Mechanism of Triplet Energy Transfer in Photosynthetic Bacterial

Article pubs.acs.org/JPCB

Mechanism of Triplet Energy Transfer in Photosynthetic Bacterial Reaction Centers Sarthak Mandal,*,† Anne-Marie Carey,† Joshua Locsin,† Bing-Rong Gao,§ JoAnn C. Williams,‡ James P. Allen,‡ Su Lin,†,‡ and Neal W. Woodbury*,†,‡ †

Center for Innovations in Medicine, The Biodesign Institute at ASU, Arizona State University, Tempe, Arizona 85287, United States School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287−1604, United States

‡

S Supporting Information *

ABSTRACT: In purple bacterial reaction centers, triplet excitation energy transfer occurs from the primary donor P, a bacteriochlorophyll dimer, to a neighboring carotenoid to prevent photodamage from the generation of reactive oxygen species. The BB bacteriochlorophyll molecule that lies between P and the carotenoid on the inactive electron transfer branch is involved in triplet energy transfer between P and the carotenoid. To expand the high-resolution spectral and kinetic information available for describing the mechanism, we investigated the triplet excited state formation and energy transfer pathways in the reaction center of Rhodobacter sphaeroides using pump−probe transient absorption spectroscopy over a broad spectral region on the nanosecond to microsecond time scale at both room temperature and at 77 K. Wild-type reaction centers were compared with a reaction center mutant (M182HL) in which BB is replaced by a bacteriopheophytin (Φ), as well as to reaction centers that lack the carotenoid. In wild-type reaction centers, the triplet energy transfer efficiency from P to the carotenoid was essentially unity at room temperature and at 77 K. However, in the M182HL mutant reaction centers, both the rate and efficiency of triplet energy transfer were decreased at room temperature, and at 77 K, no triplet energy transfer was observed, attributable to a higher triplet state energy of the bacteriopheophytin that replaces bacteriochlorophyll in this mutant. Finally, detailed time-resolved spectral analysis of P, carotenoid, and BB (Φ in the M182HL mutant) reveals that the triplet state of the carotenoid is coupled fairly strongly to the bridging intermediate BB in wildtype and Φ in the M182HL mutant, a fact that is probably responsible for the lack of any obvious intermediate 3BB/3Φ transient formation during triplet energy transfer.

1. INTRODUCTION Photosynthetic complexes of green plants, algae, and bacteria convert light energy into electrochemical potential with high quantum efficiency. When exposed to intense illumination, highly reactive and potentially damaging singlet oxygen species can be produced. To prevent this damage, photosynthetic systems have built-in photoprotection pathways that dissipate the excess energy from unwanted excited states, typically involving triplet energy transfer to carotenoids.1−3 In purple nonsulfur photosynthetic bacteria, the site of the primary photochemistry, the reaction center (RC), contains a carotenoid that can quench unproductive excited states of the bacteriochlorophyll molecules. In Rhodobacter sphaeroides, the RC cofactors include a bacteriochlorophyll dimer (P), two bacteriochlorophyll monomers (BA and BB), two bacteriopheophytins (HA and HB), two quinones (QA and QB), and a nonheme Fe that are arranged in two quasi-symmetric chains, known as the A and B chains (Figure 1).4 Only the cofactors in the A chain participate substantially in photosynthetic charge separation in the wild-type (WT) RC.5−8 A symmetry-breaking carotenoid (Car) is located 11 Å away from P and near BB in the B chain.9−11 Under semiaerobic growth conditions, this carotenoid is sphaeroide© 2017 American Chemical Society

none in its 15,15′-cis conformation. The cofactor BB lies between P and the carotenoid, and its phytol tail is in van der Waals contact with the carotenoid. In the reaction center, light absorption results in formation of the singlet excited state of P (P*), followed by electron transfer along the A chain of cofactors, from P* → P+BA− → P+HA− → P+QA− → P+QB− (black arrows in Figure 1). However, when the quinone acceptors are biochemically removed, electron transfer from HA− to QA is not possible and the charge-separated singlet radical pair, 1(P+HA−), undergoes charge recombination to the ground state (blue arrow in Figure 1) on the 10−20 ns time scale.7 The recombination process is accompanied by a competing process of spin dephasing, converting 1(P+HA−) to the triplet charge-separated state 3(P+HA−), which leads to the formation of the triplet state of P ( 3P) upon charge recombination.12−16 The 3P state is formed by charge recombination of P+HA− on a time scale of 10−20 ns. Therefore, under normal conditions in quinone-containing reaction centers, where the forward electron transfer from P+HA− to P+QA− occurs Received: April 10, 2017 Revised: June 9, 2017 Published: June 12, 2017 6499

DOI: 10.1021/acs.jpcb.7b03373 J. Phys. Chem. B 2017, 121, 6499−6510

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The Journal of Physical Chemistry B

Measurements of RCs isolated from the WT and the carotenoid-less R-26 strains of R. sphaeroides have shown that triplet energy transfer from 3P to the carotenoid takes place on the nanosecond time scale.19,22,23 The previous transient absorption studies of this process have had limited spectral and temporal resolution since the experiments made use of measurements of the kinetics at single wavelengths.22,23,37 In this work, the kinetics and spectral evolution of absorbance changes associated with the primary radical pair P+HA− charge recombination (singlet and triplet), and triplet energy transfer process from 3P to the carotenoid via BB are measured in detail at room temperature and 77 K. The quality of the measured optical changes is significantly improved compared to previous measurements as a full spectral region (400 to 910 nm) is measured for each time point. In addition, the system allows measurement over a wide temporal range (nanoseconds to microseconds), providing a complete set of temporal data for full kinetic analysis of the multistep process of energy transfer. In addition to RCs purified from the WT and R26 strains, measurements were performed on RCs isolated from M182HL as well as a carotenoid-less M182HL mutant. In addition to the mutation at M182, the carotenoid-less M182HL strain contains the alteration of Gly M71 to Leu, which changes the carotenoid binding pocket and results in the loss of the carotenoid.39 By comparing the results for RCs with either a bacteriochlorophyll or bacteriopheophytin as the intermediate energy acceptor, with and without a carotenoid, we have developed a detailed spectroscopic and kinetic picture of the interactions and dynamics of intermediates in triplet energy transfer.

Figure 1. Structure of the WT RCs showing the arrangement of the cofactors and the directional pathways of electron and energy transfer. The cofactors in the A and B chains are assigned as the suffix A and B, respectively. The quinone and nonheme iron cofactors are omitted for clarity.

at a much faster rate (200 ps) with near unity quantum efficiency, the formation of 3P by charge recombination of P+HA− is very unlikely. However, under high-light conditions, the quinone (QB) pool may become fully reduced, with the unavailability of quinone in the QB binding site leading to stability of the P+HA− state and consequently formation of 3P, producing singlet oxygen and a decrease in the cell survival rate in the absence of the carotenoid.17,18 The carotenoid can efficiently quench 3P by triplet energy transfer (red arrows in Figure 1), similar to the triplet bacteriochlorophyll to carotenoid transfer observed in bacterial light-harvesting complexes.19−33 This quenching prevents 3P from interacting with molecular oxygen and generating reactive singlet oxygen species. The triplet energy transfer to the carotenoid is thought to take place via a Dexter-type, twoelectron exchange mechanism, which requires strong electronic coupling between the donor and acceptor.34 Given the relative positions of P, BB, and the carotenoid, the triplet energy transfer almost certainly proceeds via the generation of the triplet state of BB (3BB) (red arrows in Figure 1) analogous to the pathway of singlet excitation energy transfer from carotenoid to P via BB.21,26,35,36 The triplet energy transfer from 3P to the carotenoid was first established by the observation of light-induced formation of the triplet state of the carotenoid (3Car), and an intermediate state was predicted through analysis of low-temperature measurements.20,25 Experimental verification of an essential role of 3BB as an intermediate state was shown by transient absorption measurements of RCs containing chemically modified BB cofactors.23,26 The triplet state energy of BB was previously estimated to be 200 cm−1 higher relative to the energy of 3P (7590 ± 20 cm−1).21,37 This estimation was based on phosphorescence measurements of free bacteriochlorophyll in a glass matrix. However, the energetics of 3BB inside the RC protein complex is not clear. The BB cofactor is coordinated by His M182 and the replacement of this residue with leucine in the M182HL mutant results in the incorporation of a bacteriopheophytin molecule at this position (denoted as Φ) in place of the bacteriochlorophyll molecule.38 The M182HL mutation does not significantly affect the absorption peak of the P band21,38 but the energetics of triplet energy transfer are altered compared to WT, as the cofactor change from BB to Φ alters the activation energy associated with the bridging intermediate cofactor.21

2. MATERIALS AND METHODS The carotenoid-less M182HL mutant strain was constructed, and the WT, R26, M182HL, and carotenoid-less M182HL reaction centers were isolated and purified from R. sphaeroides as described previously.38,40 In order to generate substantial 3P, it is necessary to remove the quinones (QA and QB) from the RCs, preventing forward electron transfer from HA− to QA. Both the primary and secondary quinones (QA and QB) were removed from the RCs using a modified version of the method reported earlier by Okamura et al.41 In brief, 15−20 mL of a dilute solution of RCs were loaded into a 40 mL DEAE column. The column was then washed for 2−3 h at 26 °C using 400 mL of 15 mM TrisHCl, pH 8.0, containing 4.0% lauryldimethylamine N-oxide (LDAO), 1 mM EDTA, and 10 mM o-phenanthroline. The reaction centers were eluted in buffer containing 15 mM TrisHCl, pH 8.0, 0.1% LDAO, 1 mM EDTA, and 250 mM NaCl. Finally, the reaction centers were dialyzed overnight to remove excess salt and o-phenanthroline in 15 mM Tris-HCl, pH 8.0, 0.045% LDAO, 1 mM EDTA, and 150 mM NaCl. The quinonedepleted RCs were concentrated and stored at −80 °C. This procedure resulted in 90−95% removal of QA as determined from the steady state levels of P+QA− formed upon continuous illumination (see Figure S1b). For the transient absorbance measurements, the reaction centers were diluted in 15 mM Tris-HCl, pH 8.0, 0.045% LDAO, 1 mM EDTA, and 150 mM NaCl and loaded into a 2 mm cuvette, and the samples were stirred during the measurements with a magnetic stir bar. For low temperature measurements the RCs were dissolved in a 3:1 (v/v) glycerol-buffer mixture. The final absorbance at 800 nm of the samples was about 0.8 in the 2 mm cuvette. The transient absorption measurements in the time region from nanoseconds to microseconds were performed using a 6500


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Figure 2. Two-dimensional pseudocolor contour plots of transient absorption data in the QX absorption region of quinone-depleted WT (a,c) and M182HL mutant (b,d) RCs recorded at room temperature and at 77 K after excitation at either 865 nm (room temperature) or at 880 nm (77 K). The plots highlight the excited state absorption and ground state bleaching bands corresponding to the transitions of P, H, and Car into different states involved in the triplet energy transfer dynamics as indicated in the figure.

Figure 3. Time-resolved absorption difference spectra of quinone-depleted WT RCs at room temperature (a,c) and at 77 K (b,d). The spectra are recorded at various time delays following the excitation at either 865 nm (room temperature) or 880 nm (77 K).

commercial pump−probe spectrometer (EOS VIS-NIR, Ultrafast Systems, Sarasota, FL). This system has a broadband probe light generator that covers the entire wavelength region from 400 to 910 nm. A separate femtosecond laser system is used to generate the excitation pulse as described in detail elsewhere.42 In brief, a regenerative amplifier system (consisting of a Tsunami oscillator and a Spitfire amplifier, Spectra-Physics) was used to generate 1 mJ laser pulses at a repetition rate of 1 kHz (100 fs pulse duration, 800 nm). The excitation pulse at 865 nm was generated by an optical parametric amplifier (OPA-800, SpectraPhysics) using the optical pulse from the regenerative amplifier.

Excitation pulses at 880 nm were used for the measurements at 77 K.

3. RESULTS AND DISCUSSION 3.1. Data. The ground state absorbance spectra of WT, R26, M182HL, and carotenoid-less M182HL RCs are compared in Figure S1a. At low temperature the ground state absorbance band of BB (around 810−812 nm) is distinguishable from that of BA (around 800−803 nm) in WT RCs.35,43 The maximum absorbance in the QY region of Φ in the M182HL RC is near 790 6501


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nm, about 10−15 nm blue-shifted relative to the maximum QY absorption of the B-side monomer bacteriochlorophyll in WT RCs. Therefore, direct observation of the transient absorption changes of BB (at 812 nm) in WT or Φ (at 790 nm) in the M182HL mutant can provide information regarding the interactions and dynamics of the bridging intermediate (3BB or 3 Φ) during triplet energy transfer from 3P to the carotenoid. The dynamics of 1(P+HA−)/3(P+HA−) charge recombination, 3 P formation and triplet energy transfer were monitored for quinone-depleted RCs using transient absorption spectroscopy in the nanosecond to microsecond time region with a 1 ns time resolution. The measurements were performed both at room temperature and at 77 K. Figure 2 represents two-dimensional pseudocolor contour plots of transient absorption changes in the QX region of WT and the M182HL mutant as a function of probe wavelength and pump−probe delay recorded at room temperature and at 77 K. The corresponding plots of transient absorption changes of RCs in the QY absorption region are shown in Figure S2. The initially populated 1(P+HA−) state is indicated by the ground-state bleaching bands of P and H, and excited state absorption bands of P+ and HA−. 1(P+HA−) is partially converted by a spin dephasing process to 3(P+HA−), which is spectrally identical to 1(P+HA−). The bands associated with 1(P+HA−)/3(P+HA−) largely fade away within 10−20 ns (Figure 2a−d) in WT and mutant RCs, both at room temperature and 77 K. The triplet energy transfer from 3P to the carotenoid for WT and the M182HL mutant results in excited state absorption features of 3Car at 600 nm and ground state bleaching of the carotenoid at 480 nm. The lack of an excited state 3Car absorption band at 600 nm in M182HL mutant at 77 K is consistent with a higher activation energy for triplet energy transfer in this mutant RC than in WT RC. 3.2. Time-Resolved Spectra in the Nanosecond (ns) to Microsecond (μs) Time-Region. Figure 3a,b shows timeresolved absorption spectra of WT-RCs in the 410−910 nm spectral range extracted from the whole data set (Figures 2 and S2) at several characteristic time delays, both at 298 and 77 K. The 1 ns difference spectrum has the spectral characteristics of P+HA− including ground state bleaching associated with P at 865 and 600 nm, ground state bleaching associated with HA at 760 and 545 nm, and absorbance increases near 665 and 700 nm associated with HA− and P+, respectively.42 The state 1(P+HA−) can either recombine to the ground state (P, H) or undergo a spin dephasing process to generate 3(P+HA−), which then recombines to form 3P. By 30 ns, the spectral characteristics associated with 1(P+HA−)/3(P+HA−) have largely disappeared due to charge recombination.44,45 The branching ratio between singlet recombination and 3P formation depends on the oscillation frequency of the spin dephasing and the relative rates of singlet and triplet recombination reactions. 3 P in WT RCs is quenched by the carotenoid (sphaeroidenone) through triplet energy transfer. In the 30 ns time-resolved spectra, the HA− signals at 545 and 760 nm have largely decayed, but there is still some bleaching of the P transitions at 600 and 860 nm, consistent with partial 3P population (Figure 3a,b). At the same time, a broad absorbance increase between 500 and 650 nm and ground state bleaching between 425 and 500 nm has begun to form, consistent with 3Car formation (Figure 3a,b).22,23,26,31 By 100 ns at 298 K, or 500 ns at 77 K, the spectral features associated with 3P are essentially gone (e.g., bleaching at 860 nm) and the spectrum has enhanced features associated with 3Car, particularly in the 450−650 nm region. The

Car spectrum then decays on the microsecond time scale at both 298 and 77 K (Figure 3c,d). For comparison, the formation and decay of 3P was also measured in carotenoid-less R26 RCs, with quinones removed, at both 298 and 77 K (Figure S3). The spectrum associated with P+HA− decays on the nanosecond time scale, recombining partly to the ground state and partly to 3P, and once formed, 3P decays to the ground state in tens of microseconds (298 K) to hundreds of microseconds (77 K) in the absence of carotenoid.19,23 The 3P state decays nearly 3 orders of magnitude more slowly in the absence of carotenoid than in its presence both at 298 and 77 K (compare time-dependent spectra in Figures 3 and S3), consistent with an essentially 100% triplet energy transfer efficiency from 3P to the carotenoid in WT RCs at both temperatures. Although the efficiency of triplet energy transfer is not significantly temperature dependent, 3Car formation is slower at 77 K than at 298 K, implying that the rate of triplet energy transfer in RCs decreases at low temperature.13 One possible explanation of the decreased 3P to the Car triplet energy transfer rate is that the 3BB intermediate is thermally activated. The transient absorption spectra of the M182HL mutant at several selected time delays (Figure S4a,c) showed the same processes of 1(P+HA−)/3(P+HA−) charge recombination and triplet energy transfer at room temperature, but 3Car formation through energy transfer in this mutant RC is seen to be significantly slower than in WT RCs. At 77 K, the triplet energy transfer in the M182HL mutant is thermally prohibited as revealed in the transient absorption spectra (Figure S4b,d) by the absence of the characteristic absorbance change due to 3Car excited state absorption at 600 nm. The decay of the long-lived 3P in the absence of energy transfer is essentially the same as observed in R26 RCs at 77 K. Carotenoid-less M182HL mutant RCs exhibit spectral characteristics (Figure S5) similar to that observed in R26 RCs. 3.3. Interactions of P and Car with BB. In the transient difference spectra on the hundred nanosecond to microsecond time scale, there is a narrow, pronounced absorption decrease at 812 nm accompanied by a small bleaching of the ground state P band at 865 nm, both at room temperature and low temperature (Figure 3c,d). These features remain well after one would expect both P+ and 3P to have decayed in the WT RCs. Past studies have placed the BB ground state QY transition at 812 nm.35,43 The decay kinetics of these negative absorbance changes (monitored at 812 and 865 nm) is the same as the 3Car (monitored at 600 nm) on the 2−5 μs time scale (Figure 3c,d), suggesting that an interaction of 3Car with BB and P results in changes in their ground state absorption.46 Because the carotenoid is in van der Waals contact with BB, the relative strength of interaction between 3Car and BB is likely higher than that between 3Car and P. The 812 nm spectral feature is more pronounced at 77 K (compare the spectra between Figure 3c,d). Angerhofer and coworkers19,46 reported similar negative bands in the microwaveinduced absorption spectra of the WT and R26 RCs, even at 8 K. The negative bands were previously assigned to either the formation of the triplet state of the bacteriochlorophyll monomer (3BA/3BB) or the interactions of 3P and 3Car leading to the electronic absorption changes of the nearby bacteriochlorophyll monomers. It is possible that 3BA could be formed by the 3 + (P HA−) recombination as suggested earlier.19,46 However, this is difficult to reconcile with all the data. The bleaching band at 812 nm in the WT RCs suggests that the transient absorption changes are dominated by the BB transition, not BA. Though equilibrium population of 3BB is formally a possibility, it is very 6502


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Figure 4. Time-resolved absorption difference spectra in the QY region (760−910 nm) of quinone-depleted WT and R26 RCs at several characteristic delay times (1, 100, and 500 ns) recorded at 77 K. The 1 ns spectra of both the RCs were normalized with respect to the amplitude of the P bleaching at 880 nm, and this scaling factor was then applied to the 100 and 500 ns spectra.

Figure 5. EADS spectra of quinone-depleted WT RCs at (a) 298 K and (b) 77 K. (c) Kinetics showing the recovery of P band bleaching monitored at 860 nm at 298 K and at 870 nm at 77 K. (d) Kinetics monitored at 595 nm showing the rise and decay of the 3Car signal at 298 and 77 K.

compared at selected delay times (Figure 4). The comparison of the 100 ns and 500 ns spectra between WT (in a mix of 3P and 3 Car states) and R26 RC (only 3P present) (Figure 4b,c) clearly shows an increase of the negative B-band absorption in the WT RCs, associated with the decay of 3P. This suggests that the influence of 3Car on BB is stronger than the influence of 3P on either BA or BB. Previous studies suggested that relatively strong coupling due to orbital overlap was involved in the mechanism of triplet energy transfer between 3P and Car, mediated by BB. It is thus not surprising that both 3Car and 3P would perturb BB electronic transitions to different extents depending on the relative coupling strengths.19,46,50 3.4. Global Analysis Using a Sequential Kinetic Model. 3.4.1. WT and R26 RCs. A global analysis of the whole spectraltemporal data set from each sample was performed to extract the time constants related to individual dynamic steps involved in the recombination, spin-conversion and energy transfer processes in the RCs. To estimate the spectral signature of the states associated with each kinetic component, a sequential kinetic model (A → B →···) was used. The resulting amplitude spectra associated with each time constant (evolution associated

unlikely that the negative band (at 812 nm in WT) corresponds to an actual population of the 3BB in equilibrium with 3P. This is because the relative standard free energy of 3BB is higher than that of 3P in WT RCs, yet these spectral features persist at very low temperature.46 Several other groups have previously reported similar bleaching signals in the (bacterio)-chlorophyll QY absorption region with 3Car for antenna systems of purple bacteria and plants.47−49 The carotenoid-less R26 RC also exhibits a narrow absorption decrease at 800 nm in the time-resolved spectra on the 100 ns to μs time scale (see Figure S3) at room temperature. In this RC, the decay kinetics of the negative band at 800 nm is similar to that of 3P. At 77 K, two narrow bleaching bands (at 800 and 810 nm) of relatively low intensity are observed (Figure S3, panel d). The special pair, P is in van der Waals contact with the accessory bacteriochlorophylls and therefore, the interaction of 3P with both BA and BB may lead to the perturbation of their electronic transitions in R26 RCs.4,10 To understand the perturbation of the QY electronic transition of the bacteriochlorophyll monomers by 3P and 3Car, the transient absorption difference spectra of WT and R26 were 6503


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Figure 6. (a) EADS spectra of quinone-depleted R26 RCs at 77 K. The inset shows the expanded plot of the spectra in the QY region. (b) Kinetics showing the recovery of the P band bleaching signal monitored at 870 nm at 77 K.

Figure 7. EADS of the quinone-depleted M182HL RCs at (a) 298 K and (b) 77 K. (c) Kinetics monitored at 595 and 480 nm showing the rise and decay of the 3Car signal at 298 K. (d) Kinetics showing the recovery of the P band bleaching signal monitored at 860 and 870 nm at 298 and 77 K, respectively.

characteristics of 3P and a small remaining contribution of (P+HA−)/3(P+HA−) as indicated by the presence of prominent absorbance decreases associated with P at 600 and 865 nm as well as a small HA absorbance decrease at 545 nm. Thus, at 77 K the observed 30 ns time constant includes some slow decay of P+HA−. The 0.14 μs lifetime EADS (generated by the 30 ns decay process) exhibits the spectral characteristics of both 3P (absorbance decreases at 600 and 865 nm) and 3Car (broad absorbance decrease between 400−500 nm and the 3Car absorption at 600 nm). The 0.14 μs EADS is assigned to the complete decay of 3P to the 3Car by triplet energy transfer, because the EADS generated by this process has the spectral characteristics of 3Car and a decay lifetime of 5.3 μs. The changes of the EADS spectra of WT can be further understood from the global analysis of the 77 K data of R26 RCs, which lack carotenoid. R26 RCs exhibit EADS with decay time constants of 12 ns, 34 ns, and 164 μs (Figure 6). Comparison of the time constants between WT and R26 suggests that in the absence of 3P → 3Car energy transfer, the decay of the total population of P+HA− (both singlet and triplet) via charge

difference spectra, EADS) for WT RCs at 298 and 77 K are shown in Figure 5a,b. The recovery kinetics of the P-bleaching band (860 nm at 298 K and 870 nm at 77 K) and the spectral evolution and decay kinetics of 3Car (595 nm) at both temperatures are shown in Figure 5c,d. Three exponential-fitting components adequately described the room temperature data set, and resulted in time constants of 12 ns, 25 ns, and 1.4 μs (Figure 5a). Those components are attributed to the dynamics of 1 + (P HA−) decay by both singlet recombination and by a singlet− triplet spin dephasing process, followed by triplet recombination leading to the formation of 3P, triplet energy transfer from 3P to the 3Car, and the decay of the 3Car, respectively, based on the spectral features described in the previous section (Figure 3). At 77 K, four exponential terms are required with the time constants of 12 ns, 30 ns, 0.14, and 5.3 μs (Figure 5b). The overall decay of P+HA− (both singlet and triplet forms on the 12 ns time scale) is weakly temperature dependent, consistent with previous results.51,52 Two time constants, 30 ns and 0.14 μs, are necessary to describe the kinetics for triplet energy transfer from 3P to the carotenoid at 77 K.19 The 30 ns EADS has the spectral

1

6504


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6505

164 156 152

17

13

30, 140

400, 1600

yield

77

τ1 is the observed decay rate of the total population of 1(P+HA−)/3(P+HA−), ΦRecombination is the relative yield of the individual recombination reaction steps such that ΦRecombination (T) + ΦRecombination (S) = 100%, ΦRecombination (T) is the observed yield of 3P, and τRecombination (S/T) = τ1/ΦRecombination (S/T). a

5.3

1.6

1.4

τ4 (μs) yield

0% 100% 0% 100% 0% 100% 100% 100%

τ3 (μs)

100% NA 60%, 40% NA 100% NA 0% NA

yield τ2 (ns)

25

36% 36% 40% 34% 52% 56% 59% 60%

ΦRecombination (T) τRecombination (T) (ns)

33 33 30 35 35 35 34 33 64% 64% 60% 66% 48% 44% 41% 40%

ΦRecombination (S) τRecombination (S) (ns)

19 19 20 18 45 45 49 50 12 12 12 12 12 20 20 20 298

τ1 (ns) T (K) RC sample

WT R26 M182HL Carotenoid-less M182HL WT R26 M182HL Carotenoid-less M182HL

P → PG. S. 3

P→ 3BB → 3Car 3

(P+HA−) → 3(P+HA−) → 3P 1

(P+HA−) → (P, H)G. S. 1

Table 1. Decay Lifetimes from Global Fitting and Calculated Rates and Branching Ratio of Singlet Recombination and Triplet Conversion Pathwaysa

3

Car → CarG. S.

recombination reactions is kinetically heterogeneous with two different time components of 12 and 34 ns at 77 K.52 The 164 μs component represents the decay of 3P directly to P at 77 K. 3.4.2. M182HL RCs. The rates and kinetic pathways of triplet energy transfer have been studied in a reaction center mutant, M182HL, in which the bacteriochlorophyll molecule in the BB pocket is replaced with bacteriopheophytin (Φ). The triplet energy of the bacteriopheophytin bridging state, 3Φ, in this mutant is likely higher than that of 3BB in WT, based on an estimate of the 3Φ energy from the phosphorescence of monomeric bacteriopheophytin in a glass matrix.21 At 298 K, the global fits of the room temperature data set of M182HL provide EADS with time constants of 12 ns, 0.40, and 1.6 μs (Figure 7a,b). The 12 ns EADS represents the decay of 1 + (P HA−)/3(P+HA−) and is similar to the corresponding EADS in WT and R26. The 0.40 μs EADS exhibits spectral features characteristic of 3P including a prominent bleaching at 865 nm, the absence of the bleaching at 545 nm, and an absorbance increase in the 665 nm region associated with the HA− anion. The 1.6 μs EADS has the spectral characteristics of 3Car, with some residual bleaching at 865 nm (equivalent to the 40% of the total amount of 3P generated), and is essentially the same as that in WT RCs (1.4−1.6 μs). The decay of this EADS represents a mixed decay of both 3P (due to slower energy transfer to Car) and 3Car on the time scale of 1.6 μs. This mixture suggests that the decay of 3P through triplet energy transfer in M182HL is heterogeneous where a major population is decaying in 0.40 μs and a minor population is slowly decaying in 1.6 μs mixed with the decay of 3Car. As the intrinsic lifetime of 3P without triplet energy transfer is much slower (in the 10−20 μs range at room temperature, see R26 results, Table 1, and Figure S6), the 0.40 μs lifetime thus represents a lower limit for the decay of 3P via triplet energy transfer to the carotenoid in M182HL RCs. Thus, the time scale of triplet energy transfer in M182HL is at least an order of magnitude longer than that observed in WT RCs (around 25 ns at 298 K, Figure 5b). The increased transfer time is presumably due to the higher energy of 3Φ compared to that of 3BB.21 Representative kinetic traces showing the formation and decay of 3Car at 298 K are given in Figure 7c (480 and 595 nm). These kinetic traces exhibit a slower building up of the 3Car signal by triplet energy transfer from 3P in M182HL compared to that observed in WT (Figure 5d). At 77 K, three EADS components with lifetimes of 12 ns, 30 ns, and 156 μs are obtained from M182HL reaction centers (Figure 7b). Both the spectra and lifetimes of these EADS are very similar to those observed in the carotenoid-less R26 RCs at low temperature (Figure 6). In particular, the longest EADS of 156 μs lacks the spectral signatures of 3Car in the 450−650 nm region indicating that 3P decays directly to the ground state. A comparison of the P bleaching kinetic traces between room temperature and 77 K (Figure 7d) clearly shows the significant difference in the decay rate of 3P at room temperature (through triplet energy transfer) and at 77 K (through direct decay to ground state P without any energy transfer). This temperature dependence is consistent with a thermally activated intermediate state 3Φ in triplet energy transfer between 3P and carotenoid. The observed decay kinetics of 3P in M182HL RCs vs temperature is consistent with the previous studies by Boxer and co-workers.21 The yields of 3P and the calculated singlet and triplet recombination rates of M182HL RCs (see Table 1) are similar to those observed in WT and R26 RCs. Consistent with the negative band at 812 nm near the BB absorption band observed in WT RCs (Figure 5a,b), a sharp

100% NA 100% NA 100% NA NA NA



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Figure 8. (a) Decay kinetics of R26 reaction centers monitored at 865 nm showing the recovery of the P bleaching band at different temperatures in the range of 77−298 K. (b) Arrhenius plot of the triplet energy transfer rate in quinone-depleted WT RCs. The 3P → 3Car triplet energy transfer rates at different temperatures ranging from 77−298 K were used in this plot.

absorption decrease at 790 nm near the Φ absorption region is observed in the 0.40 μs EADS of M182HL at room temperature (see inset of Figure 7a). As with WT, this absorbance decrease developed on the same time scale as 3P formation and decayed with the 3Car signal, supporting the idea that the presence of 3P and 3Car perturbs the absorbance spectrum of Φ. At 77 K, where no energy transfer to the carotenoid takes place, a bleaching band (at 790 nm) is observed in the longest-lived 156 μs EADS (Figure 7b) similar to that observed in R26 RCs, suggesting an interaction of 3P to perturb the electronic transitions of Φ. For comparison, the data of the carotenoid-less M182HL mutant RCs (Figure S7) were analyzed and the results are essentially similar to those of R26 RCs both at room temperature and 77 K. Moreover, at 77 K, the dynamics of absorbance changes of M182HL and carotenoid-less M182HL are essentially the same because triplet energy transfer does not take place. 3.5. Temperature Dependence. Analysis of the room temperature R26 data (Figure S6) resulted in a single 12 ns decay component of P+HA−, similar to that observed in WT at that temperature. The direct decay of 3P to P at room temperature in R26 occurs on the 10−20 μs time scale (Table 1). The intrinsic lifetime of 3P in the R26 RCs is temperature dependent in the range of 200−298 K.37 However, below 200 K, the decay time of 3 P (around 160 μs) is temperature independent (Figure 8a). In WT RCs, the longest-lived components are 1.4 and 5.3 μs at 298 and 77 K, respectively, and presumably represent the decay of 3 Car state, indicating a weak temperature dependence for 3Car decay. The rate of triplet energy transfer to the carotenoid has a much stronger temperature dependence (Figure 8b). At 77 K, considering the two different time components of 30 ns and 0.14 μs observed in WT RCs, the longer component appears to be purely the triplet energy transfer based on the time scale in the temperature dependence analysis. In contrast, the 30 ns component is associated with the slow decay of 1 + (P HA−)/3(P+HA−) as revealed from the R26 data at 77 K (see Figure 6). At room temperature the energy transfer time scale is 25 ns. The temperature dependence is consistent with thermally activated triplet energy transfer via 3BB. The temperature dependence of the triplet energy transfer rate of WT RCs between 77 and 298 K is linearly dependent on 1/T (Figure 8b), following an Arrhenius relationship:21

temperature-independent decay of 3P to the ground state (160 μs), which is negligible for the system considered here. The activation energy for the 3P to Car energy transfer in wild type is estimated to be 125 cm−1. This is close to the previously reported activation energy of 105 cm−1 from time-resolved EPR studies of WT RCs.53 A few other groups have reported activation energies in the range of 200 cm−1.21−23 3.6. EADS Spectra and Reaction Pathways. For any kinetic system more complex than a series of irreversible reactions, the EADS do not represent the spectrum of any pure state but rather a mixture of states that are evolving on a particular time scale. The 12 ns EADS of WT RCs at both room temperature and 77 K is dominated by the spectral characteristics of P+HA− and presumably includes both 1(P+HA−) and 3(P+HA−) (Figure 5a,b). The second EADS has a lifetime of 25 ns at room temperature and 30 ns at 77 K and exhibits a prominent 865 nm P-band bleaching. However, the anion band at 670 nm associated with HA− is largely missing. Thus, the EADS at 25/30 ns represents largely 3P. One can remove the small remaining contributions of 1(P+HA−)/3(P+HA−) in the 30 ns EADS at 77 K by subtracting a rescaled 12 ns EADS so that the 545 nm bleaching is zero (the 30 ns EADS before and after correction are shown in Figure S8). Based on the residual bleaching signal at 865 nm in the 25 ns (298 K) and 30 ns (77 K) spectra, the 3P yield is estimated to have increased from 36 to 52% as the temperature is decreased (Table 1). Similarly, from the EADS analysis of M182HL RCs, the yield of 3P was determined both at room temperature and 77 K and the values are presented in Table 1. The longest-lived EADS in WT RCs has a lifetime of 1.4 μs at room temperature and 5.3 μs at 77 K. The evolution of the spectrum from the nanosecond EADS to the microsecond EADS at either temperature involves the decay of the bleaching at 865 nm almost to zero, and emergence of an absorption decrease between 420 and 490 nm as well as an absorption increase between 550 and 650 nm. This is consistent with the formation of 3Car by triplet energy transfer from 3P. 3.7. Singlet to Triplet State Conversion and the Triplet Charge Recombination. The singlet and triplet states of the radical pair are virtually degenerate in energy.54 They oscillate back and forth due to the significant mixing of singlet and triplet levels. For the two interconverting states with the oscillation frequency (ωisc) and decay rate constants of singlet recombination (kS) and triplet recombination (kT), the total population decay of 1(P+HA−)/3(P+HA−) can be understood using the following kinetic scheme.54

⎛ E ⎞ k = c + A exp⎜ − a ⎟ ⎝ RT ⎠

where c is a temperature independent rate, A is a pre-exponential factor, Ea is the activation energy, R is the gas constant, and T is temperature (K). The constant term c accounts for the slow, 6506


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Figure 9. Kinetic model constructed based on a dynamic heterogeneity model illustrating the kinetic pathways of radical pair charge recombination (singlet and triplet), the mechanistic details of triplet energy transfer dynamics, the associated time constants of the individual evolution and decay pathways, and the yield of each state at 298 K (red) and 77 K (green).

limited by a slower interconversion process. The oscillation frequency (ωisc) between singlet and triplet radical pair states was previously reported to be on the order of 15 MHz, which corresponds to a half-life period of 33 ns.55 A similar analysis was performed using the yield of 3P (56%) and a 20 ns single decay component (see the decay component in Figure S9) of the total population of 1(P+HA−)/3(P+HA−) observed in R26 RCs at 77 K. The calculated time constant for 1 + (P HA−)→ (P, H) singlet recombination at 77 K is found to be 45 ns, which is much slower than that observed at room temperature (around 19 ns). The time constant of 3P formation limited by slower singlet to triplet radical pair interconversion followed by singlet recombination is found to be 35 ns. This time constant is similar to that observed at room temperature (Table 1), consistent with the singlet-to-triplet interconversion and triplet recombination processes being temperature independent.58 As previously suggested, the increased yield of 3P upon lowering the temperature, therefore, arises primarily due to the slower singlet recombination process.54

The oscillation frequency (ωisc) influences the yield of 3P and the relative rates of the two recombination reactions.55,56 Past studies suggested that the singlet−triplet exchange coupling interaction in reaction centers is temperature independent.57,58 In the above kinetic model, where the interconversion among states is much slower than the individual decay rates, the total population decay can be approximately represented by the sum of two exponential decays, ks and a rate limited by ωisc.54 Using the yield of 3P (35−40%) and the observed decay rate (1/12 ns−1) of the total population of 1(P+HA−)/3(P+HA−) at room temperature, one can estimate the singlet and triplet radical pair recombination rates using the equation: τRecombination(S / T ) = τobs/ΦRecombination(S / T )

where ΦRecombination is the relative yield of the individual recombination reaction steps. At room temperature, the 1

+

−

4. CONCLUSIONS In conclusion, the high-resolution broadband spectra and kinetic information on the nanosecond to microsecond time scale allowed a detailed systematic investigation that advances our understanding of the dynamics and heterogeneity associated with the primary radical pair 1(P+HA−)/3(P+HA−) charge recombination, triplet state formation, and the triplet energy transfer processes in the bacterial RCs. Table 1 summarizes the time constants obtained from global analysis using an irreversible sequential kinetic model, and the calculated microscopic kinetic rates along each reaction path for all samples at room

kS

estimated time constant for (P HA ) → (P, H)G. S. singlet recombination is found to be 19 ± 1 ns and that for the 3P formation by triplet recombination is 33 ± 2 ns (see Table 1). The calculated singlet recombination time (19 ns) agrees well with the literature value (19 ns).14 Time-resolved EPR studies show that the triplet recombination rate (1/(2−10) ns−1) is faster than the singlet recombination rate (1/19 ns−1).47,55,57,58 Therefore, the estimated 33 ns time constant is attributed to the time scale of 3P formation through radical pair interconversion followed by the triplet recombination process where the rate is 6507



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temperature and 77 K are given. Simplified kinetic models for each reaction center sample are presented in Figure 9 comparing the differences in the rates and yields of their respective kinetic paths (WT, R26, M182HL, and carotenoid-less M182HL). As shown in the kinetic models, the major kinetic pathways of charge separation and recombination (singlet and triplet) at room temperature and 77 K are the same for the WT and M182HL mutant reaction centers. However, the triplet energy transfer kinetics and pathways are significantly different presumably due to the elevated energetics of 3Φ in M182HL mutant RCs compared with 3BB in WT RCs. The mechanism and energetics of triplet energy transfer have been further delineated by observing the temperature dependence of the reaction center samples. As can be seen from the kinetic scheme presented in Figure 9, in the quinone-depleted RCs, about 35−40% of the singlet charge-separated state 1(P+HA−) is converted to 3(P+HA−) at room temperature, and then generates 3P via triplet state charge recombination. A deeper insight into the radical-pair chargerecombination dynamics via singlet and triplet pathways was obtained from a more quantitative calculation of the experimentally observed transient absorption data for the decay of the total population of 1(P+HA−)/3(P+HA−). The analysis shows that with decreasing temperature, the yield of 3P increases to 55−60% at 77 K, mainly due to a slower rate of singlet recombination at low temperature (77 K). However, on the time scale (30−35 ns) of 3P formation, where the spin dephasing between the singlet and triplet states of the P+HA− radical-pair is the rate-limiting step, the process remains temperature independent. This is consistent with what has been previously reported from EPR studies.55,57,58 A comparison of the temperature dependence of the rate of 3Pto-Car triplet energy transfer in the WT RCs and the M182HL mutant RCs shows that the mutant has a higher triplet state energy of the 3Φ intermediate compared to 3BB in WT, suggesting a thermally activated mechanism of triplet energy transfer via 3BB/3Φ. In particular at room temperature, the rate of triplet energy transfer is significantly slower (15 times slower) in M182HL mutant RCs than in the WT RCs, although both the RCs exhibit nearly 100% efficiency of the energy transfer. Moreover, at 77 K, unlike WT RCs, which display 100% efficient energy transfer, the M182HL mutant RCs do not exhibit a triplet energy transfer process. Additionally, detailed time-resolved spectral analysis of P, Car, and BB (Φ in the M182HL mutant) reveals that the triplet state of the carotenoid is coupled fairly strongly to the bridging intermediate BB in WT and Φ in M182HL mutant, a fact that is probably responsible for the lack of any obvious intermediate 3BB/3Φ transient formation during triplet energy transfer.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Sarthak Mandal: 0000-0002-5592-9664 Anne-Marie Carey: 0000-0001-6409-6580 Present Address §

State Key Laboratory of Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, China 130012 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by NSF grants MCB-1157788 and CHE1505874. B.R.G. thanks National Science Foundation of China for funding through No. 21473076.

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ABBREVIATION: RCs, reaction centers; EADS, evolution associated difference spectra; Car, carotenoid; WT, wild type REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b03373. Ground state absorption spectra and light-minus-dark difference absorption spectra of all RC samples; twodimensional contour plots of transient absorption spectra of WT and M182HL RCs; the time-resolved absorption spectra of R26, M182HL, and carotenoid-less M182HL RCs; and the EADS spectra of R26 and carotenoid-less M182HL RCs (PDF) 6508


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