Charge Recombination Time Distributions in Photosynthetic Reaction

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Charge Recombination Time Distributions in Photosynthetic Reaction Centers Exposed to Alternating Intervals of Photoexcitation and Dark Relaxation Anthony J. Manzo,†,‡,§ Alexander O. Goushcha,†,^ Nataliya M. Berezetska,^ Valery N. Kharkyanen,^ and Gary W. Scott†,* †

Department of Chemistry, University of California, Riverside, Riverside, California 92521, United States Department of Physics, University of California, Riverside, Riverside, California 92521, United States § Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States ^ Institute of Physics, National Acadamy of Science of Ukraine, Kyiv, Ukraine ‡

ABSTRACT: The charge recombination lifetime of photosynthetic reaction centers (RCs) increases significantly upon lengthy illumination, revealing nonequilibrium structural transitions in the protein
cofactor system. This paper analyzes the charge recombination kinetics measured in isolated RCs following a systematic variation of actinic illumination times (pulses) from 0.1 s to hundreds of seconds. The maximum entropy method (MEM) was utilized for optimizing the fitting procedure to retrieve the relaxation spectrum from the experimental recombination kinetics curves. The MEM-assisted analysis reveals that each relaxation curve contains at least three peaks in the relaxation time-distribution domain. Two peaks are always observed, one near 0.1 s and the other near 1 s recombination times. A third peak appears after prolonged photoexcitation with a relaxation time significantly greater than 1 s, and the time of this peak increases further in recombination time as the photoexcitation pulse duration is increased. In addition to the shifts of the time constant distributions, the amplitudes of the distributions in the time domain spectrum demonstrate a variation in the quinone occupancy of the RCs. The results reported here support our previous claim that accumulation of slow conformational changes, triggered by charge separation events in the RCs, controls system dynamics and favors stabilization of more efficient functioning regimes of the RCs.

’ INTRODUCTION Light-induced electron transfer (ET) events in photosynthetic reaction centers (RCs) have been proposed to initiate structural changes in RCs through charge separation and transfer, effectively acting as a source of stochastic forces within RC proteins and driving conformational changes and nonequilibrium dynamic behavior in the RC protein structure.1,2 Studies suggest that the rate of photoinduced ET over the cofactor chain as well as the photoelectron localization time on a terminal acceptor in a photosynthetic RC depend upon the history of prior ET events,1,2 thus serving as an example of a “memory conductor” in a macromolecular system. This means that an RC may be considered as an element of (bio)molecular electronics, in which the charge transfer rate and survival time of the charge separated state are controlled by the structural rearrangements and structural memory effects initiated by the previous ET events. Numerous studies have shown that charge separation events in RCs are accompanied by processes such as proton transfer and uptake, water uptake and binding,3,4 and reorientations of protein residues and cofactors within RCs.5,6 Studies of lightinduced structural changes in RCs from purple bacteria are especially interesting because of the wealth of information known r 2011 American Chemical Society

about the RC structure, charge separation events, and biochemical processes involved in its function. This includes basic light interactions, charge transfer events, and kinetics for the bacterial RC in various environments that have been extensively studied and thoroughly reported in the literature, making it a model system for studying the relationship between electron transfer processes and protein conformational dynamics.7
11 Understanding conformational changes induced by these processes as well as the feedback of conformational changes on charge transfer kinetics remains an important active area of research. On a broader scale, such studies contribute to a comprehensive understanding and modeling of the effects of fluctuating light on the photosynthetic process, its relation to other environmental factors, and its evolution.12 Moreover, recent advances in nanotechnology demonstrate exciting properties of memory resistors (“memristors”), whose operation principles are based upon a dependence of the current flow through the nanometer-scale device due to structural changes induced by the preceding events Received: December 4, 2010 Revised: May 4, 2011 Published: June 15, 2011 8534

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The Journal of Physical Chemistry B of charge flow through the device.13
15 Obviously, such a dependence of charge conductivity upon the history of current flow through the device may provide a straightforward analogy with RCs, in which structural changes triggered by charge transfer events and the feedback of those structural changes on ET kinetics have been described in terms of nonequilibrium dynamic effects in our recent work.1 The results of the present paper demonstrate more experimental evidence in support of previously developed theories of the functioning of photosynthetic RCs. The isolated photosynthetic RCs used in this study were prepared from the purple bacterial strain Rhodobacter sphaeroides, consisting of a primary electron donor (the bacteriochlorophyl dimer) and a series of electron acceptors embedded in a protein matrix. The electron transfer reactions within RCs have been studied and characterized extensively, and are discussed in several reviews.4,8,16,17 Briefly, upon illumination of a photosynthetic RC at room temperature, the bacteriochlorophyll dimer P is excited and charge separation occurs followed by electron transfer along the active branch of electron acceptors. ET initially occurs from the excited dimer to the monomeric BChl (bacteriochlorophyl), or accessory BChl, in ∼3 ps. ET from the accessory BChl to the bacteriopheophytin BPh occurs in ∼1 ps and subsequent ET from the BPh to the primary quinone acceptor QA occurs in ∼200 ps. This is followed by ET to the secondary quinone acceptor QB within ∼100 μs. The efficiency of the forward charge separation process is essentially 1. For RCs that lack a quinone at the secondary acceptor site, charge recombination from QA
to the photo oxidized Pþ, PþQA
f PQA, proceeds with a rate constant of approximately 10 s
1. In the absence of a secondary donor to reduce the oxidized dimer Pþ, direct charge recombination from QB
to Pþ is negligible, with recombination from the secondary quinone site, PþQAQB
f PQAQB, occurring predominantly through QA
in ∼1 s in the dark-adapted state.18
20 The absorption band of the primary photoelectron donor P (λmax = 865 nm) bleaches upon photoexcitation, signaling the creation of the radical pair Pþ(QAQB)
and providing a convenient method for monitoring the charge separation, electron transfer, and charge recombination kinetics.21 As is well-known, appreciable amounts of the quinones at the QB site may also be lost during the RC isolation procedure.22 The overall transmittance recovery kinetics following photoexcitation of RCs with short actinic pulses reflects the heterogeneity of the sample and is usually analyzed by fitting with a biexponential decay function with the components describing charge recombination in two types of RCs —those with no quinone (fast component) and those containing a quinone (slow component of ∼1 s duration) in the QB site. When considering experiments performed under steady-state illumination with the intensity Iexp, the effective forward ET rate for each RC is affected by the frequency of photoexcitation, which is dependent upon the light flux (intensity) and the oscillator strength of the chromophores. The dependence on photoexcitation frequency may be considered as a kind of memory effect controlled by the photoinduced structural changes, in which the rates of forward electron transfer and charge recombination depend on the history of previous ET events in the macromolecule. It is important to emphasize that we discuss here conformational changes arising in RCs following multiple consecutive events of photoexcitation/recombination (turnover) of each RC, not just structrural changes induced by a single event of photoinduced charge separation and transfer. It

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has been previously shown that increasingly prolonged continuous wave (CW) illumination of the bacterial reaction center results in increasingly longer charge recombination lifetimes, particularly in RCs having an active secondary quinone acceptor QB.2,9 Recently, a maximum entropy method (MEM) analysis of such affects has been carried out, probing the distribution of relaxation times of preilluminated RCs,23 and illustrating several distinct peaks in the lifetime distribution associated with equilibration. Further exploration of this effect in isolated bacterial RCs at room temperature using a wider range of continuous wave illumination periods, systematically cycling illumination from short periods to longer periods, and vice versa, should allow for a more comprehensive picture and understanding of the process. Analyzing the relaxation kinetics of the RCs using the maximum entropy method should enable the distinction between various lifetime components and their respective amplitudes through a systematic variation of the illumination time. In addition to observing a general systematic variation in the recombination lifetimes of the RCs, the MEM method can retrieve amplitudes of the components attributed to charge recombination from the primary (QA) and secondary (QB) quinone acceptors, showing how recombination rates are shifted and correlated with increasing or decreasing photoexcitation time cycles.

’ MATERIALS AND METHODS Samples. The RCs used for this study were obtained from the photosynthetic bacteria Rhodobacter (Rb.) sphaeroides strain R26, which lack the carotenoid found in wild type RCs.24 The absence of the carotenoid, a known charge transfer and redox reaction species, avoids any possible interference in the charge transfer processes of isolated RCs.25,26 Lauryl dimethylamine oxide (LDAO) buffered RCs were isolated and prepared as described previously.27,28 RCs were then suspended in a final buffer solution of 10 mM HCl
Tris (pH = 8.0), 1 mM EDTA, and 0.025% LDAO. RC concentrations were determined from their absorption using the molar absorption coefficient of 2.88  105 M
1 cm
1 at 802 nm29 and ranged from 1 to 2 μM. The absorbance ratio (A280)/(A800) for isolated RCs ranged from 1.25 to 1.35, demonstrating high purity.29 Triton X-100 buffered RCs were prepared from photosynthetic membranes using the detergent LDAO and a poly histidine tag for rapid isolation according to the procedure described previously.27,30,31 Following purification on a column of oxiapatite, RCs were suspended in 10 mM Tris
HCl buffer with 0.05% LDAO, pH 7.5. The RC suspension was then dialyzed against an excess of the detergent Triton X-100 (0.05%, pH 7.5) according to conventional methods. No quinone reconstitution procedure was applied to any of the samples. Preillumination and Relaxation Kinetics Experimental Methods. Transient absorption experiments were carried out as described previously.32 Briefly, absorbance changes of RC samples in a quartz cuvette were probed with a monitoring light beam from a quartz tungsten
halogen (QTH) lamp filtered by monochromators set at 865 nm (slit bandwidth = 20 nm) and placed before and after the cuvette. The monitoring light was additionally filtered with a red long pass filter (>630 nm), and neutral density filters to adjust the intensity to 10s) if the exposure time is longer than several seconds and the photoexcitation light intensity is relatively high (at least several mW/cm2, such as the ∼20 mW/cm2 used in this study). As an example, parts a and b of Figure 1 show kinetics traces illustrating typical transient absorption kinetics of RCs upon applying a photoexcitation pulse followed by dark relaxation to the ground state (the dark adapted state). Traces are shown for ∼200 to 600 s CW photoexcitation pulses applied to RCs at various dark adapted states at room temperature, with their traces partially overlapping during photoexcitation/bleaching periods. Relaxation periods for dark adaptation of the RCs were long enough to allow for sufficient charge recombination to occur and initial prephotoexcitation transmittance levels of the RCs to be reached. In all experiments, recording of the dark recovery kinetics traces was stopped at least 300 s after the monitoring signal reached within ∼2.5% (or ∼3
4 standard deviations) of the initial signal level that was measured before photoexcitation was applied. Additional periods of dark relaxation were allowed at the end of data acquisition for various experimental curves, subsequent to initiating the proceeding photoexcitation period, to allow for RCs to further structurally relax. Note that the recovery of the initial absorption levels in the transient RC kinetics measurements does not necessarily indicate complete structural relaxation of the RCs to the initial dark-adapted state, since light-induced structural changes in the RCs do not necessarily relax within the same time frame of electronic relaxations (or complete charge recombination of the primary

bacteriochlorophyl donor). We have found that the structural relaxation times, necessary for complete structural relaxation, may be as long as several hours or more for especially long photoexcitation pulses and high light intensities (see below). These times may dramatically exceed the time required to complete the electronic recombination in RCs. The samples that were used in this study contained a mixture of QB-active and QB-depleted RCs. Prolonged photoexcitation can therefore create a broad population of long-lived light adapted states for the QB-active and possibly QB-depleted RCs, resulting in a distribution of relaxation times, or a multiphasic relaxation. Similar long-lived charge recombination kinetics were reported recently in the work by Andreasson et. al for both QB-active and QB-depleted RCs.9 As was previously shown, the “average survival time”, or AST (τAST),42,43 of the charge separated state is a convenient quantity to characterize the charge recombination kinetics of RCs photoexcited with long actinic pulses. τAST may be considered as an effective time constant for the charge recombination process in the ensemble of RCs, and is determined by the area under the normalized relaxation kinetics curve: Z ¥ σðtÞ dt ð3Þ τAST ¼ 0

Here σ(t) is the probability of charge separation between a donor and acceptor molecule, σ(t) = 1
F(t,D), and F(t,D) is the normalized charge occupied donor population.1,43 For multiphasic relaxation kinetics, the probability of charge separation, σ(t), can be approximated as a sum of exponential decays with a set of relaxation rate constants γi with amplitudes Ai as σðtÞ ¼

∑i Ai e
γ t i

ð4Þ

Figure 1c shows a plot of τAST versus CW photoexcitation periods measured at room temperature for isolated RCs as areas under the normalized relaxation kinetics curves in accord with eqs 3 and 4. The AST of the charge separated state becomes longer with increasing CW photoexcitation time (ascending periods) and begins to level off at τAST ∼ 80 s, or fluctuate 8537

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Figure 3. (a) Dark recovery kinetics following a single laser flash activation of a dark-adapted sample. (b) Spectrum of relaxation times for the kinetic trace of panel (a) obtained using MEM assisted procedure (see text). (c) Details of the relaxation spectrum around the shortest relaxation time peak of 0.1 s.

around τAST values in the range of 60
85 s, with photoexcitation pulse durations of 1000 s or greater. Two pronounced drops in the τAST data occur at 120 and 300 s CW photoexcitation periods. These drops can be considered as artifacts of the experimental procedure of substituting a fresh, unused (and, hence, true darkadapted) aliquot of an otherwise identical RC sample into the sample cuvette at these times. These discontinuities in data are because of sample differences due to accumulated, light-induced structural changes that were not completely relaxed during the dark intervals between consecutive photoexcitation pulses. The scatter in τASTvalue for photoexcitation pulse duration beyond 1000 s is thought to be caused mainly by the dependence of the overall recombination kinetics on the dark resting period between two consecutive experimental runs and a varying waiting time beyond completion of electronic relaxation. The AST analysis allows for a straightforward qualitative description of relaxation dynamics of photoexcited RCs. However, MEM analysis provides valuable quantitative information and insight into relaxation process physics. A formal decomposition of the recombination kinetics traces into a number of components like those of eq 4 usually results in increasing the number of weighted exponents and the need to provide an explanation for each of them. In contrast, the MEM method provides a more reliable approach to fit theory with experiment and avoids the drawbacks of both the AST method and a formal decomposition of the relaxation kinetics into numerous relaxation curves. The MEM method gives a more accurate determination of the distribution of relaxation time constants resulting

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from each photoexcitation period. It allows an accurate tracing of the evolution of different components of recombination spectra with light intensity and duration of photoexcitation. MEM analysis should be considered as an alternative to simple fitting procedures used ubiquitously, providing at the same time a much more reliable and physically substantiated result for extracting the function sought from a fitted curve. Analysis of Photoexcitation Kinetics and Recovering the Distribution of Relaxation Times. The recombination time distributions following RC photoexcitation were retrieved using the MEM method for the optimization of the fitting procedure as described above. Representative relaxation curves with corresponding fits from the MEM analysis are shown in Figure 2, parts a and b for several photoexcitation pulse durations within the range from 1 s through 1000 s. MEM fitting of experimental data was very consistent and resulted in excellent χ2 values smaller than 10
6. Dark recovery kinetics following a single actinic laser flash applied to a dark adapted sample are shown in Figure 3a. Retrieving the relaxation time distribution using MEM assisted optimization procedure resulted in two distinct peaks, one near 0.1s and the other near 1s. These peaks can be attributed to the well-known charge recombination components from QA (QB-depleted RCs) and QB (QB-occupied RCs), respectively— see Figure 3b. Note the logarithmic scale of the x-axis in Figure 3b. A linear scale view of the 0.1 s distribution peak is detailed in Figure 3c. Laser flash activated kinetics always revealed only two peaks in the relaxation spectrum if the sample was properly dark adapted. Moreover, the same two peaks were always observed for several consecutive flashes applied 10 s or more apart from each other. However, if the dark period between the actinic flashes was shorter, the resulting relaxation spectrum contained a small portion of longer relaxation components (not shown in Figure 3). Parts a and b of Figure 4 show the relaxation time distributions obtained for several trials of applied CW photoexcitation pulses from 1 to 20 s in duration. Each pair of Figure 4, parts c and d, parts e and f, and parts g and h, presents relaxation spectra for photoexcitation pulse durations within the range from 20 through 100 s, 120 through 250 s, and 290 through 1400 s, respectively. The MEM-assisted analysis always retrieved recombination time distributions with peaks around 0.1 and 1 s. The axes of Figure 4 parts b, d, f, and h have been adjusted to emphasize the portions of relaxation spectra centered around the 0.1 s peak. The 0.1 s peak broadened and also shifted to slightly longer times with increasing CW photoexcitation pulse duration. A third peak was observed for increasingly longer photoexcitation periods. This third peak, with characteristic times longer than 1 s, appears in the relaxation spectra for samples subjected to a photoexcitation time duration longer than 0.1 s, and it shifts steadily toward longer recombination times with increasing photoexcitation time duration. For photoexcitation periods greater than 20 s, the relaxation spectra became even more complex with additional peaks emerging at yet longer relaxation times. For example, we observe two rather broad peaks around 10 and 60 s in the relaxation spectrum of RCs subjected to a photoexcitation pulse of durations between approximately 150 and 250 s, whereas these peaks shift gradually toward ∼15 and ∼150 s, respectively, when the pulse duration exceeds 500 s (see panels e and g in Figure 4). The location of the 1-s peak in the relaxation spectrum shifts slightly toward longer times when the pulse duration exceeds ∼130 s. Since all relaxation spectra peak consistently around ∼0.1 and ∼1.0 s, we find the time constants 8538

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Figure 4. Relaxation time distributions obtained using MEM assisted analysis of RC relaxation kinetics following various trials of applied CW photoexcitation pulses of (a) 1
20 s duration, (c) 20
100 s duration, (e) 120
250 s duration, and (g) 290
1400 s duration. Panels b, d, f, and h show the details of the corresponding distributions around the 0.1 s peak.

for these two time domains of 0.1 and 1.0 s and plot them versus photoexcitation time duration in Figure 5a. Fairly constant and consistent ∼0.1 and ∼1.0 s values of time constants were observed for each trial. Closer examination of the data corresponding to those two relaxation times revealed their slight variation between 0.5 and 1.5 s for the longer component and between 0.09 and 0.18 s for the shorter component. The “long component” time constant in Figure 5a is the peak time constant for the time domain longer than 1.5 s in the relaxation spectra. The time constant of this long recombination component exhibits a significantly more pronounced change with increasing photoexcitation time. The discontinuities in the data trend of Figure 5a at ∼120 and ∼300 s are the signatures of a fresh sample aliquot in the cuvette; at these points a fresh, unused aliquot from the same original RC preparation was replaced into the cuvette. As suggested above, these drops in lifetimes for a fresh sample indicate accumulation of light-induced structural changes in heavily illuminated samples used in the preceding experimental trials. These structural changes did not have enough time to relax completely during the dark intervals between consecutive photoexcitation pulses. Figure 5b shows the time constant for the long component time domain obtained from a single RC batch, as opposed to several different batches (as in Figure 5a). The two sets of data in Figure 5b

correspond to the ascending durations of photoexcitation pulses (solid triangles) and descending durations of photoexcitation pulses (open squares). These two sets of data were collected consecutively, first ascending pulse durations data followed by the descending pulse durations data all using the same RC sample aliquot over a 7 day period of experimentation and data acquisition. Comparing experimental results for several (four) alternating batches of RC samples (Figure 5a) versus a single batch of RCs (Figure 5b) demonstrates the reproducibility of results, effects of different dark adaptation periods, and possible degradation of RC properties during periods of lengthy CW photoexcitation. Figure 6 shows more results for the time constant of the long component on a log
log scale. The whole range of photoexcitation times was covered with experiments using four different batches of the same RCs sample. Batch 1 was used in experiments with photoexcitation time durations from 0.5s up to 110s. Batches 2 and 3 were used in experiments with time durations between 110 and 290 s. Batch 4 was used in experimental runs with the longest pulses from 300 to 1400 s. Batches 2, 3, and 4 were allowed to sit overnight at 4 C after exposure to several initial experimental trials, with further CW excitation experiments continued with each of those samples for one or more days afterward. Of particular note is the overlap of the results using batches 2 and 3. For comparison purposes, Figure 6 also shows 8539

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periods are shown in Figure 7, parts a, c, and e. Parts b, d, and f of Figure 7 show these normalized amplitudes for the case of decreasing photoexcitation periods. Figure 7 shows the amplitudes that correspond to the time components shown in Figure 5a. The amplitudes of both 0.1 and 1 s components of the relaxation spectrum decrease with increasing photoexcitation time simultaneously with an increase in amplitude of the long relaxation component of the relaxation spectrum. From the descending dependencies (panels b, d, and f of Figure 7), it is probable that at the end of experimental runs most of the RCs had lost ubiquinone Q10 from the secondary quinine acceptor binding site QB.

Figure 5. (a) Time constants (peaks from MEM derived distributions) of RC relaxation kinetics plotted versus select ascending CW photoexcitation periods and using three different aliquots of the same RC sample. (b) Average survival times (from integrating under the relaxation kinetics curves) for a single RC portion exposed to ascending and descending photoexcitation periods.

Figure 6. The time constants (peak values from time constant distributions) of the long relaxation time domain for four batches of the same RC sample with ascending photoexcitation pulses applied (see text for details). The AST values for batches 1, 3, and 4 are also shown for comparison purposes.

the AST values calculated using the relaxation kinetics curves for batches 1, 3, and 4. The normalized amplitudes of the components of the relaxation spectra for RCs exposed to increasing photoexcitation

’ DISCUSSION In a number of previous studies we reported a dramatic increase of a time constant for charge recombination kinetics of RCs subjected to lengthy actinic illumination. The current study is unique since it provides valuable additions to already available results by applying very lengthy periods of photoexcitation (up to 1400 s) to RCs and uses a rigorous MEM-based analysis of the relaxation kinetics. The analysis of relaxation spectra of RCs subjected to lengthy and high-intensity illumination reported in this study clearly reveals the emergence of an additional broadly distributed time constant component that is peaked at a much longer time domain than the well-known 0.1 and 1 s time constants reported for flash-induced relaxation kinetics of dark-adapted RCs. The distribution of this long relaxation component broadens and shifts toward still longer time with longer periods of actinic photoexcitation. Moreover, we found that this long relaxation component disappears as revealed in subsequent single-flash activated charge recombination experiments when the RCs sit in the dark for a sufficiently long time. Recent work by different authors relate the emerging longlived charge recombination kinetics in RCs to light-induced structural transitions between distinctly different conformations in the protein
cofactor system of RCs.6,9,10,44 In particular, Andreasson et al.9 attributed the long-lived relaxation kinetics to a new conformation formed due to multiple consecutive events of charge separation events in RCs. The authors showed that the same long-lived relaxation kinetics is observed when monitoring at 865 and 450 nm, proving therefore its origin as due to the long-lived charge separated state Pþ(QAQB)
. Recent theoretical and experimental works suggested that such structural changes in RCs can be attributed to self-consistent behavior of the transferred charges and protein, revealing a kind of memory effect due to a strong charge-conformational interaction in RCs.1,2 In a simplified model, those works formalized such effects as a diffusion process along the surface of an effective lightintensity-controlled, nonequilibrium, bistable potential Veff I (x) with respective dark and light adapted state distributions PP(t,x) and PB(t,x), where x corresponds to a single generalized structural reaction coordinate and t is the time. The theoretical modeling and its comparison with experimental results provided evidence for the Gibbs free energy change for states with the photoelectron localized on QA and QB, which was shown to lead to the formation of a long-lived, charge separated state and ETinduced structural memory in RC proteins. These light-induced structural changes may accumulate within the RC protein
cofactor system due to the repeated, light-induced charge separation events, and these changes are reversed after cessation of the 8540

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Figure 7. Normalized amplitudes of relaxation time domains of the RCs versus ascending and descending photoexcitation periods. Part a shows amplitudes for each relaxation component (∼0.1 s, ∼1 s, and long component) separately with ascending photoexcitation periods, whereas part b shows the amplitudes for each component for descending photoexcitation periods (note the ascending and descending values on the time axes, respectively). Parts c and d combine amplitudes of the ∼0.1 and ∼1 s components, and parts e and f combine the amplitudes of the ∼1 s and long relaxation components.

repeated charge separation events for sufficiently long wait times. The wait time for this structural relaxation is determined mainly by protein
cofactor rearrangement back toward the dark adapted state, which, using the simplified formalism developed previously, is attributed to system drift toward one of the two (bistable) potential wells of the effective potential Veff I (x)). As was discussed previously in a simplified adiabatic model,1 upon prolonged photoexcitation and charge flow within the RC, the Veff I (x) is instantly shifted to either a bistable potential with two minima or a single minima potential with a relatively higher structural coordinate value. Diffusive rearrangements in the structure occur on relatively slower time scales, during either light or dark adaptation. Such diffusive rearrangements may return to the genuine dark adapted state if the RCs are left for a long time in the dark. In contrast, increasingly longer excitation times and/or shorter dark intervals produce more significant structural rearrangement thus altering markedly the RC charge recombination kinetics, as is observed in this work with the application of long CW photoexcitation periods in either ascending and descending sequences (Figures 4 and 5). Hysteretic effects observed in the recombination lifetimes with ascending and descending CW photoexcitation, particularly for photoexcitation times below 150 s (see Figure 5b), indicate that the dark relaxation periods may not have been long enough during the descending branch of the curve due to the increased recombination lifetime of the long-lived component relative to that for the ascending curve. Gradual modification of the recombination time spectrum with increasing illumination periods is obvious from the analysis of Figure 4. These results clearly show a transition from the usual 0.1 and 1.0 s charge recombination components, characteristic for the dark-adapted RC recombination kinetics, to a charge recombination with shifted and broadened components as well

as the emergence of at least one additional recombination component with a characteristic recombination time of several seconds to hundreds of seconds. While ascending branch photoexcitation cycles demonstrate the overall shift of recombination lifetimes to longer time domains (Figure 5a and solid triangles in Figure 5b), the descending CW photoexcitation cycles shown with open squares in Figure 5b and clearly reveal the decrease of the long-lived recombination lifetime and return of the usual 0.1 and 1.0 s recombination components. The exact values of wait times between CW photoexcitation periods (the dark adaptation periods) are not reported here, and it is most likely that full dark adaptation was not achieved in many of the experimental trials. Full dark adaptation may take longer than several hours, depending upon photoexcitation period and light intensity. As stated in the Results above, all CW photoexcitation periods were followed by dark relaxation periods immediately upon cessation of photoexcitation and continued for at least 5 min after the monitoring intensity level reached within 2.44% of the initial preillumination monitoring intensity level. As a rule, we applied longer dark adaptation time for longer photoexcitation periods. However, structural relaxation times are not necessarily the same as electronic relaxation measured by charge recombination curves. The effect of the incomplete conformation relaxation is most likely the cause of the apparent jitter or oscillations in the time constant values observed in the time constant curve for descending photoexcitation periods (Figure 5b). Recall from the Results section that the discontinuities in the AST plots (Figures 1c and 6) and in the time constant plots (Figure 5a and Figure 6) occur at points where the sample aliquots in the cuvette were replaced with unused aliquots that had not been previously used in any photoexcitation 8541

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The Journal of Physical Chemistry B experiments. Applying long equal photoexcitation pulses to an RC aliquot just before and immediately after dark adaptation overnight at 4 ˚ C revealed that the long relaxation component was essentially the same in both cases, with no significant discontinuity in the lifetimes. In contrast, the fresher, less used aliquots of RCs generally showed shorter relaxation times for the long-lived components than the aliquots already subjected to multiple lengthy photoexcitation pulses of the same duration. This effect is readily observed in Figure 6 and demonstrates the long lasting conformational memory of RCs exposed to long photoexcitation periods. There was no significant shift or discontinuity in the time constant curves resulting from multiple ascending and/or descending photoexcitation periods (see Figure 5b) where a single RCs batch was used over several days for the entire cycle of experiments. This is expected because the RCs were subject to shorter and shorter photoexcitation periods in combination with the dark adaptation periods, allowing them to recover more completely from previous, much longer photoexcitation pulses. The period for complete protein structural relaxation, or dark adaptation for a single long photoexcitation pulse, was not fully tested for and would require an excessively long time interval in excess of several days. Detergent effects should normally be considered when discussing the charge
conformation behavior of RCs. Previous studies2 showed that RC samples prepared and isolated with different detergents exhibit distinctly different structural relaxation diffusion constants after application of long photoexcitation pulses to thoroughly dark adapted samples. In contrast, RC samples isolated in LDAO, prepared by different laboratories, that underwent the same experimental examination in different laboratories, exhibited the same distinct diffusive properties in their relaxation. The current study presents the case of RCs in a single detergent concentration with a fixed CW photoexcitation intensity applied for varying periods. We do not present any comparisons with RCs in various detergent preparations, but note that detergents may impose structural constraints that can be altered over repeated CW photoexcitation periods, and that the opposite effect is possible, where structure and volume changes of the RC alter the distribution of detergent lipids binding to the RC.45,46 An interesting feature that the MEM analysis reveals is a change in quinone occupancy at the QB site that depends on the photoexcitation duration. The amplitudes of the time constants from the MEM analysis show dynamic variations as the photoexcitation periods increase and decrease (Figure 7). For an RC sample with a fixed concentration of QB-occupied and QB-depleted RCs, the amplitude of the 0.1 s time constant is expected to remain constant, and the amplitude of the very long time component(s) is expected to increase as the 1 s component decreases with ascending photoexcitation periods. The shifts in the amplitudes of all three relaxation components in Figure 7 indicate more complex dynamics is occurring, as there is a shift in the QB-occupancy in addition to a transition of the RCs into a light adapted conformational state with increasing light pulse duration. However, Figure 7a shows that the amplitude of the long relaxation component steadily increases with respect to the 0.1 and 1 s components for the photoexcitation periods of ∼200s or more, with the 0.1 and 1 s amplitudes both decreasing in a correlated manner. This is seen more clearly when the 0.1 and 1 s component amplitudes are added together (Figure 7c,d). This result shows that the increase in the long-lived recombination component in RCs is not simply due to quinone uptake at the QB

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site within the RCs. Regardless of these amplitude shifts, simple changes in quinone occupancy at the QB site are insufficient to explain any long-lived recombination kinetics in RCs. In general, the binding of quinone at the QB site in RCs is not fully saturated in detergent sample preparations, and quinones may freely diffuse in and out of the QB pocket.22,47 Quinone binding at the QB site and the true lifetime of the ∼1 s charge recombination component depend upon the concentrations of detergent and quinones. This is because of quinone exchange between the detergent phase and the QB binding site within the same RC
detergent micelle (detergent-solubilized RCs)22,47 and because of quinone exchange between RC micelles and all-detergent micelles.22 Because the binding constant for the secondary quinone is increased in its reduced state, QB
,17 under conditions of CW photoexcitation, the QB binding equilibrium may be allowed to shift allowing quinones more time to diffuse around and bind in unoccupied QB sites in the RCs. As mentioned at the beginning of the Discussion, the theoretical modeling of RC nonequilibrium dynamics focuses on the redox conditions that affect the charge transfer equilibrium between the QA and QB sites and how charge is favored at either site. The sharing of electrons between the QA and QB sites and the many factors that affect this equilibrium (such as pH, temperature, proton interactions, water uptake and regulation, the surrounding protein matrix and various charges and conformations of this matrix) are extensively probed and discussed in the literature, though many questions remain.4,17 Repeated charge transfer cycles and prolonged light interactions add to the complexity of the problem.48 However, the present work, in combination with nonequilibrium dynamics theories developed for RCs earlier,1,49 supports a simple mechanism of RC transition between “dark-adapted” and “light-adapted” conformations as self-consistent structural changes in the protein
cofactor matrix, dependent on a history of previous ET events in RCs. Further detailed analysis of our experimental results will be required to separate and to determine to what extent light adaptation, dark relaxation, binding equilibrium shifts, and/or redox shifts (or any of the factors involved in electron equilibrium) play a role in the charge recombination kinetics and distributions. Recent X-ray diffraction studies of light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis showed that light-induced protein conformational changes participate in the function and regulation of photosynthetic reaction centers.6 That study revealed rather pronounced light-induced structural rearrangements within the QA binding pocket, whereas no significant structural changes within the QB binding pocket were reported. However, those X-ray structural studies were made using single actinic laser flashes, which allowed capturing effects 3 ms after the light activation was over. Our current studies show that light-induced structural changes in RCs may become very pronounced and cause a dramatic modification in the charge recombination kinetics only if multiple consecutive photoexcitation (turnover) events to the RC are applied. In addition, no modification in the RC relaxation kinetics is observed if only a single or a few ultra short actinic pulses are applied to the sample. In a recent work by Andreasson et al.,9 the slow relaxation kinetics following prolonged illumination of RCs was attributed to creation of a new, light adapted conformation with an electron localized on QA. Similarly to the conclusions of our earlier work1,49 the authors explained appearance of that new conformation as due to multiple, consecutive charge separation events 8542

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The Journal of Physical Chemistry B that accumulate structural changes in the protein matrix of RCs. However, the suggested description did not analyze the dynamics of formation of different conformations; in contrast, our present work, in line with previously developed theories,1 discusses lightinduced changes in RCs relaxation kinetics as occurring in a selfconsistent manner due to dynamic regulation of accumulated structural changes caused by transferring photoelectrons and reverse action of modified structure on the rate of ET reaction. In addition, our consideration was based on the importance of structural changes caused by photoelectron localization on QB binding site, whereas Andreasson et al. indicated the prime importance of the PþQA
state in creation of a long-lived conformational state. Obviously, more studies will be required to better understand complex dynamic behavior of photosynthetic RCs under lengthy illumination conditions.

’ CONCLUSION The Bayesian analysis technique of the maximum entropy method was used to recover the relaxation rate distributions of photoexcited isolated RCs under various excitation conditions and allowed for improved separation of kinetic distributions compared to that from simple multiexponential fits. The results reported here help support previous claims that the accumulation of slow conformational changes, triggered by charge separation events in the RCs control system dynamics, depend upon illumination conditions and their history. In addition, we have demonstrated here that the charge recombination lifetimes and amplitudes are generally recoverable after application of ascending and descending CW excitation pulses. We demonstrate that there is an increased quinone occupancy in the QB site with increased photoexcitation periods. This increase in QB-site occupancy is not necessarily the cause of the longer charge relaxation period. The longer charge relaxation period is most likely due to other structural changes within the RC, and possibly contributes to the increased QB-site occupancy. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: (951) 827-4713.

’ ACKNOWLEDGMENT The authors would like to thank Dr. N. W. Woodbury for Triton X-100 isolated RCs, and Drs. G. Feher and M. Y. Okamura for the LDAO isolated RCs that they each generously provided for these studies. ’ REFERENCES (1) Goushcha, A. O.; Kharkyanen, V. N.; Scott, G. W.; Holzwarth, A. R. Biophys. J. 2000, 79, 1237. (2) Goushcha, A. O.; Manzo, A. J.; Scott, G. W.; Christophorov, L. N.; Knox, P. P.; Barabash, Y. M.; Kapoustina, M. T.; Berezetska, N. M.; Kharkyanen, V. N. Biophys. J. 2003, 84, 1146. (3) Paddock, M. L.; Feher, G.; Okamura, M. Y. FEBS Lett. 2003, 555, 45. (4) Wraight, C. A. Biophys. J. 2004, 86, 12a. (5) Stowell, M. H. B.; McPhillips, T. M.; Rees, D. C.; Soltis, S. M.; Abresch, E.; Feher, G. Science 1997, 276, 812. (6) Wohri, A. B.; Katona, G.; Johansson, L. C.; Fritz, E.; Malmerberg, E.; Andersson, M.; Vincent, J.; Eklund, M.; Cammarata, M.; Wulff, M.; Davidsson, J.; Groenhof, G.; Neutze, R. Science 2010, 328, 630.

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