Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial

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Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial Photosynthetic Reaction Center From Rhodobacter Sphaeroides Chang Sun, Anne-Marie Carey, Bingrong Gao, Colin A. Wraight, Neal W. Woodbury, and Su Lin J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b05082 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 3, 2016

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The Journal of Physical Chemistry

Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial Photosynthetic Reaction Center from Rhodobacter sphaeroides

Chang Sun†, Anne-Marie Carey‡, Bing-Rong Gao¶, Colin A Wraight†, Neal W. Woodbury‡,§, Su Lin‡,§* †

Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois

61801, United States ‡

The Biodesign Institute, Arizona State University, Arizona State University, Tempe, Arizona

85287-5201 §

School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287-1604

¶

State Key Laboratory of Integrated Optoelectronics, College of Electronic Science and

Engineering, Jilin University, China 130012

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ABSTRACT

It has become increasingly clear that dynamics plays a major role in the function of many protein systems. One system that has proven particularly facile for studying the effects of dynamics on protein-mediated chemistry is the bacterial photosynthetic reaction center from Rhodobacter sphaeroides. Previous experimental and computational analysis have suggested that the dynamics of the protein matrix surrounding the primary quinone acceptor, QA, may be particularly important in electron transfer involving this cofactor. One can substantially increase the flexibility of this region by removing one of the reaction center subunits, the H-subunit. Even with this large change in structure, photoinduced electron transfer to the quinone still takes place. To evaluate the effect of H-subunit removal on electron transfer to QA, we have compared the kinetics of electron transfer and associated spectral evolution for the LM dimer with that of the intact reaction center complex on picosecond to millisecond time scales. The transient absorption spectra associated with all measured electron transfer reactions are similar, with the exception of a broadening in the QX transition and a blue shift in the QY transition bands of the special pair of bacteriochlorophylls (P) in the LM dimer. The kinetics of the electron transfer reactions not involving quinones are unaffected. There is, however, a four-fold decrease in the electron transfer rate from the reduced bacteriopheophytin to QA in the LM dimer compared to the intact reaction center and a similar decrease in the recombination rate of the resulting charge-separated state (P+QA–). These results are consistent with the concept that the removal of the H-subunit results in increased flexibility in the region around the quinone and an associated shift in the reorganization energy associated with charge separation and recombination.


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INTRODUCTION There are three distinct architectures known so far for type II photosynthetic reaction centers (Figure 1a). The core of the water-evolving photosystem II1 and bacterial reaction centers from Chloroflexi2,3 are homologous heterodimers, comprising D1/D2 and L/M protein subunits, respectively. Each monomer polypeptide is composed of five transmembrane alpha helices with the N terminus in the cytosol. Although the water-evolving photosystem II from higher plants and algae contains many additional protein subunits in the vicinity of the D1/D2 proteins, the

Figure 1. (a) The three different architectures reported for type II photosynthetic reaction centers: the core of the photosystem II from Thermosynechococcus vulcanus (left), the reaction center from Rhodobacter sphaeroides (center), and the reaction center from Blastochloris viridis, formerly known as Pseudomonas viridis (right). Atomic coordinates were obtained from the protein data bank files: 3WU2, 1DV3 and 2X5U, respectively. The gray rectangular box behind the structures represents the lipid bilayer membrane. (b) Electron transfer cofactors of reaction center from Rhodobacter sphaeroides. The orientation of the membrane is the same as that in Figure 1a. There are four bacteriocholorophyll molecules, two of which are excitonically coupled and known as the special pair (P), two bacteriopheophytin (HA and HB) molecules, two ubiquinones (QA and QB) and one divalent metal ion (typically an iron). These cofactors are arranged in a two-fold symmetry (branches A and B). Only branch A is active in electron transfer. The electron transfer pathway is 3 ACS Paragon Plus Environment


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two subunits bind all the electron transfer cofactors, as do the L and M protein subunits in the reaction centers of Chloroflexi.2,3 In contrast, all known reaction centers from Proteobacteria, such as Rhodobacter (Rb.) sphaeroides4 used in this work, have, in addition to the two core subunits, an H subunit consisting of a single transmembrane helix and a globular domain in the cytosol. Some reaction centers from the same phylum, such as Blastochloris viridis,5 have a tetraheme subunit on the periplasmic side to facilitate rapid reduction of the photo-oxidized primary donor. The cofactors involved in electron transfer are bound within the core heterodimer with similar structures, positions and orientations as the homologous heterodimer structures like PSII6,7 (Figure 1b). Despite the structural variations, all type II photosynthetic reaction centers reduce quinone to quinol via sequential electron transfer reactions. Although some type II reaction centers inherently lack the H subunit, it is essential for photosynthetic growth in Rb. sphaeroides.8 It is not entirely clear why evolution resulted in an additional H subunit in some reaction centers and not others although it has been hypothesized that the H subunit is essential for reaction center assembly9,10. In fact, recent work has indicated that removal of the H-subunit in Rb. Sphaeroides reaction center results in increased protein flexibility in the region of the primary quinone acceptor, QA.11 This, in turn, suggests that reaction centers lacking the H-subunit may provide a useful system for better understanding the role of protein dynamics in reaction center function, and in particular electron transfer to QA. Both our recent work and work of others has shown the critical role that protein dynamics plays in defining the kinetics and thermodynamics of electron transfer.12–14 The role protein plays in affecting the rate and yield of each electron transfer step has been found to be largely dependent upon the time scale of that reaction. Fundamentally, the protein represents a non-ergodic bath, in which energy coupling and dissipation is highly dependent on


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the reaction time scale.15 Because of this, the reorganization energy of the reaction is dependent on the time scale of the reaction and the type and scale of protein motion available. Our recent studies have shown that protein dynamics has the most profound effects on reaction energetics14– 16

on the hundreds of picosecond to microsecond time scale. With this in mind, we have recently

focused on the transfer of an electron from the bacteriopheophytin (HA–) to the primary quinone acceptor (QA) in the reaction center of Rb. sphaeroides, a reaction that takes place in a few hundred picoseconds (see Figure 1b for a schematic of the electron transfer pathway). Many of our studies have involved altering the amino acids near the HA and Q cofactors to perturb the dynamics. However, it is difficult to introduce large scale flexibility into the system with single amino acid changes while maintaining the overall stability of the protein. H-subunit removal does not block electron transfer to QA, though it is known to prevent the reduction of the terminal quinone acceptor (QB).10–12 Several studies on the kinetics of the multistep electron transfer in the LM dimer have been published,17–21 but there is no time-resolved spectral data available for the P* → P+HA– reaction in the LM dimer, and the published results for HA– → QA reaction are only from single wavelength kinetic measurements and reaction kinetics reported in the literature for this reaction are inconsistent18,21 (see Results and Discussion). In addition, the kinetics of P+HA– recombination, and formation of the carotenoid triplet state, have not been determined. Here, a systematic comparison of electron transfer kinetics (at 298K) between the intact reaction center complex and the LM dimer from Rb. sphaeroides strain SMpHis22 has been performed. The study reveals the kinetics of electron transfer with associated spectral evolution from femtoseconds to microseconds, providing an understanding of not only the overall kinetics but details of kinetic heterogeneity, which is critical to understanding the role of protein dynamics.



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MATERIALS AND METHODS LM Dimer Preparation. Rb. sphaeroides strain SMpHis was used for reaction center expression.22 No difference in reaction center function has been observed between the SMpHis reaction center and the reaction center isolated from 2.4.1, in terms of spectral and electron transfer properties.22 Bacteria were grown photosynthetically under anaerobic conditions in Sistrom medium.23 Reaction center isolation was performed as described previously,22 with the exception that 1%, rather than 0.5%, LDAO (lauryldimethylamine N-oxide) was used in the membrane solubilization step. The LM dimer was prepared from purified reaction centers as described in Debus et al.19 The detergent was then exchanged from 0.025% cholate to 0.05% DDM (n-dodecyl β-D-maltoside) by dialysis. Exchange into DDM was chosen based on several considerations: i) cholate, as an anionic detergent, may interact with the LM dimer electrostatically, especially considering the relatively large area of positive surface charge on the LM dimer (Figure S1, Supporting Information); ii) the LM dimer was difficult to concentrate when solubilized in cholate; iii) the LM dimer is less stable in the detergent LDAO that is commonly used in reaction center preparations.19 The LM dimer reaction centers were concentrated to approximately 120 µM and stored at -80 °C until use. For consistency, intact reaction centers (reaction centers containing the L, M and H subunits) were also detergent exchanged into 0.05% DDM. Quinone Extraction. We followed the protocol reported in Okamura et al.24 to extract quinones from the intact reaction center. The quinones in the LM dimer were extracted using a similar approach. First, 10 mL of LM dimer reaction centers (~ 10 µM) in 20 mM Tris pH 8 were loaded onto a 20 mL DEAE column connected to a FPLC instrument. The column was then 6 ACS Paragon Plus Environment

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washed with 200 mL of 0.5% LDAO in 20 mM Tris pH 8 at 4 mL/min. It was unnecessary to include o-phenanthroline in the wash buffer because quinone binding is much weaker in the LM dimer than it is in intact reaction center complexes. After the washing step, quinone extracted LM dimer was eluted with 0.03% LDAO in 20 mM Tris 200 mM NaCl pH 8. Fractions were selected based on their spectra and pooled and dialyzed overnight to remove excess salt and to exchange the detergent into 0.05% DDM. Millisecond Flash Spectroscopy. The percentage of the quinones extracted from the intact reaction center and the LM dimer was determined using an locally constructed flash spectrophotometer, as described previously.25 QA extracted reaction centers do not exhibit an absorption change that is detectable on the time scale of the experiment (millisecond), however QA can be fully reconstituted with the addition of a saturating amount of ubiquinone (~ 20 µM). The percentage of reaction centers with bound QA was calculated by dividing the amplitudes of the absorbance change at 430 nm before and after quinone addition. The absorbance change at 430 nm between P+QA– and PQA is the largest observed in the blue region.26 Typical assay conditions were ~ 1 µM protein in 20 mM Tris pH 8 with detergent only from the protein stock (final concentration 5 ppm). The quinone stock used was 1.2 mM Q10 (Sigma-Aldrich), prepared by stirring Q10 crystals in 20 mM Tris (pH 8), 10% deoxycholate at 60 °C for one hour. Femtosecond Transient Absorption Spectroscopy. The detailed setup of ultrafast transient absorption spectroscopy has been reported previously.16 Briefly, 1 mJ laser pulses at a repetition rate of 1 kHz (100 fs pulse duration at 800 nm) were generated from a regenerative amplifier system (Tsunami and Spitfire, Spectra-Physics). Part of the pulse energy (600 µJ) was used to pump an optical parametric amplifier (OPA-800, Spectra-Physics) to generate 865 nm excitation pulses. Excitation at 865 nm was used for all measurements as this directly generates the lowest



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excited singlet state of P. Transient absorption changes were measured using a locally constructed broadband transient spectrophotometer coupled with a charge-couple device camera (DU420, Andor Technology). The polarization of the pump pulses was set to the magic angle (54.7°) with respect to that of the probe pulses. Measurements were performed in two overlapping spectral regions: visible (500−760 nm) and near-IR (680−980 nm), in order to investigate the QX and QY transitions of the reaction center bacteriochlorins. Data was collected from 0.5 picosecond (ps) before to 7 nanoseconds (ns) after the excitation pulse. The light path of the spinning wheel was 1.2 mm and ~ 20 µM reaction centers were prepared in 20 mM Tris pH 8, 1 mM o-phenanthroline, 0.05% DDM. Ferrocene (20 µM) and ferrocyanide (1 mM) were added to help minimize the steady state amount of oxidized P+ (Figure S3b, Supporting Information). All measurements were carried out at room temperature. The accumulated raw data were corrected for the wavelength dispersion inherent in the optical system. Processed data were then loaded into Matlab and analyzed by ASUFIT 3.2. A sequential kinetic model was used to fit the data in the global analysis. Nanosecond-to-Microsecond Transient Absorption Spectroscopy. Absorbance changes as a function of time on nanosecond to microsecond time scales were measured using a multichannel pump-probe transient absorbance spectrometer (EOS, Ultrafast Systems, Sarasota, FL). The system utilized a photonic fiber-based continuum generator to create probe light between 360 – 914 nm. The excitation source and sample cuvette were the same as used for femtosecond transient spectroscopic measurements.

RESULTS AND DISCUSSION


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LM Dimer. The LM dimer was prepared from intact reaction center complexes as described in Methods. SDS-PAGE analysis confirmed successful H-subunit removal (Figure S2, Supporting Information). Metal analysis showed there was ~ 0.7 Fe per LM dimer. The long-lived charge separated state P+QA– recombined with a lifetime of 360 ms in the LM dimer (Figure S3, Supporting Information), consistent with previous reports.17,19 Millisecond flash spectroscopy determined that approximately 90% of QA was retained in LM dimer reaction centers. Determination of the QB content of the LM dimer reaction center from spectroscopic observables is hampered due to the very slow rate of electron transfer from QA– to QB (estimated to be 1000fold slower than in intact reaction centers).19 Past work has shown that substantial occupation of the QB site requires the presence of a large excess of free quinone (~ 20 µM Q10).19 Thus, it is expected that nearly all LM dimer reaction centers isolated as described above have only a single functional quinone acceptor (QA). Absorption Spectra Comparison. The ground state absorption spectrum of the LM dimer at room temperature (Figure 2a) is nearly identical to that of the intact complex though there are small differences, as described previously.17,19 Briefly, 1) all three absorption bands of the carotenoid molecule (spheroidene) between 440 and 500 nm are blue shifted by about 2 nm, 2) the QX band that encompasses the two Bphs near 540 nm is sharpened, and 3) the QY band for P is blue shifted about 4 nm (Figure S4, Supporting Information). Additionally, we find that the QX transition band of bacteriochlorophyll is broader and shallower in the LM dimer (Figure 2a, inset). The full-width at half maximum (FWHM) of this peak is 28 nm for the LM dimer, while that for the intact reaction center is 23 nm. This observation has previously been reported by Agalidis et al.17 and has been interpreted in terms of an increased degree of dynamic freedom for the bacteriochlorophyll molecules in the LM dimer relative to intact reaction centers and an



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associated increase in structural heterogeneity. This heterogeneity will be addressed below in terms of the time dependent spectral changes. Note that the 3 absorbance peaks in the carotenoid absorbance region (420 nm to 550 nm) near 442 nm, 471 nm and 503 nm match the reported absorption peaks of spheroidene in 2.4.1 reaction center.27 In order to study the P+HA– recombination kinetics, it is necessary to remove both quinone molecules (QA and QB). The extent of quinone extraction was determined using millisecond flash spectroscopy as described in the Materials and Methods section. Quinone extraction was

Figure 2. Absorption spectra of intact reaction centers containing all three subunits (black) and the LM dimer (red) for (a) quinone-containing and (b) quinone-depleted reaction centers. Spectra were normalized at the absorption maxima at 800 nm. Insets show spectra of the 600-nm band with a linear baseline subtracted from 560 nm to 640 nm. ACS Paragon Plus Environment

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performed as described above and typically removed ~ 90% of QA from the LM dimer. The ground state absorption spectrum of the quinone-depleted LM dimer is compared with that of the quinone-depleted intact reaction center in Figure 2b, and shows a more pronounced blue-shift of the P band in the LM dimer, in agreement with previous reports.17,19 Apparently, either the lack of ubiquinone in the QA site or the process of quinone removal in the LM dimer alters the environment of P. Orientation of the QX transition of HA and QY transition of P. The relative orientation between the QX transition of HA and the QY transition of P were estimated following the method of Vermeglio et al.28 In this study, time resolved difference spectra for quinone-depleted intact

Figure 3. Calculated angles between QX transition of HA and QY transition of P using time resolved spectra at various delay times (from 0.2 ns to 6.7 ns). For this analysis, spectra from 500 nm to 570 nm are corrected using a linear baseline between 500 nm and 570 nm. Absorption changes observed parallel or perpendicular to the direction of the QY transition of P are calculated with equations described previously28. Six different wavelengths around the peak maximum (540 nm, 541.2 nm, 542.4 nm, 543.6 nm, 544.7 nm, 545.9 nm) are used to estimate the statistical error of this analysis (shown as the error bars). The protein in a micelle is sufficiently large so that the rotational correlation time is long enough (~50 ns ACS Plus Environment based on the Perrin equation) for thisParagon polarization based measurement.

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reaction centers and for the LM dimer were recorded as a function of time using polarization parallel and perpendicular relative to the polarization of the 865 nm excitation pulse. The resulting time-resolved spectra recorded at 3 ns, along with the calculated polarized absorption changes for both intact and LM dimer reaction centers are shown in Figure S8. The magnitude of peaks in parallel and perpendicular spectra are directly proportional to cos2α and sin2α, respectively, with α being the angle between the dipolar transition of the peak of interest and QY transition of P.28 As shown in Figure 3, the calculated angles between QX transition of HA and QY transition of P between time delay of 200 ps to 6.7 ns essentially invariant. The differences between the two reaction centers are also within 5% of the total value, suggesting that there is no significant difference in the angle between the QX transition of HA and QY transition of P in the LM dimer compared to the intact reaction center. Initial Charge Separation. The H subunit is an integral part of the purple bacterial reaction center. Its globular soluble domain interacts extensively with both the L and M subunits via salt bridges (Figure S1, Supporting Information) and its transmembrane helix stacks on top of helix 5 of the M subunit. Extraction of the H subunit from the reaction center would, therefore, be expected to result in both static and dynamic changes in the protein environment of the electron transfer cofactors in the LM dimer, and possibly changes in their relative positions/orientations as well. To investigate the effect on the primary electron transfer (P* forming P+HA–), timeresolved spectra for the LM dimer and the intact reaction center were collected as a function of time after the 865 nm excitation. The time resolved spectra at several representative time delays are plotted in Figure 4a and 4b. At 0.5 ps, P has been excited to form P*, and the absorption difference spectra at 600 nm and 865 nm are due to bleaching of the ground state bands of P. The apparent absorbance decrease in the 950 nm region is due to stimulated emission for P*. As


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charge separation takes place, the stimulated emission from P* diminishes, resulting in a recovery of the bleaching signal between 865 and 950 nm. Simultaneously, bleaching bands at 545 nm and 760 nm develop and there is an increase in the absorbance near 665 nm (Figure 4a and 4b, 10 ps trace). These features are due to the ground state bleaching of QX (545 nm) and QY (760 nm) transitions of the bacteriopheophytin at the HA binding pocket, and the absorption



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increase of the newly formed HA– signal (665 nm). Therefore these spectral changes (Figure 4a and 4b) reflect the electron transfer of P* → P+HA–. The early-time spectral changes in the LM dimer are very similar to those observed in the intact reaction center, except that the amplitude of the bleaching at 600 nm is only ~ 70% of that of the intact reaction center. In the meantime, the 600 nm bleaching is significantly broader for

Figure 4. Time-resolved absorption difference spectra for (a) the intact reaction center and (b) the LM dimer recorded within the first 10 ps following laser excitation at 865 nm. Kinetic comparison of intact reaction centers and LM dimers at (c) 545 nm and at (d) 950 nm, showing formation of HA– and the decay of P*, respectively. The smooth curves in these two panels represent mono-exponential fittings. 14 ACS Paragon Plus Environment

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the LM dimer: at 10 ps, the FWHM of this peak is 27 nm for the LM dimer, while that for the intact reaction center is 20 nm. The integral areas for this peak are comparable. The broadening observed in the time resolved spectra is greater than that observed in the ground state absorption spectra (Figure 2, insets). The time resolved spectra show only the contribution from the QX band of P, while the ground state absorption spectra have contributions from both P and monomeric bacteriochlorophylls (Bs), implying that it is the P ground state band that is responsible for the spectral broadening. In contrast, the FWHM of the QY band of P remains more or less the same (56.7 nm in the LM dimer vs 55.6 nm for the intact reaction center). The kinetics of the intact 3-subunit reaction center and the LM dimer are identical for both the HA– formation (545 nm, Figure 4c) and the decay of the P* (950 nm, Figure 4d). Apparently, the primary electron transfer from P to HA is not disturbed when the H-subunit is extracted from the reaction center. Electron transfer from HA– to QA. In intact reaction centers, the electron is transferred from HA– to QA with a time constant of 200 ps.29–33 For the LM dimer, this electron transfer time constant has been less clear based on past literature, ranging from 200-450 ps, based on measurements near 670 nm.

18, 21

To better understand this process, a more comprehensive

analysis was performed over a broad spectral range as a function of time (Figure 5), making it possible to simultaneously monitor absorbance changes for both ground and ionic states. Looking at the time dependent spectra, one can see that the spectral changes associated with HA ground state bleaching recovery near 545 nm and decay of the HA– anion signal near 665 nm are much slower in the LM dimer than in the intact reaction center (the spectral evolution is essentially complete by 800 ps in Figure 5a, but still continues between 800 ps and 4 ns in Figure 5b). Figure 5c shows a particular kinetic trace at 545 nm, again demonstrating that the HA



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ground state recovery is several fold slower in the LM dimer. As discussed below, fitting of the spectral evolution gives rise to an 860-ps time constant for the HA to QA electron transfer in the LM dimer. Notably, and in contrast to what is observed for the intact reaction center, the bleaching detected for the LM dimer at 865 nm and 4 ns is ~95% of that present at 20 ps (Figure

Figure 5. Transient absorption spectra collected for (a) the intact reaction center containing all three subunits and (b) the LM dimer. Kinetics comparison at (c) 545 nm and (d) 865 nm for HA– and P+ signals of the LM dimer (red) and the intact reaction centers (black) on nanosecond time scale. The first data point for all kinetics traces was recorded at 20 ps after excitation. The kinetics trace for the LM dimer in (c) has been up shifted by 3 mOD for better comparison. Fitting with a single exponential model (c) revealed a rate constant of 220 ps for the reaction center and 860 ps for the LM dimer. 16 ACS Paragon Plus Environment

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5d). Apparently there is a small amount of P ground state recovery on this time scale, a process that will be explored on longer timescales below. Global analysis of the QA containing LM dimer spectral changes. Time resolved spectral surfaces were fit for the entire spectral region between 400 and 914 nm and analyzed using a sequential, irreversible kinetic model (A→B→C→…). The amplitude spectra corresponding to each kinetic component obtained in this way are referred to as evolution-associated decay spectra (EADS).34 Although almost certainly not an accurate representation of the actual states present at

Figure 6. EADS for (a) intact reaction centers and (b) LM dimer reaction centers generated by global analysis with a sequential model. Refer to main text for details.



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any given time, this sequential kinetic model provides a simple means of dissecting the progression of spectral changes and associated time constants. The sequential model is suitable for electron transfers in the reaction center due to the much larger forward rate constants compared to the corresponding backward rate constants. The rate constants for the P* → P+HA– and P+HA– → P+QA– reactions were determined assuming a sequence of two irreversible reactions. For both samples studied, three kinetic components adequately fit the data for the entire QX and QY wavelength regions. The EADSs for intact and LM reaction centers are shown, together with associated decay time constants, in Figure 6a and 6b, respectively. For a strictly sequential reactions, EADS describes the evolution of the absorbance change spectra, for example, the first EADS evolves with its associated time constant to the second EADS. The EADS associated with the shortest time constant in the LM dimer (black curve in Figure 6b) shows spectral features typical of P*. P* evolves to P+HA–, with a time constant of 3.4 ps. This P+HA– state further evolves to P+QA– with a time constant of 860 ps. In contrast, the intact reaction center containing the H subunit has corresponding time constants of 3.2 ps and 210 ps, in good agreement with previous reports.35 Charge Recombination of P+HA–and Associated Triplet Kinetics. Transient absorption spectra were recorded for quinone-extracted samples, from 400 nm to 915 nm with a 1-ns time resolution for 10 µs following excitation at 865 nm (Figure S6, Supporting Information). The scenario laid out based on previous studies on the ns to µs involves the recombination of P+HA– radical pair and the formation of the triplet state of P. When the primary quinone acceptor is reduced or removed, the P+HA– radical pair lives for nanoseconds before decaying via recombination. Part of it recombines when the radical pair electrons are still in the singlet state, either directly or via the excited singlet P*. Another part interconverts to an almost isoenergetic


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triplet 3[P+HA–], which can further form the triplet state of P (3P) by charge recombination. In carotenoid containing reaction centers, the dominant decay pathway of 3P is to transfer the triplet energy to the bound carotenoid.36–38 Global analysis reveals three kinetic components with lifetimes of 12 ns, 22 ns and 1.4 µs for intact reaction center and 12 ns, 23 ns and 1.4 µs for the LM dimer. The 12-ns EADS (Figure 7a and 7b) resembles that of P+HA– observed in the ps transient measurement and the 12 ns time constant presumably represents the decay of that state. The spectral profile and the associated lifetime are in good agreement with previous studies.39–42 The kinetics at 545 nm and 865 nm, due to the recovery of HA– and P+, respectively, are essentially identical in both intact and LM dimer reaction centers (Figure 7c and 7d, respectively). After decay of the 12 ns component, 30– 40% of the P band bleaching at 865 nm remains. Previous studies have reported that P+HA– recombines to form the triplet state, 3P, after spin dephasing in the P+HA– state. This dephasing process requires nanoseconds to take place and thus a noticeable amount of 3P can only be observed when forward electron transfer from HA is blocked by removal or reduction of QA.27,43 The 22-ns/23-ns EADS is consistent with the expected spectra and kinetics of triplet P. There is a dominant bleaching band at 865 nm, but the spectral characteristics of P+HA–, such as the positive and negative bands at 790 and 810 nm, the bleaching at 545 nm, and the absorbance increase 665 nm, are largely diminished. The spectral comparison of the 12-ns and 24-ns EADS normalized at the P-band at 865 nm reveals that less than 30% of the P+HA– state remains in the 22-ns/23-ns EADS (Figure S7, Supporting Information). Given the depletion of ground state P, the lack of P+HA– spectral signature and the long live time P-triplet state (3P) is assigned to this component. In the absence of carotenoid, 3P has a life time of 10 µs at room temperature.44 As a photoprotection mechanism, the carotenoid quenches the triplet energy of P to prevent it from



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reacting with singlet oxygen.43 The 1.4 µs EADS (blue) shows a bleaching between 420 – 490 nm and emergence of a narrow absorption band at 545 nm, indicative of the formation of carotenoid excited state, likely resulting from triplet-triplet energy transfer from 3P to Carotenoid via BA.37 It should be noted that the EADS with time constants of 12- and 23-ns were obtained from global analysis based on an irreversible sequential reaction model, and therefore do not

Figure 7. EADS for quinone extracted reaction centers of (a) intact complexes and (b) LM dimer, resulting from global analysis of transient absorbance change spectra recorded on µs time scale. Comparison of the kinetics of decay of the state P+HA- for quinone extracted intact and LM dimer reaction centers (c) at 545 nm measuring the disappearance of the ground state bleaching of HA and arise of carotenoid triplet state , (d) at 865 nm measuring the20 ACS Paragon of Plus disappearance of the ground state bleaching P. Environment

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necessarily represent pure kinetically resolved states. A lower limit of the time constants of the actual microscopic reactions was estimated assuming that the 23-ns EADS is dominated by the 3

P state. Taking the P-band bleaching in the 23-ns EADS as the maximum yield of 3P formed, it

is calculated that τelectron transfer =τobs/φelectron transfer, where φelectron transfer is the relative population undergoing an individual reaction path, τobs is the observed time constant associated with the EADS obtained in the fit. This analysis yields a time constant of 19 ns for P+HA– recombination, both in the intact reaction center and the LM dimer, which has previously been determined to be 15-20 ns45–52 in the intact reaction center. On the other hand, multi-exponential kinetic fitting of the carotenoid triplet peak at 545 nm resulted in a rise-time of 26 ns (LM dimer) and 25 ns (intact reaction center) and decay of 1.3 µs (both LM dimer and intact reaction center), representing the formation and decay of the carotenoid triplet state, respectively. These values agree well with the reported value of 27 ns for triplet carotenoid (3Car) formation.53 A 4-µs time constant was reported previously for the decay of the triplet carotenoid in reaction centers isolated from Rb. sphaeroides strain 2.4.1 when measured anaerobicly,54 almost 3 times longer than our value. This discrepancy is likely due to the oxygen presented in our samples, which is known to accelerate carotenoid triplet decay.55 The key feature of the above analysis is that 1

[P+HA–] → 1P and 3[P+HA–] → 3P → 3Car kinetics in intact reaction centers and the LM dimer is

essentially the same. Kinetics Model. All time constants determined in this study for electron transfer reactions in the LM dimer and the intact reaction center are summarized in Table 1. The time constants for the forward electron transfer P* → P+HA–, P+HA– → P+QA– and the 3P → 3Car triplet energy transfer were obtained directly from the global analysis since these processes are associated with well-defined spectral features. The time constants for P+HA– recombination, and P+HA– → 3P are



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estimated based on their relative yields and their combined rate from global analysis, as described above. The P+QA– recombination was measured directly at 430 nm using millisecond

Figure 8. Schematics of photochemical reaction pathways and time constants (at 298K) for the intact reaction centers (black numbers) and the LM dimer (red numbers) with QA present (a), and in the absence of QA (b). Triplet P formation and triplet energy transfer to carotenoid are only observed when QA is removed. *Time constant for direct 3P decay via intersystem conversion without carotenoid was taken from ref 27. flash induced spectroscopy (Figure S3, Supporting Information). Figure 8 presents a kinetic scheme incorporating the information from Table 1.

This is based on a kinetic scheme

previously presented in deWinter et al37 and Laibel et al. 43

Table 1. Comparison of electron transfers kinetics at 298 K between the LM dimer and the intact reaction center. P*→P+HA–

P+HA–→P+QA–

P+HA–→PHA P+QA–→PQA

P+HA–→3P

3

P→3Car


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Intact RC

3.2 ps

210 ps

19 ns

100 ms

29 ns

25 ns

LM dimer

3.4 ps

860 ps

19 ns

360 ms

30 ns

26 ns

Kinetics Changes in the LM Dimer. Both forward and backward electron transfer kinetics between P and HA are very similar in intact and LM dimer reaction centers. This implies the extraction of the H subunit from the reaction center protein only has a minor influence on the arrangement of cofactors. However, the P+HA– spectra on both ps and ns time scales show a broadening of the QX band of P and a blue shift of the QY band of P, suggesting some changes in the local environment. Note that the blue shift of the QY band of P is more obvious in the quinone extracted LM dimer reaction centers, likely due to a further perturbation of the protein matrix during quinone extraction. To determine whether these absorption spectral changes reflect a variation in the orientation of the cofactors, the linear dichroism of transitions associated with the P+HA– state has been measured. The average angle between the QX transition of HA and the QY transition of P, following the protocol of Kirmaier56 and Vermeglio,28 was calculated to be 52.5 and 53.5 degree in the quinone extracted intact reaction center and the quinone extracted LM dimer, respectively (Figure 3). The similarity of these angles suggests, though indirectly, that any change in the orientation of HA with respect to P caused by removal of H subunit is not significant enough to explain the observed change in absorption for P. Therefore, the spectral broadening and blue shift of the P band in the QX and QY transitions, respectively, are more likely induced by protein environmental changes around P. From the fact that the HA– → QA electron transfer and P+QA– charge recombination are both significantly slower in the LM dimer than those in intact reaction centers, it appears that only electron transfer reactions involving QA are affected in the absence of the H subunit. One



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possible explanation is that upon removal of the H subunit, the quinone in the QA pocket adopts one or more alternate conformations. This has been addressed in a separate study using pulsed electron paramagnetic resonance.11 The experimental results imply that the hydrogen bonds around QA– are significantly weakened in the LM dimer relative to intact reaction centers. However, data from the same study shows the midpoint potential for QA/QA– in the LM dimer doesn’t change significantly, suggesting the degree of weakening in binding of the fully oxidized QA in the LM dimer is similar to that for the semiquinone QA–. Both driving force and reorganization energy play a major role in determining the kinetics of electron transfer. When the H subunit is extracted from the reaction center protein, a large portion of the protein surface that is normally buried becomes exposed to the surrounding aqueous environment. The increased protein-water interaction can affect the activation barrier of the reaction through dynamic restriction of the configurational space sampled by the protein– water solvent on the hundreds of picosecond to nanosecond reaction time-scale.14 Proteins as dynamic media thus allow dynamic tuning of the reaction rates, in contrast to the commonly assumed thermodynamic tuning achieved by adjusting the reaction free energy (redox potentials) alone. These ideas have been applied to the electron transfer between HA and QA cofactors previously for the mutant M214LG to explain an observed decrease in transfer rate and increased reaction heterogeneity.14,16 In this regard, it is interesting that the electron transfer rate constant for HA– to QA transfer in a related reaction center from Chloroflexus aurantiacus, which has no H subunit naturally and has a large exposed protein surface around the quinone pocket as the LM dimer, has been reported to be 330 ps.57 Of course, an alternate explanation is that the change in protein structure or dynamics near HA and QA in the LM dimer may change the relative geometry between the QA and HA in a way that is less favorable for HA– → QA electron transfer.


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CONCLUSION This study compares the electron transfer kinetics of the LM dimer of Rb. sphaeroides strain SMpHis with those of the intact reaction center containing all three (L, M and H) subunits. A comprehensive picture of the electron transfer reactions in the LM dimer is presented, together with the associated spectral evolution. The results indicate that, while the rates of initial charge separation and charge recombination of P+HA– remain essentially the same upon removal of the H subunit, the rates of forward electron transfer from HA– to QA and charge recombination of P+QA– are reduced by a factor of four. Though some broadening and shifting of transitions associated with P are observed, there is no significant difference in the angle between the QX transition of HA and QY transition of P in the LM dimer compared to the intact reaction center. These results suggest that in terms of electron transfer functionality, the primary effects of Hsubunit removal involve changes in interactions between the quinone in the QA pocket and the surrounding protein. Recently theoretical treatments of the role of the protein environment on the HA to QA electron transfer reaction have shown that the available reorganization energy depends strongly on the rate of the reaction, due to the fact that the nature of the vibrational motions that are able to couple with the reaction are dependent on the time scale of the reaction.15 This is consistent with the current findings: removing the H-subunit likely changes the dynamics of the interactions between QA and the surrounding protein bath which in turn alters the reorganizational energy and thus the rates of the reactions involving the quinone.



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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author: * Su Lin Email: [email protected]

ACKNOWLEDGEMENTS This work was funded by NSF grants MCB-0818121 at UIUC and MCB-1157788 at ASU. BRG thanks National Science Foundation of China for funding through No. 21473076. CS thanks Dr. Antony R Crofts for helpful discussion.

Supporting Information The document contains details regarding electrostatics calculations, SDS gel, millisecond flashinduced spectroscopy, ground state absorption comparison, picosecond peak assignments, nanosecond kinetics comparison and linear dichroism analysis (PDF with link). The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.


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Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial

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