited state of P700

e.g. 6, 13, 17-20

.

The so-called accessory eC2 Chls (Chlacc) do not act as redox active chromophores but would be either an assisting bridge in the transfer from P700 to A0 or a constituent of the A0 acceptor itself by forming a functional redox dimer (Chl eC2:eC3). The latter scenario would agree with the original suggestion of Shuvalov and coworkers of A0 being a Chl dimer 56,57. In the “two-step” mechanism, [P700+A0-] is populated via two consecutive radical pairs instead (Figure 1). In this case, the eC2 Chls have to act as proper ET intermediates51 and it has been proposed that they may represent the site of primary charge separation51, 53. The primary radical pair was considered to be populated in ~3-6 ps, followed by the formation of P700+A0- in about 20 ps51,53. This suggestion was advanced, mainly, by the analysis of ultrafast transient absorption data recorded on the PSI core complex isolated from the green alga Chlamydomonas reinhartii51 as well as by the comparative study of site directed mutants affecting the coordination of A053. Yet, recent reinvestigation of PSI core complex isolated from cyanobacteria concluded that charge separation is characterized by lifetimes of ~150 fs55, in agreement with the previous suggestion of Beddard and co-workers20, who investigated the core complex isolated from higher plants.


Page 5 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Some of the above-mentioned discrepancies might relate either to the fact that the investigated PSI supercomplexes are isolated from different organisms or to the application of different spectroscopic approaches. This, in turn, might affect the modeling of the experimental data required to extract the actual kinetic rates from the measured decay of either absorption or fluorescence. For the case of higher plants, the whole PSI-LHCI supercomplex has been principally studied employing fluorescence lifetime techniques6, 7, 22, 24, 29, 36, 37, 39, 40, whereas transient absorption has been most commonly applied to the PSI core complex alone28, 43, 44. Since, differently from cyanobacteria, the core complex of higher plants and green algae is almost completely depleted in red Chl form content8, the impact of low energy states in the antenna matrix is not fully understood in the PSI-LHCI supercomplex. In this study, to address some of these controversies, we investigate the excited state dynamics in the PSI-LHCI supercomplex isolated from spinach monitoring both time-dependent fluorescence and transient absorption. The combination of these two spectroscopic approaches yields complementary as well as common pieces of information. Transient absorption measurements have a significantly higher temporal resolution (≈100 fs vs. ≈10 ps); in addition, they allow us to observe processes that are only indirectly monitored by fluorescence, such as the formation of radical pairs, that possess close-to-null fluorescence yield. Instead, fluorescence detection has a higher sensitivity for excited state de-activation kinetics in the antenna bed, where the excited state resides, statistically, for most of its lifetime. This is even more the case in PSI, due the presence of the red forms that, being the lowest energy states, have an overall higher population probability with respect to the bulk of the antenna chromophores. We took advantage of the complementary information resulting from these approaches, developing unified kinetic models that simultaneously reproduces transient absorption and time-dependent fluorescence data sets. These models are then capable of describing both the excited state dynamics in the antenna and the charge-separation reactions. We found that the model that most satisfactorily describes PSI-LHCI of spinach considers charge separation occurring through a sequential two-step process, when the two active ET chains of PSI are not considered explicitly. When bidirectional ET is considered explicitly instead, the population of two parallel, secondary, radical pairs describes the data, albeit less satisfactorily. Irrespectively of the exact charge separation model considered, the excited state dynamics appears to be mainly trap-limited, although longer components due to de-excitation of the red forms are present.

Experimental Methods



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Purification of PSI-LHCI. PSI-LHCI was purified from spinach leaves bought at the local market. The leaves, washed in distilled water and dark adapted for at least one hour before the preparation, were grinded in an ice cold buffer containing 0.4 M Sucrose, 30 mM Tricine-NaOH (pH 7.8), 10 mM NaCl, with a ratio of 1:10 w/v. The slur was filtered through eight layers of cheesecloth, and, successively through a 20 µm nylon membrane. The filtrate was centrifuged at 1500 g for 5 minutes at 4 °C. The pellet was resuspended and incubated in 20 mM Tricine-NaOH (pH 7.8), 10 mM NaCl, 1 mM EDTA (sodium salt) for two minutes, after which twice the volume of 0.4 M Sucrose, 30 mM Tricine-NaOH (pH 7.8), 10 mM NaCl was added to the suspension. The material was then centrifuged at 1500 g for 10 minutes and the last step repeated. The pellet contained unstacked thylakoid membranes, which were then suspended in ice cold double distilled water, and diluted to an equivalent Chl concentration of 2 mg ml-1. All procedures were performed in the dark, or in the presence of very dim green light, in a ventilated cold room at 4 °C. Unstacked thylakoids were solubilized at concentration of 1 mg ml-1 with 1.5 % w/v OctylGlucopyranoside (OGP). The suspension was gently stirred for 20 minutes, in complete darkness and on ice. The solubilised material was centrifuged for 20 min at 5500 g at 4 °C, to remove nonsolubilized material, which was anyway less than 2% of the total. The supernatant was then loaded on a 0.1-1.1 M sucrose density gradient (5 mM Tricine-NaOH pH 7.8 and 0.8% w/v OGP buffer), layered on a 2 M sucrose cushion and centrifuged for 24 h at 40000 RPM in a SW41 swinging bucket rotor (Beckmann) at 4 °C. The lowest band containing the PSI-LHCI supercomplex (see Supporting Information Figure S1) of the gradient was collected with a syringe, immediately diluted in 5 mM Tricine NaOH, 5 mM NaCl buffer and then concentrated by several gravity assisted microfiltration steps (Amicon, Centricon). The samples were either used directly for further biochemical and spectroscopic characterisations or flash frozen in liquid nitrogen and stored at -80 °C. Bidimensional denaturing electrophoresis. 2D-PAGE of purified PSI-LHCI was performed as described in Croce et al.58: the separation in the first dimension was performed in SDS-6 M ureaPAGE using the Tris-Glycine/Tris-Sulphate buffer system IV described by Machold et al.59, on a 12-18% acrylamide gradient gel (160 × 120 × 1.5 mm). For the second dimension a slice of the Tris-Sulphate-PAGE was excised and fitted on a 12-18% acrylamide gradient gel, run using the Tris-Tricine buffer system described by Schägger and von Jagow60. Gels were stained with blue Coomassie (0.25% w/v Coomassie Brilliant Blue R-250 in 10% v/v ethanol and 10% v/v glacial acetic acid). Steady State Spectroscopy. Absorption and fluorescence emission spectra were recorded in a laboratory-assembled spectrofluorimeter previously described10, equipped with a liquid nitrogen6 Environment ACS Paragon Plus

Page 6 of 41

Page 7 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cooled CCD camera (Princeton Applied Research, LN/CCD-ST138), coupled to a spectrometer (SpectraPro300i), as a detector. For fluorescence emission spectra, the excitation wavelength was, unless otherwise specified, 435 nm (full width at half maximum, FWHM, 2 nm), which was further filtered by a band-pass filter (Corning CS 4-96) and the CCD detector was protected from residual stray light by a cut-on filter (OG550, Schott). The spectrometer was calibrated with a Neon lamp (Cathodeon) and the spectra were corrected for the wavelength sensitivity of the detector. For absorption measurements, the sample was diluted to an optical density of 0.7 cm-1 at the maximal absorption in the Qy band (680 nm) and reduced to 0.05 cm-1 in fluorescence measurements to avoid artifacts due to re-absorption. Low-temperature emission spectra were acquired using a flow cryostat (Oxford mod. Optistat CF). The temperature was controlled by an ICT-503 (Oxford Instruments) unit. The sample, placed in either a 1-cm path-length quartz (room temperature) or Perspex (low temperature) cuvette, was suspended in a buffer with 10 mM Tricine pH 7.8, 5 mM NaCl, which for lowtemperature measurements also contained 70% (w/v) glycerol to obtain transparent matrixes upon cooling. The sample was added to the glycerol-containing buffer immediately before being placed in the cryostat to avoid possible perturbations of the supercomplex61. Time-Resolved Fluorescence. The excited state decay kinetics were recorded in a laboratory assembled set-up, which has been previously described in detail62,63, using the Time-Correlated Single-Photon Counting (TCSPC) technique. In brief, the detector was a cooled microchannel plate photomultiplier (MCP-PMT, Hamamatsu, R5916U-51). The excitation source was a pulsed diode laser (PicoQuant 800B), centered at 632 nm and operated at a repetition rate of 20 MHz, yielding an energy of 2 pJ/pulse. The instrument response function (IRF), measured using 1,1’-Diethyl-2,2’carbocyanine Iodide, was 110 ps FWHM63, which allows us to resolve lifetimes of ~10 ps, after numerical deconvolution. The samples, at an optical density of 0.1 cm-1 at 680 nm, were placed in a 3 mm pathlength cuvette, held at 10° C. Phenazine Methosulfate PMS (10 µM) and Ascorbate (Sodium salt, 300 µM) were added to the incubation buffer to ensure that P700 remains in its neutral state during the measurements. Acquisition of TCSPC traces was performed following two strategies: i) keeping the acquisition time constant at each detection wavelength, in which case the number of integrated counts under the time traces is directly proportional to those detected in steady-state measurements, and ii) adjusting the acquisition time to acquire approximately the same number of counts (3.5-4 104) at the peak channel, with the aim of producing the same level of residual statistics during data analysis. In the latter case, normalization to the acquisition time rescales the amplitude to the steady state fluorescence emission, since photon counting is operated under linear regime and the number 7 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of background (dark) counts is several orders of magnitude lower than the number of actual photons collected. Transient absorption. The laser system employed for the transient absorption measurements is based on Ti:Sapphire chirped-pulse amplified source (Coherent Libra), with maximum output energy of ~4 mJ, 1 kHz repetition rate, central wavelength of 800 nm and pulse duration of ~100 fs. Tunable excitation pulses were obtained by a home-built optical parametric amplifier, thus allowing the selection of narrowband (~10 nm) pump spectra in the 480-650 nm wavelength range. Most of the experiments were performed using as pump wavelength either 620 nm, thus exciting preferentially the Qy vibronic (0,1) transition bands (unselective excitation), or 490 nm, thus exciting preferentially carotenoids and Chl b. The white-light continuum, used as a broadband probe pulse covering the 450-740 nm wavelength range, was generated by focusing the fundamental beam into a thin sapphire plate. The pump and probe pulses were time delayed with respect to each other via a motorized computercontrolled translation stage. The relative polarizations of the pump and probe pulses were set to magic angle. They were then recombined and focused into the sample cell to a ~150 µm diameter spot, after which the probe beam was sent to the spectrometer equipped with a CCD sensor64. In order to obtain the differential transmission spectrum, the pump beam was modulated at 500 Hz by a chopper wheel, synchronized with the laser repetition rate. Differential transmission spectra were obtained from CCD readings with ( Ton (λ , t ) ) and without ( Toff (λ ) ) the pump pulse hitting the sample at a given pump-probe delay ( t ), and presented as ∆T / T (λ , t ) = [Ton (λ , t ) − Toff (λ )] / Toff (λ ) . The samples, at an optical density of ~8 cm-1 at 680 nm, were placed in a 1 mm pathlength quartz cuvette and held at room temperature. The pump pulse energy was attenuated down to 10-20 nJ to avoid non-linear processes such as singlet-singlet annihilation, as tested by the excitation power-dependence in the 5-40 nJ/pulse energy range. Moreover, 10 mM Ascorbate (Sodium salt) and 60 µM PMS were added as redox agents and the cuvette was continuously translated in a plane perpendicular to the laser beam, to ensure that the primary donor P700 remains in its neutral state during the measurements. Data Analysis. TCSPC time traces were fitted by convoluting the decay model function, consisting in a sum of weighted exponential functions, with the measured IRF. The global analysis approach, which considers the decay lifetimes, (τ i ), as wavelength-independent parameters and the pre-exponential amplitudes, Ai (λ ) , as wavelength-dependent parameters, was used and implemented by a laboratory developed software24, 63. The algorithm minimizes the (global) reduced

χ 2 and allows for the analysis of the errors associated with the fit parameters. Ai (λ ) describes the 8 Environment ACS Paragon Plus

Page 8 of 41

Page 9 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Decay Associated Spectrum (DAS) of the i-th lifetime. The DAS presented are corrected for the MCP-PMT detector spectral sensitivity. The correction function was determined by comparing the reconstructed fluorescence emission spectra for a range of dyes having simple monoexponential decay kinetics with their corrected steady-state fluorescence emission measured in the CCD set-up described above. This procedure yields good agreement between reconstruction of the steady-state spectra from TCSPC analysis and measured steady-state emission, provided that the fit description is adequate22-24, 65. The latter is expected when the fit of TCSPC traces contains only decay components (i.e. positive amplitudes). Deviations can be observed when rise components (i.e. negative amplitudes) are present, particularly when these are large, as they might lead not only to a discrepancy between measured and reconstructed steady-state spectra, but even to reconstructed negative fluorescence emission. When these kinds of unphysical fit solutions were retrieved, they were discarded, albeit yielding a good numerical description of the TCSPC traces. Transient absorption traces were also fitted by a global fit approach66, 67, considering the convolution with a 100-fs Gaussian IRF, by a laboratory written software based on the one previously described24, but which included a correction for probe pulse chirp, implemented as a wavelength-dependent time shift approximated by a quadratic function. Before fitting, the experimental maps were binned initially in 5 nm wavelength intervals, and then refined to 2.5 nm. The software minimizes the sum of the squared residues weighted, at each monitored wavelength (after binning), for the statistical noise in the pre-pulse time window. The outputs of the fit are the wavelength-independent lifetimes (τ i ) and their associated, wavelength dependent, pre-exponential amplitudes ( ∆Ai (λ ) ), which define the Decay Associated Difference Spectra (DADS). Kinetic Modeling. The kinetics of excited-state relaxation and photochemical electron transfer obtained from the analysis of TCSPC and transient absorption data were modeled by a unified spectral/kinetic model, based on the compartmental approach (ref. 67 and citations therein). The modeling considers compartments associated to functional sections of the photosystem antenna, as well as compartments associated to photochemically generated radical pair states. These compartments are connected by pair-wise (forward/backward) rate constants, describing either energy transfer (for excited state antenna compartments) or electron transfer processes (for primary photochemistry and successive electron transfer reactions). The excited state pair-wise rates are detailed-balance constrained, as described in ref. 24. The population changes for all the compartments are described by a system of linear differential equations, the solution of which has the form of a linear combination of time-dependent exponential functions, which define the time evolutions of the fractional population (molar fraction) of each compartment, determined by the initial excitation conditions (eigenvectors of the kinetic system). On the contrary, the lifetimes are 9 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

independent from the excitation conditions (eigenvalues of the kinetic system). The combined analysis of the fluorescence decay and transient absorption measurements permits, in principle, to describe both the fluorescence and differential absorption spectrum of each compartment. The spectral properties of the compartments are obtained by weighting the population evolutions by either their emission spectra (SAS, species associated spectra) or differential absorption spectra (SADS, species associated difference spectra). Hence, the only difference between simulations of fluorescence or absorption monitored kinetics relates to these spectra, whereas the rate constants are held equal. We consider that the radical pairs have a negligible fluorescence yield, therefore their SAS were set to zero. The simulations were performed in order to reproduce semi-quantitatively both sets of experimental results. Further details on kinetic model simulation, including all the most relevant mathematical equations, are presented in the supplementary information, section 4.1. Mean decay parameters. Useful information concerning excited state dynamics and ET events could be obtained by the calculation of macroscopic parameters describing the overall kinetics. In particular, we have extracted both from the experimental and the modeled kinetics the average, τ av = ∑ Aiτ i / ∑ Ai and mean, τ m = ∑ Aiτ i2 / ∑ Aiτ i , decay lifetimes, which relate to the i

i

i

i

zero- and first-order moments of the decay distributions, respectively. These parameters are, in general, computable for experimental dataset. However, the calculation of τ av associated to the j-th modeled compartment, requires that

∑A

j

≠ 0, i.e. that the compartment is populated by the initial

j

excitation. Since the radical pair states are not populated initially, two additional parameters, the average rise ( τ av ,r ) and decay (τ av ,d ) times were also considered68. These are calculated as described above, but separating within each j-th eigenvector the negative sign elements (which are interpreted as a population rises) from the positive ones (which correspond to a population decay).

Results 1. Characterization of the PSI-LHCI supercomplex isolated from spinach. Figure 2A shows the steady-state absorption (solid line) and fluorescence emission (dashdotted line) spectra recorded for the PSI-LHCI supercomplex close to the room temperature. The absorption spectrum peaks at 681.5 nm, and displays a broad long-wavelength tail extending well over 700 nm. This is characteristics of the presence of the so-called “red chlorophyll spectral forms”. The presence of Chl b gives rise to an asymmetric broadening in the wavelength region of 650-660 nm. The presence of red spectral forms in the absorption tail of PSI is further highlighted in the steady-state fluorescence spectrum recorded upon preferential excitation of Chl a at 435 nm. 10 Environment ACS Paragon Plus

Page 10 of 41

Page 11 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The emission spectrum (Figure 2A) is characterized by a rather broad and unstructured bandshape, having maximum intensity at 725 nm, and displaying only a shoulder centered at about ~690 nm. Further characterization of steady-state properties at room temperature and a cryogenic temperature is presented in Figures S2 and S3 of the supporting information, respectively. Figure 2B shows an analysis by means of bidimensional electrophoresis (2D-PAGE) of the isolated PSI-LHCI, which resolves several low-weight protein subunits of PSI, as well as the different complexes composing the LHCI complement. The overall pattern observed in 2D-PAGE is very similar to those previously reported for the PSI-LHCI supercomplex of Zea mays, which was isolated employing a very similar approach to that utilized here, based on membrane solubilization using the mild detergent OGP58. The assignment of the subunits observed in the 2DPAGE is therefore based on those reported for the Z. mays photosystem. As a conclusion, both the biochemical composition and the room temperature steady-state spectroscopic characteristics of spinach PSI-LHCI supercomplex are remarkably similar to those of the complex isolated previously from Z. mays 8, 58 which together with the complex purified from

Arabidopsis thaliana e.g.23, 24, 37, 39, 40, represents the best characterized PSI-LHCI supercomplex of higher plants. The similarity of the biochemical composition and the steady-state spectroscopic properties of these purified complexes allows us to discuss and compare the results shown here in the context of previous studies in the literature.

2. Excited state relaxation and electron transfer kinetics in spinach PSI-LHC. Time resolved fluorescence. Excited state decay in spinach PSI-LHCI was investigated by the TCSPC technique, upon unselective excitation at 632 nm. The excited state decay is satisfactorily fitted considering three main lifetimes characterized by values of 12±2, 33±4 and 77±4 ps, and a small amplitude component, whose lifetime is 650±40 ps. A moderately long-lived component (200-800 ps) was also observed in previous studies22-24, 36, 40, 58, 69. However, because it possesses a rather low amplitude and its origin is not well established, it will not be further considered in the analysis. The four DAS resulting from the global analysis are shown in Figure 3A. The fractional contribution of each decay component, defined as Fi , SS (λ ) = Aiτ i (open symbols in Figure 3B), to the estimated steady-state emission, given by FSS (λ ) = ∑ Aiτ i (half-filled symbols), i

is shown in Figure 3B. The comparison of the reconstructed (half-filled circles) and measured (solid line) steady-state emission spectra also shown in Figure 3B, showing a very good agreement. The results of TCSPC data analysis summarized in Figure 3 are fully consistent with those previously reported for maize PSI-LHCI22, 36. In particular, the DAS of the shorter lifetime (12±2 11 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ps, black symbols and lines Figure 3A) is the most blue-shifted, having maximal fluorescence contribution at 690 nm and being characterized by a relatively narrow bandwidth. It also shows small amplitude on the long-wavelength emission tail, which becomes negative over 730 nm, indicating some contribution from energy transfer processes. The DAS associated to the 33±4 (red symbols and lines Figure 3A) and 77±4 ps (green symbols and lines Figure 3A) lifetimes have positive amplitude at all investigated wavelengths. The DAS of the 77±4 ps component appears redshifted with respect to the 33±4 ps one, having maximum at ~740 nm with respect to ~725 nm. The resolution of three, relatively rapid and closely lying decay components was possible, even considering that the IRF of our instrumental set up has a FWHM of ~100 ps, because of their different relative contribution at each wavelength, as can be seen by the clearly distinct associated DAS. We notice, furthermore, that the fastest component retrieved here (12±2 ps), is in good agreement with the analysis obtained from set-ups having higher temporal resolution, such as streak cameras e.g. 6,7,40 . In these studies e.g. 6,7 an ever faster lifetime, characterized by values of about 3-5 ps is commonly detected. This was not resolvable in our analysis. On the other hand, the two 33±4 and 77±4 ps components, which are individually resolved here, are most commonly reported as a single decay contribution having a lifetime of about 40-50 ps, and a DAS which resembles the sum of the two detected in this study 6,7,40. This is likely due to fact that, although streak-camera detection coupled to CCD cameras guarantees a higher temporal and spectral resolution , it has a lower signal-to-noise ratio, particularly in the tail of decay of the traces, with respect to TCSPC. The difference in spectral bandshapes of the DAS associated to the three lifetimes resolved in the present analysis gives rise to a marked wavelength dependence of the average lifetime ( τ av ), which attains values of ~20 ps in the 670-690 nm, and increases progressively in the longwavelength emission tail, reaching values of ~80 ps at wavelengths longer than 740 nm (see Figure S4 Supporting information). This has also been commonly observed in isolated PSI-LHCI of other higher plants. Since τ av was shown to represent a good estimator of the effective trapping time33, its wavelength dependence can be taken as an indication of the presence of excited state diffusion bottlenecks from the external antenna red forms. The almost monotonic increase of τ av towards long emission wavelengths is due to a pronounced spectral diffusion. Therefore, the evolution of the excited state distribution is enriched in the short wavelength emission, at early times after the excitation, and tends at later times to a long wavelengths emission enrichment, that almost approaches the Boltzmann weighted distribution. On the other hand, in the case of pure trap-limited kinetics, spectral evolution should extinguish more rapidly than the trapping dynamics. In turn, the


Page 12 of 41

Page 13 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


trapping-dominated kinetics would be associated with a very limited wavelength dependence of τ av , rather than the almost four folds increase observed in PSI-LHCI (Figure S4 and refs. 22-24, 33, 36).

Transient absorption. Figure 4 shows the ∆T (t ) / T kinetics monitored at selected wavelengths, together with the ∆T (t ) / T spectra at selected time points of the kinetics. Data are shown for excitation at 490 nm (Figure 4A and B), which preferentially excites carotenoids and Chl b (620 nm), and 620 nm (Figure 4C and D), which is unselective pumping Chl vibrational bands. The ∆T (t ) / T kinetics acquired at other pump wavelengths, in the 520-640 nm interval, are presented in the supporting information. It can be seen that, after ∼100 ps, the ∆T (t ) / T spectra converge to an asymptotic bandshape (Figure 4B and D) for both excitation wavelengths. Figure 4 also shows the fits to the datasets according to a linear combination of exponential decay functions (see Experimental Methods). The kinetics are described by lifetimes of 0.78±0.22, 4.2±1.1, 15.5±2.5, 32.7±4.5 and 64.5±6.5 ps, when exciting at 490 nm, and 0.58±0.28, 4.4±1.4, 13.5±3.1, 32.8±6.5 and 65.2±6.5 ps upon excitation at 620 nm. A long-lived component, > 5 ns, which can be considered as non-decaying within the instrumental time window, is also present. These results show that the values of the retrieved lifetimes are little affected by the initial site of excitation, except for the fastest component (< 1 ps) that shows a moderate difference. This holds true for the whole range of the pump wavelengths investigated (Figures S5 and S6 of the Supporting Information). The comparison of these lifetimes with those retrieved from TCSPC analysis shows a broad agreement for the ∼16 ps lifetime, a very good agreement for the ∼33 ps lifetime and a slightly faster ∼65 ps lifetime. It should be also taken into account that the higher temporal resolution of pump-probe with respect to TCSPC measurements renders it possible to resolve three components in the time range < 20 ps. The DADS resulting from the global fit analysis are displayed in Figure 5. The nondecaying component corresponds, according to both the long-delay ∆T (t ) / T spectrum (Figure 4) and to its associated DADS (Figure 5), to the P700+ – P700 difference spectrum, as expected for measurements performed under “open” reaction center conditions. The DADS associated to the three longer lifetimes, 16, 33 and 65 ps, are little affected by the initial site of excitation, 490 nm (Chl b and carotenoids) or the unselective 620 nm (Figure 5). This holds true for the whole range of the pump wavelengths investigated (Figures S5 and S6). These DADS are dominated by ground-state bleaching (GSB) at wavelengths longer than 680 nm, and display excited state absorption signature at wavelengths shorter than 660 nm. The DADS related to the 15.5 ps lifetime shows the largest amplitude and is dominated by GSB centered at



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

∼690 nm, with a shoulder at 710 nm. The DADS of the ~33 ps lifetime has a main GSB peak at 698 nm and a clear shoulder at 723 nm while that of the ~65 ps component shows a more red shifted GSB maximum, centered at ~ 710 nm, accompanied by a secondary peak at ~690 nm. On the other hand, the DADS associated to the two shorter lifetimes show a moderate dependence on the excitation wavelength, with the bandshape associated to the shortest lifetime being the most dependent. The DADS associated to the two fastest components (0.58-0.7 and 4.2-4.4 ps) have a characteristic negative/positive amplitude pattern, with overall almost null integrated amplitude. The negative amplitudes in the short-wavelength region are dominated by GSB. The positive amplitude above 695 and 700 nm, for ~0.4 and ~4 ps components, respectively, can be interpreted as GSB rises, instead, even though contributions from both excited state absorption and stimulated emission may be present in this spectral region. Therefore, the spectral shape of these DADS can, in general, be interpreted as being dominated by energy transfer from short to long wavelengths that, in turn, lead to spectral diffusion occurring at short pump-probe delay times. Although the DADS of the ~4 ps component appears to be largely dominated by energy transfer to the antenna red-forms, the maximum of its positive feature centered at ~705 nm (see Figure 5), corresponds also to the maximum bleaching of the electron donor P700. The broadness of the ~4 ps DADS might therefore suggest that it contains contributions not only from the dominant energy transfer process towards red-shifted Chl forms, but also from radical pair population, including band-shift due to local charge formation (Stark effect).

Kinetic Modeling. In order to better interpret the kinetics and to resolve the contribution of excited state equilibration and ET events associated with primary photochemistry and successive charge stabilization processes in PSI-LHCI, kinetic models capable to describe simultaneously time-resolved fluorescence and transient absorption were developed. The model which provides the most satisfactory description of the experimental results is presented in Figure 6A. It considers seven compartments. Three of them describe the energy transfer processes in the antenna and are associated to the bulk of the chromophores excited states (Bulk*) and of the two low-energy Chl forms excited states, denoted as Red1* and Red2*. Three other compartments, describing primarily charge separation and ET reactions had to be considered. One describes the RC excited state (RC*), which is considered as reversibly populated by the bulk of the antenna. The others describe two consecutive radical pair states, RP1 and RP2, which are considered as reversible. An additional compartment describing the ternary radical pair RP3, which represents an exit from the system, is included. Moreover, a channel for natural de-excitation is 14 Environment ACS Paragon Plus

Page 14 of 41

Page 15 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


present for all excited state compartments, and it is set to 0.5 ns-1, corresponding to a natural lifetime of 2 ns, which is similar to the average fluorescence decay of isolated LHCI complexes e.g .36, 40

. Also shown are the SAS (Figure 6B) and SADS (Figure 6C) required to reproduce the

experimental DAS (Figure 3A), and DADS (Figure 5). Since the RP states are not fluorescent, they have no corresponding SAS in Figure 6B. The model yields seven simulated lifetimes (τ i , sim , i=1, 2…7) with values of τ 1,sim =0.4, τ 2,sim = 4.2, τ 3,sim =11, τ 4,sim = 32, τ 5,sim =63, τ 6,sim =79 ps, and τ 7,sim > 2 ns, that satisfactorily reproduce the experimental observables. Since the model contains more compartments than the number of lifetimes retrieved from the experiments, the simulated DAS (DASsim) and DADS (DADSsim) have been grouped to reduce their number to the experimental one. Therefore, the spectra associated to the τ 1−3,sim of 0.4, 4.2, 11 ps components are pooled in the fluorescence DASsim, whereas the 63 (τ 5,sim ) and the 79 ps ( τ 6,sim ) are pooled both in the modeled DASsim and DADSsim. The >2 ns component has no amplitude in the DAS simulation (see Figure S8 supporting information for further information) and corresponds to the experimental non-decaying DADS. The thus retrieved DASsim and DADSsim spectra, shown in Figure 7A and 7B, respectively, are in very good agreement with the DAS and DADS retrieved from the fits of the experimental kinetics, shown in Figures 3A and 5, respectively. The population evolution associated to each compartment comprised in the model is shown in Figure 8, both on a linear scale (Figure 8A), to better discern rapidly decaying processes, and on a logarithmic timescale (Figure 8B), to better appreciate the full temporal evolution. An alternative model which was also capable, albeit less satisfactorily, to describe the timeresolved fluorescence and absorption measurements, is discussed in the Supplementary information. The kinetic model of Figure 6 does not therefore consider explicitly the population of radical pairs on two active ET branches of PSI14-16,21,53. Thus, the population kinetics associated to RP1, RP2 and RP3 describe collectively primary charge separation and successive ET events on both branches. Parallel population of the two ET branches in PSI is instead explicitly considered in the alternative model described in detail in the supplementary information, Figure S9. The same excited state compartments as in Figure 6 are considered, yet two parallel RP1 states (RP1A and RP1B) are populated from RC*, each leading to the population of the respective RP2A and RP2B radical pair states. This model does not require the presence of a ternary radical pair, RP3, but requires to consider, in total, one more component. Therefore, in order to limit the number of adjustable parameters, equal charge separation rates on both active branches (which are not explicitly distinguished) was imposed. Moreover, to obtain a reasonable simulation of the experimental results 15 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a 3 to 4 fold difference in the rate constants associated to the (irreversible) populations of the secondary RP2A and RP2B had to be considered. Such difference is however larger than the one reported when the RPs populated on the two ET branches were distinguished experimentally, also based on cofactor coordination mutational analysis53. Thus based on the criterion of constraining the number of free parameters, and because of the overall better agreement with the experimental results provided by the model reported in Figure 6 with respect to the alternative explicitly bidirectional scheme (see supplementary information, section 4.3 and Figures S9-S11), we tend to favor it over the alternative descriptions. Assignment of radical pair states. Based on the kinetic modeling (Figure 6) it is possible to discuss and assign the experimental DADS (Figure 5) on the basis of their simulations (Figure 8) and the requirement to consider the presence of two decaying (RP1 and RP2) and one meta-stable (on the measurement time scale) radical pair (RP3). The latter is straightforwardly identified as [P700+A1-], since the DADS (Figure 5), the SADS (Figure 6C) as well as the long lifetime agree with previously reported P700+ –P700 difference spectra e.g. 17, 18, 47-49. Moreover, the kinetics of A1oxidations fall in the tens of nanosecond scale13-16, giving rise to a non-decaying state in the time window investigated here. In addition, A1, being a phylloquinone, is not expected to give rise to any significant transient absorption in the investigated spectral window, as its principal spectral signature falls in the near UV13-15. The SADS (Figure 6C) associated to RP2 shows significant similarity to that of RP3. Still, it is broader and shows additional differential absorption in the 685+ 695 nm window. Similar broadening of the SADS spectrum of the species preceding [P700 A1− ] , i.e.

RP2, has been previously reported by Müller and coworkers 51, 53, although with some differences in the exact bandshape. This is likely associated to the different organism from which PSI was purified, i.e. the green alga C. reinhardtii51, 53. These authors assigned RP equivalent to RP2 in the + present model to [P700 A 0− ] . This assignment is consistent with both the main bleaching attributed to

the A 0− − A 0 difference being located at ~690 nm17, 18, 47-49 and the average reduction time of A1− being 20-40 ps17-20. The SADS (Figure 6C) of the primary radical pair, RP1, is further broadened and less structured than that of RP2. Whereas it is characterized by a GSB centered at around 705 nm, which corresponds rather nicely to the P700 bleaching, it has an overall amplitude which is significantly larger than the one of either RP2 or RP3. We attribute this discrepancy to two, independent but additive factors. The first is that, even though we have taken great care to operate under “open center” conditions, we cannot exclude that a fraction of closed centers, with an upper limit estimated between 10-15% on the basis of pump intensity dependence, is still present during the measurements. As the overall excited state de-excitation kinetics are not much affected in PSI when P700 is oxidized during the measurements30,38 and since a primary ET step can still occur 16 Environment ACS Paragon Plus

Page 16 of 41

Page 17 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


under these conditions38, the spectral signatures from all steps preceding RP2 and RP3 formation would have larger relative intensities. The second factor which reasonably brings additional amplitude to the RP1 SADS is the contribution of a local Stark effect, associated to charge separation (also called electrochromic bandshift). As this will depend on the charge distance, the local Stark contribution is expected to be less prominent for the successive RPs than for RP1. The relative sharp positive feature (in −∆T / T scale) centered at ~684 nm could be, for instance, interpreted along these lines. A counterpart of this signal having opposite sign and similar amplitude is expected, thereby, to increase the overall apparent bleaching by about 30-35%. By considering the two aforementioned processes, the actual amplitude of the RP1 GBS would therefore become, albeit reasonable, still somewhat larger than expected on the basis of RP2 and RP3 SADS. Thus, based on the assignments of RP2 and RP3, RP1 shall then contain some + eC 2− ] contribution from the so-called accessory Chls (eC2), and be therefore assignable to either [P700

or [eC+2 A 0− ] . Müller and coworkers51, also based on the analysis of site-directed mutants affecting A0- coordination53, favored a role for the accessory Chls as primary electron donor. Nevertheless, the red-shifted SADS of RP1 (Figure 6C) and the observation of a rise component in the 4 ps DADS having maximal intensity at 705-710 nm (Figure 5) appears to suggest that P700 pigments might have a direct involvement in primary radical pair formation. Although the present data could be + taken as an indication that the primary radical pair might be the [P700 eC 2− ] pair, the possibility that

the eC(2− ) might contribute to a red-shifted transition, possibly by virtue of coupling with the A0 Chl, cannot be excluded. This suggestion reconciles the apparent discrepancy concerning the assignment of the SADS of the primary radical pair (RP1) in the present work, i.e. in PSI of higher plants, with the proposition advanced by Müller and coworkers for the PSI of a model green alga51,53. In fact, species-specific differences of the site energies and exciton interactions amongst the pigments participating to the RC of PSI have been already reported and analysed70. It is however worth noticing that the exact SADS bandshape and intensity depend on the kinetic model details. For a complex system such as the PSI-LHCI supercomplex, the functional graining associated might be too coarse, so that some caution should be maintained in the interpretation. For instance, when considering the alternative “bidirectional” kinetic scheme of Figure S9, the assignment of the radical pair state is instead straightforward, with RP2A/B + − + − corresponding to [P700 A1A/B ] and RP1A/B to [P700 A 0A/B ] , in which the assignment to a particular ET

branch is only formal, as the present data do not allow to distinguish between them. Yet, as discussed before, the overall description of the kinetics provided by this model is less satisfactory (section 4.3 of the supplementary information). It should be expected that a model that combines



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the basic features of the ones here discussed separately, and experimental approaches having finer spectral/temporal resolution (e.g. such as two-dimension electronic spectroscopy) will aid in gathering a better understanding of the primary charge separation and stabilization reactions in spectrally crowded biological supercomplexes.

Discussion In the present study we have investigated the excited-state dynamics in the PSI-LHCI supercomplex isolated from spinach. We measured, on the same preparation and under comparable experimental conditions, both fluorescence and absorption kinetics. A unified kinetic model taking into account the overlapping as well as complementary information derived from the two experimental approaches has been developed. This integrated model delivers information on processes that have been previously mainly analyzed by either one of the two methodologies independently, such as the effect of energy spread in the antenna, mainly investigated by timeresolved fluorescence, and primary charge separation and successive ET steps, primarily investigated by transient absorption. The combined kinetic modeling of fluorescence and transient absorption, in addition to being more restrictive on the model variables, gives the possibility to investigate the interplay of these factors on the overall excited state dynamics. For instance, in order to describe satisfactorily the fluorescence data, it was mandatory to consider at least two long-wavelength (Red) states. These appear to be almost isoenergetic, with mean energies of Red1 and Red2 state being 1.68 eV (736 nm) and 1.69 eV (732 nm), respectively. The macroscopic energy transfer from these red forms to the bulk of PSI antenna appears to be amongst the slowest processes, being characterized by rates of k1 = 14 ns-1 (~ (70 ps)-1) and k2 = 43 ns-1 (~(23 ps)-1). As a comparison, the rate describing energy transfer from the bulk of the antenna to RC* is about one order of magnitude higher (k3 ~285 ns-1, ~(3.5 ps)-1). Moreover, the (macroscopic) back transfer rate of this process is rather remarkably large ( k−3 ~1700 ns-1), due to the larger number of pigments, as observed in biochemical and structural data1-12, increasing thereby the site degeneracy of the antenna compartment (nbulk=160, where n is the number of Chls in each compartment) with respect to RC (nRC=6), which is only partially compensated by the difference in the mean energies of the states, being ERC =1.77 eV (700 nm) and EBulk =1.73 eV. (715 nm). The overall rate for the depopulation of RC* by primary charge separation, k pc , was found to be 632 ns-1, i.e. ~(1.5 ps)-1. When considering bidirectional ET explicitly (Figure S9) we found similar values for the overall de-excitation of photochemical RC*, of 750 ns-1, resulting from charge separation rates of 375 ns-1 on each active branch. These values 18 Environment ACS Paragon Plus

Page 18 of 41

Page 19 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for the primary charge separation rate falls in the middle of a rather broad range already reported in the literature, being faster for instance than the ~350 ns-1 proposed by Müller and coworkers51, 53, who also considered a reversible radical pair reaction scheme, but slower than the value suggested by Shelaev and coworkers54, 55 and Beddard20, who reported rates exceeding 1000 ns-1. Reversible primary charge separation (pc) is accounted by a RC* repopulation constant,

k− pc , of 38 ns-1, i.e. ~ (25 ps)-1, yielding an apparent equilibrium constant ( k pc / k− pc ) of ~16, which is in excellent agreement with what reported in refs.51, 53. The charge stabilization (cs) of the primary radical pair (RP1), resulting in the population of RP2, is characterized by a macroscopic overall constant, kcs1 , of ~190 ns-1, i.e. ~(5.5 ps)-1, representing an average value over the two active ET branches. This value is also roughly twice than the value suggested by Müller and coworkers for C. reinhardtii PSI, whereas the back-population rate k− cs1 is in this case of minor relevance, being ~150 times smaller than the forward ET reaction. The successive ET step, leading to the population of RP3, which is the long-lived metastable state, observed as a non-decaying component in transient absorption, is described by an overall rate of kcs 2 =(62.5 ps)-1 that is comparable, in terms of speed, to the slowest energy transfer process from the Red1 to the bulk of the antenna described by k1 = ~ (70 ps)-1. Under the utilized experimental conditions, the excitation is largely unselective, i.e. the initial population is proportional to the site degeneracy of the excited state compartments. The population evolution of all states considered, shown in Figure 8, indicates that the excited state evolution is largely overlapped to the primary charge separation and charge stabilization events, and extinguishes in concomitance with the maximal population of RP3, which, in the analyzed spectral interval, contains uniquely contribution from the P700+ – P700 difference spectrum. It is therefore apparent that, although the secondary radical pair attains a much larger population than the antenna excited state for delays longer than ~ 20 ps , electron transfer and excited state relaxation appear to be in kinetic competition. To better understand these aspects, we compared the modelled unselective excitation with idealized conditions in which the initial population in the kinetic simulation is set either entirely in the RC* compartment (Figure 9A and B), or in the two red-most states (Figure 9C and D). Comparison of the simulated population evolution evidences the profound effect of the initial site of excitation on both energy transfer, primary charge separation, as well as on successive ET reactions. Useful parameters to compare the different initial excitation scenarios are the average rise ( τ av ,r ) and decay times ( τ av , d ) of the RC* and the RPs, as well as the mean decay lifetime (τ m ) of these states. These parameters, calculated from the modeling of the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

population evolution of the four compartments RC*, RP1, RP2 and RP3, are shown in Table 1 for initial conditions considering either unselective, red-forms or RC initial excitation, respectively. These kinetic simulations show that excitation in the red-most states imposes a severe excited-state diffusion bottleneck. The impact of this kinetic limitation dilutes, however, as the electron transfer proceeds. In fact, the differences in the population rise and decay times, as well as in their mean decay times, become progressively smaller, although remaining sizable. The ratios of

τ m for the two “ideal” limit conditions, i.e. exciting either the photochemically active pigments or the external antenna red forms only, are 3.5, 2.8, 1.6 for RC*, RP1 and RP2, respectively, and 1.2 when comparing the rise time of RP3. Instead when ideal excitation of RC* and the unselective excitation conditions are compared, the τ m ratios have values of 1.3, 1.2, 1.1 and 1, respectively. We found the same trend for selective excitation when performing the simulations in the frame of an explicitly bidirectional model (Figure S11), although the details are unavoidably model dependent, the main conclusion appear to be rather robust and independent from the model details. The results obtained from kinetic simulations based on compartmental modeling should not be considered as an exhaustive description, despite reproducing on a close to quantitative basis the experimental observables. Nonetheless, it is clear that excited state diffusion from the bulk of the antenna, even in presence of the red forms, has an overall limited impact on the trapping kinetics. The excited state trapping dynamics are the dominant process in establishing excited state relaxation, unless excitation is selective, or at least largely preferential, for the red-most states. In the latter case, the dominant process becomes the overall kinetics of energy transfer to the trap (i.e. transfer-to-trap limited). This leads also to an increase in the non-photochemical losses, and, therefore, to a decrease of the maximal photochemical efficiency, which is the result of a lengthening of the overall excited state relaxation. In turn, the modeled maximal population level of the meta-stable charge separated state, RP3, decreases from 0.92 to 0.89, comparing ideal RC and red-form excitation, respectively (Figure 9C). Yet, as observed previously also for other processes modulating the excited state dynamics (such as the antenna size), the overall impact of energy transfer bottlenecks on the stabilized RP state is remarkably small, i.e. only about 4% e.g. 23, 24, 69,71.

Conclusions The analysis of excited state dynamics in PSI-LHCI of spinach by an approach combining fluorescence and transient absorption kinetics, as well as a unified model of energy and electron transfer reactions indicates that, in this photosystem: i) upon unselective excitation, the primary radical pair is populated in ~4 ps, but its population dynamics is significantly dependent on the initial site of excitation; ii) the overall primary charge separation constant is estimated to be ~ 65020 Environment ACS Paragon Plus

Page 20 of 41

Page 21 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


750 ns-1; iii) the mean time required for the stabilization of the secondary radical pair(s) is ~ 60 ps; iv) this time is broadly overlapped with the mean excited state de-excitation of the long-wavelength Chl forms, i.e. ~ 70 ps. We conclude that the overall trapping in PSI contains contributions of both excited state diffusion in the antenna and primary charge separation. The interplay of these two processes depends significantly on the site that is initially excited. Yet, for the most probable excitation condition, i.e. the bulk of the antenna system, the excited state dynamics appear to be primarily trap-limited.

Supporting Information The supporting information presents 1. an additional characterization of the biochemical (Figure S1) and steady-state spectroscopic properties (Figures S2 and S3) of PSI-LHCI isolated from spinach. 2. additional information concerning the TCSPC data analysis, in terms of average lifetimes (Figure S4) and photochemical quantum yield. 3. additional analysis of transient absorption data using pump wavelengths in the 520-640 nm range (Figure S5), and the results of global fit analysis of these datasets (Figure S6). 4. additional information on kinetic modeling including the pre-exponential amplitudes associated to each compartment for different simulated initial conditions (Figure S7), the full set of simulated DASSim and DADSSim (Figure S8), the alternative model considering bidirectional charge separation explicitly (FigureS9), associated SAS, SADS, (FigureS9), DASSim and DADSSim (FigureS10) and compartment population evolutions (FigureS11). Also shown in 4 are temporal evolution ratio of the RC* and [Red1*+Red2*] populations, calculate from linear two-step charge separation scheme, with respect to the other excited state compartments populations (Figure S12), as well as the direct comparison of population/depopulation kinetics of RP1, RP2 and RP3 simulated under different initial conditions. This information is available free of charge via the Internet at http://pubs.acs.org

Acknowledgments We thank Prof. R.C. Jennings (Univerità di Milano) for stimulating discussion on energy transfer and trapping in photosystem I.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References (1) Fromme, P.; Jordan, P.; Krauss, N. Structure of Photosystem I. Biochim Biophys Acta

2001, 1507, 5–31. (2) Nelson, N.; Yocum, C. F. Structure and Function of Photosystems I and II. Annu. Rev. Plant Biol. 2006, 57, 521–565. (3) Caffarri, S.; Tibiletti, T.; Jennings, R.C.; Santabarbara, S. A Comparison between Plant Photosystem I and Photosystem II Architecture and Functioning. Curr. Protein Pept. Sci. 2014, 15, 296–331. (4) Amunts, A.; Toporik, H.; Borovikova, A.; Nelson, N. Structure Determination and Improved Model of Plant Photosystem I. J. Biol. Chem. 2010, 285, 3478–3486. (5) Qin, X.; Suga, M.; Kuang, T.; Shen, J.R. Photosynthesis. Structural Basis for Energy Transfer Pathways in the Plant PSI-LHCI Supercomplex. Science 2015, 348, 989–995. (6) Gobets, B.; van Grondelle, R. Energy Transfer and Trapping in Photosystem I. Biochim. Biophys. Acta-Bioenerg. 2001, 1057, 80–99. (7) Croce, R.; van Amerongen, H. Light-Harvesting in Photosystem I. Photosynth. Res.

2013, 116, 153–166. (8) Croce, R.; Zucchelli, G.; Garlaschi, F.M.; Jennings, R.C. A Thermal Broadening Study of the Antenna Chlorophylls in PSI-200, LHCI, and PSI Core. Biochemistry 1998, 37, 17355– 17360. (9) Croce, R.; Morosinotto, T.; Castelletti, S.; Breton, J.; Bassi, R. The Lhca Antenna Complexes of Higher Plants Photosystem I. Biochim. Biophys. Acta 2002, 1556, 29–40. (10) Jennings, R.C.; Zucchelli, G.; Engelmann, E.; Garlaschi, F.M. The Long-Wavelength Chlorophyll States of Plant LHCI at Room Temperature: a Comparison with PSI-LHCI. Biophys. J.

2004, 87, 488–497. (11) Wientjes, E.; Croce, R. The Light-Harvesting Complexes of Higher Plant Photosystem I: Lhca1/4 and Lhca2/3 form Two Red-Emitting Heterodimers. Biochem. J. 2011, 433, 477–485. (12) Jordan, P.; Fromme, P.; Witt, H.T.; Klukas, O.; Saenger, W.; Krauss, N. Threedimensional Structure of Cyanobacterial Photosystem I at 2.5 Å Resolution. Nature 2001, 411, 909–917. (13) Brettel, K. Electron Transfer and Arrangement of the Redox Cofactors in Photosystem I. Biochim. Biophys. Acta. 1997, 1318, 322–373. 22 Environment ACS Paragon Plus

Page 22 of 41

Page 23 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(14) Santabarbara, S.; Heathcote, P.; Evans, M.C.W. Modelling of the Electron Transfer Reactions in Photosystem I by Electron Tunnelling Theory: the Phylloquinones Bound to the PsaA and the PsaB Reaction Centre Subunits of PS I are Almost Isoenergetic to the Iron-Sulfur Cluster FX. Biochim. Biophys. Acta 2005, 1708, 283–310. (15) Rappaport, F.; Diner, B.A.; Redding, K.E. in Photosystem I: The Light–Driven Plastocyanin:Ferredoxin Oxidoreductase; Golbeck J.H. Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands; 2006; pp 223–244. (16) Srinivasan, N.; Golbeck, J.H. Protein-Cofactor Interactions in Bioenergetic Complexes: the Role of the A1A and A1B Phylloquinones in Photosystem I. Biochim. Biophys. Acta 2009, 1787, 1057–1088. (17) Hecks, B.; Wulf, K.; Breton, J.; Leibl, W.; Trissl, H.-W. Primary Charge Separation in Photosystem I: a Two-Step Electrogenic Charge Separation Connected with P700+A0- and P700+A1Formation. Biochemistry 1994, 33, 8619–8624. (18) Nuijs, A.M.; Shuvalov, V.A.; van Gorkom, H.J.; Plijter, J.J.; Duysens, L.N.M. Picosecond Absorbance Difference Spectroscopy on the Primary Reactions and the Antenna Excited States in Photosystem I Particles. Biochim. Biophys. Acta. 1986, 850, 310–318. (19) Melkozernov, A.N. Excitation Energy Transfer in Photosystem I from Oxygenic Organisms. Photosynth. Res. 2001, 70, 129–153. (20) Beddard, G.S. Excitations and Excitons in Photosystem I. Phil. Trans. R. Soc. Lond. A.

1998, 356, 421–448. (21) Santabarbara, S.; Galuppini, L.; Casazza, A.P. Bidirectional Electron Transfer in the Reaction Centre of Photosystem I. J. Integr. Plant. Biol. 2010, 52, 735–749. (22) Jennings, R.C.; Zucchelli, G.; Santabarbara, S. Photochemical Trapping Heterogeneity as a Function of Wavelength in Plant Photosystem I (PSI-LHCI). Biochim. Biophys. Acta 2013, 1827, 779–785. (23) Galka, P.; Santabarbara, S.; Khuong, T.T.; Degand, H.; Morsomme, P.; Jennings, R.C.; Boekema, E. J.; Caffarri, S. Functional Analyses of the Plant Photosystem I-Light-Harvesting Complex II Supercomplex Reveal that Light-Harvesting Complex II Loosely Bound to Photosystem II is a Very Efficient Antenna for Photosystem I in State II. Plant Cell 2012, 24, 2963–2978. (24) Santabarbara, S.; Tibiletti, T.; Remelli, W.; Caffarri, S. Kinetics and Heterogeneity of Energy Transfer from Light Harvesting Complex II to Photosystem I in the Supercomplex Isolated from Arabidopsis. Phys. Chem. Chem. Phys. 2017, 19, 9210–9222.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(25) Owens, T.G.; Webb, S.P.; Mets, L.; Alberte, R.S.; Fleming G.R. Antenna Size Dependence of Fluorescence Decay in the Core Antenna of Photosystem I: Estimates of Charge Separation and Energy Transfer Rates. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1532–1536. (26) Jean, J.M.; Chan, C.K.; Fleming, G.R.; Owens T.G. Excitation Transport and Trapping on Spectrally Disordered Lattices. Biophys J. 1989, 56, 1203–1215. (27) Turconi, S.; Schweitzer, G.; Holzwarth, A.R. Temperature Dependence of Picosecond Fluorescence Kinetics of a cyanobacterial Photosystem I Particle. Photochem. Photobiol. 1993, 57, 113–119. (28) Hastings, G.; Kleinherenbrink, F.A.M.; Lin, S.; Blankenship, R.E. Time-Resolved Fluorescence and Absorption Spectroscopy of Photosystem I. Biochemistry 1994, 33, 3185–3192. (29) Croce, R.; Dorra, D.; Holzwarth, A.R.; Jennings, R.C. Fluorescence Decay and Spectral Evolution in Intact Photosystem I of Higher Plants. Biochemistry 2000, 39, 6341–6348. (30) Byrdin, M.; Rimke, I.; Schlodder, E.; Stehlik, D.; Roelofs, T.A. Decay Kinetics and Quantum Yields of Fluorescence in Photosystem I from Synechococcus elongatus with P700 in the Reduced and Oxidized State: are the Kinetics of Excited State Decay Trap-Limited or TransferLimited? Biophys. J. 2000, 79, 992–100. (31) Gobets, B.; van Stokkum, I.H.M.; Rogner, M.; Kruip, J.; Schlodder, E.; Karapetyan, N.V.; Dekker, J.P.; van Grondelle, R. Time-Resolved Fluorescence Emission Measurements of Photosystem I Particles of Various Cyanobacteria: a Unified Compartmental Model. Biophys. J.

2001, 81, 407–424. (32) Kennis, J.T.M.; Gobets, B.; van Stokkum, I.H.M.; Dekker, J.P.; van Grondelle, R.; Fleming, G.R. Light Harvesting by Chlorophylls and Carotenoids in the Photosystem I Core Complex of Synechococcus elongatus: a Fluorescence Upconversion Study. J. Phys. Chem. B.

2001, 105, 4485–4494. (33) Jennings, R.C.; Zucchelli, G.; Croce, R.; Garlaschi, F.M. The Photochemical Trapping Rate from Red Spectral States in PSI-LHCI is Determined by Thermal Activation of Energy Transfer to Bulk Chlorophylls. Biochim. Biophys. Acta 2003, 1557, 91–98. (34) Gobets, B.; Valkunas, L.; van Grondelle, R. Bridging the Gap Between Structural and Lattice Models: A Parameterization of Energy Transfer and Trapping in Photosystem I. Biophys. J.

2003, 85, 3872–3882. (35) Holzwarth, A.R.; Müller, M.G.; Niklas, J.; Lubitz, W. Charge Recombination Fluorescence in Photosystem I Reaction Centers from Chlamydomonas reinhardtii. J. Phys. Chem. B. 2005, 109, 5903– 5911.


Page 24 of 41

Page 25 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36) Engelmann, E.; Zucchelli, G.; Casazza, A.P.; Brogioli, D.; Garlaschi, F.M.; Jennings, R.C. Influence of the Photosystem I-Light Harvesting Complex I Antenna Domains on Fluorescence Decay. Biochemistry 2006, 45, 6947–6955. (37) Slavov, C.; Ballottari, M.; Morosinotto, T.; Bassi, R.; Holzwarth, A.R. Trap-Limited Charge Separation Kinetics in Higher Plant Photosystem I Complexes. Biophys J. 2008, 94, 3601– 3612. (38) Giera, W.; Ramesh, V.M.; Webber, A.N.; van Stokkum, I.H.M.; van Grondelle, R.; Gibasiewicz, K. Effect of the P700 Pre-oxidation and Point Mutations Near A0 on the Reversibility of the Primary Charge Separation in Photosystem I from Chlamydomonas reinhardtii. Biochim Biophys Acta 2010, 1797, 106–112. (39) Wientjes, E.; van Stokkum, I.H.; van Amerongen, H.; Croce, R. Excitation-energy Transfer Dynamics of Higher Plant Photosystem I Light-Harvesting Complexes. Biophys. J. 2011, 100, 1372–1380. (40) Wientjes, E.; van Stokkum, I. H.; van Amerongen, H.; Croce, R. The role of the Individual Lhcas in Photosystem I Excitation Energy Trapping. Biophys. J. 2011, 101, 745–754. (41) van Stokkum, I.H.; Desquilbet, T.E.; van der Weij-de Wit, C.D.; Snellenburg, J.J.; van Grondelle, R.; Thomas, J.C.; Dekker, J.P.; Robert, B. Energy Transfer and Trapping in RedChlorophyll-Free Photosystem I from Synechococcus WH 7803. J. Phys. Chem. B 2013, 117, 11176–11183. (42) Giera, W.; Szewczyk, S.; McConnell, M.D.; Snellenburg, J.; Redding, K.E.; van Grondelle, R.; Gibasiewicz, K. Excitation Dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative Studies of Isolated Complexes and Whole Cells. Biochim Biophys Acta

2014, 1837, 1756–1768. (43) White, N.T.H.; Beddard, G.S.; Thorne, J.R.G; Feehan, T.M.; Keyes, T.E.; Heathcote, P. Primary Charge Separation and Energy Transfer in the Photosystem I Reaction Center of Higher Plants J. Phys. Chem. 1996, 100, 12086–12099. (44) Beddard, G.S. Exciton Coupling in the Photosystem I Reaction Center. J. Phys. Chem. B 1998, 102, 10966–10973. (45) Melkozernov, A.N.; Lin, S.; Blankenship, R.E. Excitation Dynamics and Heterogeneity of Energy Equilibration in the Core Antenna of Photosystem I from the Cyanobacterium Synechocystis sp. PCC 6803. Biochemistry 2000, 39, 1489–1498. (46) Melkozernov, A.N.; Lin, S.; Blankenship, R.E. Femtosecond Transient Spectroscopy and Excitonic Interactions in Photosystem I. J. Phys. Chem. B. 2000; 104, 1651–1656.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(47) Savikhin, S.; Xu, W.; Chitnis, P.R.; Struve, W.S. Ultrafast Primary Processes in PSI from Synechocystis sp. PCC 6803: Roles of P700 and A0. Biophys. J. 2000, 79, 1573–1586. (48) Kumazaki, S.; Ikegami, I.; Furusawa, H.; Yasuda, S.; Yoshihara, K. Observation of the Excited State of the Primary Electron Donor Chlorophyll (P700) and the Ultrafast Charge Separation in the Spinach Photosystem I Reaction Center. J. Phys. Chem. B 2001, 105, 1093–1099. (49) Gibasiewicz, K.; Ramesh, V.M.; Melkozernov, A.N.; Lin, S.; Woodbury, N.W.; Blankenship, R.E.; Webber, A.N. Excitation Dynamics in the Core Antenna of PSI from Chlamydomonas reinhardtii CC 2696 at Room Temperature. J. Phys. Chem. B. 2001, 105, 11498– 11506. (50) Savikhin, S.; Xu, W.; Martinsson, P.; Chitnis, P.R.; Struve, W.S. Kinetics of Charge Separation and A0 →A1 Electron Transfer in Photosystem I Reaction Centers. Biochemistry 2001, 40, 9282–9290. (51) Müller, M.G.; Niklas, J.; Lubitz, W.; Holzwarth, A.R. Ultrafast Transient Absorption Studies on Photosystem I Reaction Centers from Chlamydomonas reinhardtii. 1. A New Interpretation of the Energy Trapping and Early Electron Transfer Steps in Photosystem I. Biophys. J. 2003, 85, 3899–3922. (52) Sarkisov, O.M.; Gostev, F.E.; Shelaev, I.V.; Novoderezhkin, V.I.; Gopta, O.A.; Mamedov, M.D.; Semenov, A.Y.; Nadtochenko, V.A. Long-Lived Coherent Oscillations of the Femtosecond Transients in Cyanobacterial Photosystem I. Phys. Chem. Chem. Phys. 2006, 8, 5671–5678. (53) Müller, M.G.; Slavov, C.; Luthra, R.; Redding, K.E.; Holzwarth, A.R. Independent Initiation of Primary Electron Transfer in the Two Branches of the Photosystem I Reaction Center. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4123–4128. (54) Semenov, A.Y.; Shelaev, I.V.; Gostev, F.E.; Mamedov, M.D.; Shuvalov, V.A.; Sarkisov, O.M.; Nadtochenko, V.A. Primary Steps of Electron and Energy Transfer in Photosystem I: Effect of Excitation Pulse Wavelength. Biochemistry (Moscow) 2012, 77, 1011–1020. (55) Shelaev, I.V.; Gostev, F.E.; Mamedov, M.D.; Sarkisov, O.M.; Nadtochenko, V.A.; Shuvalov, V.A.; Semenov, A.Y. Femtosecond Primary Charge Separation in Synechocystis sp. PCC 6803 Photosystem I. Biochim. Biophys. Acta 2010, 1797, 1410–1420. (56) Shuvalov, V.A. ; Klevanik, A.V.; Sharkov, A.V.; Kryukov, P.G.; Ke, B. Picosecond Spectroscopy of Photosystem I Reaction Centers. FEBS Lett. 1979, 107, 313–316. (57) Parson, W.W.; Ke, B. Primary Photochemical Reactions. In Energy Conversion by Plant and Bacteria; Govindjee Ed.; Academic Press: London, U.K., 1982; pp. 331–386.


Page 26 of 41

Page 27 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(58) Croce, R.; Zucchelli, G.; Garlaschi, F.M.; Bassi, R.; Jennings, R.C. Excited State Equilibration in the Photosystem I -Light-Harvesting I Complex: P700 is Almost Isoenergetic with its Antenna. Biochemistry 1996, 35, 8572–8579. (59) Machold, O.; Simpson, D.J.; Lindberg Møller, B. Chlorophyll-Proteins of Thylakoids from Wild-Type and Mutants of Barley (Hordeum vulgare L.). Carlsberg Res. Commun. 1979, 44, 235–254. (60) Schägger, H.; von Jagow, G. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa. Anal Biochem. 1987, 166, 368–379. (61) Santabarbara, S.; Bordignon, E.; Jennings, R.C.; Carbonera, D. Chlorophyll Triplet States Associated with Photosystem II of Thylakoids. Biochemistry 2002, 41, 8184–8194. (62) Engelmann, E. C. M.; Zucchelli, G.; Garlaschi, F.M.; Casazza, A.P.; Jennings, R.C. The Effect of Outer Antenna Complexes on the Photochemical Trapping Rate in Barley Thylakoid Photosystem II. Biochim. Biophys. Acta 2005, 1706, 276–286. (63) Tumino, G.; Casazza, A.P.; Engelmann, E.; Garlaschi, F.M.; Zucchelli, G.; Jennings, R.C. Fluorescence Lifetime Spectrum of the Plant Photosystem II Core Complex: Photochemistry Does Not Induce Specific Reaction Center Quenching. Biochemistry 2008, 47, 10449–10457. (64) Polli, D. ; Lüer, L.; Cerullo, G. High-Time-Resolution Pump-Probe System with Broadband Detection for the Study of Time-Domain Vibrational Dynamics. Rev. Sci. Instr. 2007, 78, 103108–9. (65) Rizzo, F.; Zucchelli, G.; Jennings, R.C.; Santabarbara, S. Wavelength Dependence of the Fluorescence Emission Under Conditions of Open and Closed Photosystem II Reaction Centres in the Green Alga Chlorella sorokiniana. Biochim Biophys Acta 2014, 1837, 726–733. (66) Beechem, J.M.; Gratton, E.; Ameloot, M.; Knutson, J.R.; Brand, L.The Global Analysis of Fluorescence Intensity and Anisotropy Decay Data - 2nd-Generation Theory and Programs. In Topics in Fluorescence Spectroscop; Lakowicz, J.R. Ed.; Plenum Press: New York, 1991;Vol. 2, pp 241–305. (67) Straume, M.; Frasier-Cadoret, S.; Johnson, M.L. Least-Square Analysis of Fluorescence Data. In Topics of Fluorescence Spectroscopy; Lakowicz, J.R., Ed.; Plenum Press: New York, U.S.A., 1991; Vol. 2, pp 177–240. (68) Santabarbara, S.; Galuppini, L. Electron and Energy Transfer in the Photosystem I of Cyanobacteria: Insight from Compartmental Kinetic Modelling. In Handbook of Cyanobacteria; Gault, P.M., Marler H.J., Eds.; Nova Publisher: New York City, U.S.A., 2009; pp 1–50.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(69) Wientjes, E.; van Amerongen, H.; Croce, R. LHCII is an Antenna of Both Photosystems After Long-Term Acclimation. Biochim. Biophys. Acta 2013, 1827, 420–426. (70) Witt, H.; Bordignon, E.; Carbonera, D.; Dekker, J.P.; Karapetyan, N.; Teutloff, C.; Webber, A.; Lubitz, W.; Schlodder E. Species-Specific Differences of the Spectroscopic Properties of P700: Analysis of the Influence of Non-Conserved Amino Acid Residues by Site-Directed Mutagenesis of Photosystem I from Chlamydomonas reinhardtii. J. Biol. Chem. 2003, 278, 46760– 46771. (71) Le Quiniou, C.; van Oort, B.; Drop, B.; van Stokkum, I.H.M.; Croce, R. The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II. J. Biol. Chem. 2015, 290, 30587–30595.

Figure Legends

Figure 1. Arrangement of redox active cofactors in Photosystem I. The redox-active cofactors are labeled on the PsaB-branch only, for simplicity, even though both cofactor chains are active in electron transfer reactions. Also shown are the two reaction schemes describing photochemical primary charge separation and charge stabilization, as currently discussed in the literature. For simplicity the schemes describe a mono-directional charge separation/electron transfer model. The same schemes can however be applied to a bi-directional scenario considering that only RC* is common to both active chains.

Figure 2. A: Steady state optical characterization of PSI-LHCI from spinach at room temperature. Absorption (solid line) and fluorescence emission spectra (dashed-dotted line) upon excitation at 435 nm. Spectra are normalized to their maxima. B: Two-dimensional denaturing electrophoresis of purified PSI-LHCI. The overall polypeptide composition observed in 2D-SDS PAGE is very similar to the one reported by Croce et al.58. The spots were therefore assigned accordingly. Labels refer to polypeptides composing the LHCI complement and to small RC subunits. Furthermore, broad protein spots corresponding to the large PsaA and PsaB RC subunits can also be distinguished in the high molecular weight region.

Figure 3.. Results of the global analysis of TCSPC traces of PSI-LHCI of spinach. A: Decay Associated Spectra (DAS). Solid black squares: 12 ps; solid red circles: 33 ps; solid green triangles: 77 ps; solid blue diamonds: 650 ps. Dash-dotted lines are a guide for the eye. B: Fractional 28 Environment ACS Paragon Plus

Page 28 of 41

Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


contributions of each decay component ( Fi ,SS (λ ) = Ai (λ ) ⋅τ i ) to the steady-state emission ( FSS ,tot (λ ) = ∑ Ai (λ ) ⋅τ i ). Symbols as in panel A, but open, joined by dash-dotted lines as guide for i

the eye. Also shown is the reconstruction of the steady-state fluorescence spectrum from TCSPC data (half-filled diamonds), compared with the measured one (thick solid line). The amplitudes are normalized to yield an integrated steady-state emission of 1. Both the DAS amplitudes and the steady-state emission spectra have been corrected for the respective wavelength-dependent detector sensitivity.

Figure 4. Ultrafast transient absorption of PSI-LHCI of spinach. Differential transmission ( ∆T (t ) / T ) kinetics (A and C) and spectra (B and D) are extracted from the time/wavelength differential intensity matrixes. A: Representative experimental (symbols) and fitted (lines) kinetics, pumped at 490 nm. B: Representative experimental (symbols) and fitted (lines) ∆T (t ) / T spectra, pumped at 490 nm. C: Representative experimental (symbols) and fitted (lines) kinetics, pumped at 620 nm. D: Representative experimental (symbols) and fitted (lines) ∆T (t ) / T spectra , pumped 620 nm. Pump energy was ~20 nJ/pulse, 0.5 kHz repetition rate.

Figure 5. Decay associated difference spectra (DADS) resulting from the global fitting analysis of transient absorption kinetics obtained upon pumping at 490 nm (A) and 620 nm (B). Before fitting the data were binned in 2.5 nm intervals. The fit results are presented as symbols (open 490 nm, closed 620 nm) whereas the solid lines are interpolations. To allow direct comparison between the two pump wavelengths, the DADS amplitudes resulting from the two excitation datasets were normalized, using the long-lived non-decaying signal (attributed to (P700+ – P700)) as an internal standard. By this procedure, in both DADS sets, the ground state bleaching of the non-decaying (> 2 ns) component at 705 nm has an amplitude of (-1). All other DADS, within a given dataset, are scaled accordingly.

Figure 6. Combined compartmental kinetic modeling of TCSPC and transient absorption data. A: Reaction scheme, comprising four excited state compartments (Red1*, Red2*, Bulk* and RC*) and three radical pair states (RP1-RP3); B: Species Associated Spectra (SAS) of the excited state compartments (RP are considered non-fluorescent) required to simulate the experimental DAS of Figure 3A. C: Species Associated Difference absorption Spectra (SADS) required to simulate the DADS of Figure 5. The excited state compartment site degeneracy, i.e. the number of Chls in each compartment, was, according to available biochemical and structural information on pigment 29 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

binding: nRed1* = 2, nRed2* = 2, nBulk* = 160, and nRC* = 6. Because of unselective excitation in both type of measurements the initial population was considered to be proportional to the number of sites in each compartment. The rate constants were: k1(-1): 13.8 (6) ns-1; k2(-2): 43 (16) ns-1; k3(-3): 285 (1669) ns-1; kpc(-pc): 632 (38) ns-1; kcs1(-cs1): 189 (1.3) ns-1; kcs2: 16 ns-1; k cs3: 0.2 ns-1. An additional relaxation rate equivalent to 0.5 ns-1 accounting for the natural singlet state decay processes was considered for all excited state compartments. The outcome of the simulations is shown in Figure 7 and 8. Extended information on the kinetic scheme are shown in Figure S7 of the supporting information.

Figure 7. Simulated DAS (A) and DADS (B) from the kinetic models and SAS and SADS shown in Figure 6. The lifetimes are those retrieved from the kinetic model of Figure 6 after pooling the appropriate compartments to reduce their number to the experimental ones (see text and for further detail and Figure S8 of the supporting information the full sets of simulated DAS and DADS). The spectra should be compared to those retrieved from the experiments, shown in Figure 3A and Figure 5, respectively.

Figure 8. Population evolution of the states considered in the combined transient fluorescenceabsorption modeling according to the kinetic scheme of Figure 6 for unselective (degeneracy weighted) excitation in the excited state compartments. A: fast portion of the population evolutions shown on linear timescale. B: full population evolution shown on logarithmic timescale. The ratio of the RC* population and that of the other excited state compartment is presented in Figure S12 of the supporting information

Figure 9. Simulated population evolutions of the states considered in the combined transient fluorescence-absorption modeling for preferential excitation in the RC* compartment (A and B) or in the [Red1*+Red2*] compartments (C and D). The two red states were considered as equally initially populated in the simulations. Panels A and C: fast portion of the population evolutions shown on linear timescale. B and D: full population evolution shown on logarithmic timescale. The ratio of the RC* population and that of the other excited state compartment, as a function of the initial excitation site is presented in Figure S12 of the supporting information. Figure S13 shows the direct comparison of RPs evolution for the different initial conditions tested.


Page 30 of 41

Page 31 of 41

Table 1. Average rise and decay lifetimes and mean lifetime obtained from kinetic modeling. Compartment i.c. (ps) RC RP1 RP2 RP3 1.7 20 69 τav,r 1.9 15 55 τav,d 24 32 90 τm unselective

τav,r τav,d τm

0.4 17 31

4 16 39

21 62 98

70

Red forms

RC

*

Excitation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


τav,r τav,d τm

11 82 83

12 38 90

45 72 148

87

Values of the simulated average rise (τ av ,r ), decay ( τ av , d ) and mean ( τ m ) lifetime resulting from the kinetic modeling for initial conditions (i.c.) under which excitation is equally distributed on all pigments sites composing the model compartments (unselective, kinetics shown in Figure 8), set entirely in the RC* compartment (RC-selective, Figure 9), or in set in the red forms only (Red1*+Red2* compartments, Figure9). All values are in ps.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PsaB ET Chain

PsaA ET Chain

P700 Chlacc (eC2) A0 (eC3) A1 FX

Page 32 of 41

One-step mechanism

RC*

RP1

RP1= [P700+A0-]

Two-step mechanism

RC*

RP1

RP2

FA FB

a: RP1= [P700+Chlacc-], RP2= [P700+A0-] b: RP1= [Chlacc+A0-], RP2= [P700+A0-]

Figure 1


Page 33 of 41

TRIS-SULPHATE SDS - 6M UREA PAGE 1.0

A

B

0.8

TRIS-TRICINE SDS PAGE

Absorption/Fluorescence (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

0.4

0.2

0.0 600

650

700

750

PsaA/PsaB

LHCI Small RC Subunits

800

Wavelength (nm)

Figure 2



2.0

DAS

A

12±2 ps 33±4 ps 77±4 ps 650±50 ps

1.0

-4

Ai x 10 (a.u.)

1.5

0.5

0.0

-0.5

6.0

B

4.0

-3

Ai*τ i x 10 (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.0

0.0 675

700

725

750

Wavelength (nm)

Figure 3


Page 34 of 41

Page 35 of 41

0.2

A

B

-∆T/T 10

-2

0.0

-0.2 L640 L660 L680 L700 L720 L735

-0.4

-0.6

C

D

0.0 0.5 ps 1 ps 2.5 ps 5 ps 7.5 ps 10 ps 20 ps 40 ps 100 ps 200 ps

-2

-∆ T/T 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-0.1

-0.2

0

50

100

Time (ps)

150

200 640

660

680

700

720

Wavelength (nm)

Figure 4



-∆ T/T (a.u.)

1

A

0

-1

-2

1

-∆ T/T (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DADS 0.78 ps 4.2 ps 15.5 ps 32.7 ps 64.7 ps > 5 ns

λ490

B

0

-1

-2

DADS 0.58 ps 4.4 ps 13.5 ps 32.8 ps 65.2 ps > 5 ns 650

λ620 675

700

725

Wavelength (nm)

Figure 5


Page 36 of 41

Page 37 of 41

*

*

Red1 k1

Red2 k-1 k-2

A

0.03

k2

Red1* Red2* Bulk* RC*

0.02

k3

Φi

Bulk

0.01

k-3 *

RC

0.00 625

kpc k-pc

RP2 kcs2

RP3 kcs3

650

675

700

725

750

C

0.4

RP1 kcs1 k -cs1

B

SAS

*

0.0

∆ε i (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-0.4

-0.8

-1.2

SADS Red1* Red2* Bulk* RC* RP1 RP2 RP3

625

650

675

700

725

750

Wavelength (nm)

τsim: 0.4, 4.2, 11, 32, 63, 79 ps and > 2 ns

Figure 6



A

4

DAS sim 11 ps 32 ps 80 ps

2

1

0

-2

-4 0 -6 675

700

725

Wavelength (nm)

750

B

2

(∆A/A)i (a.u.)

3

Ai (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DADS sim 0.4 ps 4.2 ps 11 ps 32 ps 80 ps >2 ns 650

675

700

725

Wavelength (nm)

Figure 7


Page 38 of 41

Page 39 of 41

Compartment Red1* Red2* Bulk* RC* RP1 RP2 RP3

1.0

0.8

Population

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

A

B

0.4

0.2

0.0 0

20

40

60

Time (ps)

80

100

120 0.1

1

10

100

1000

Time (ps)

Figure8



*

RC excitation Red1* Red2* Bulk* RC* RP1 RP2 RP3

1.00

Population

0.75

0.50

A

B

C

D

0.25

0.00 *

*

Red1 +Red2 excitation

1.00

Red1* Red2* Bulk* RC* RP1 RP2 RP3

0.75

Population

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.50

0.25

0.00 0

10

20

30

40

Time (ps)

50

60 0.1

1

10

100

1000

Time (ps)

Figure 9


Page 40 of 41

Page 41 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Image


1 The Trapping Dynamics in Photosystem I- Light Harvesting Complex

Recommend Documents