Spectrally Silent Light Induced Conformation Change in Photosynthetic Reaction Centers

FEBS Letters 582 (2008) 3657–3662

Spectrally silent light induced conformation change in photosynthetic reaction centers

La´szlo´ Nagya,*,Pe´ter Maro´tia, Masahide Terazimab a Institute of Medical Physics and Biophysics, University of Szeged, 6720 Szeged, Rerrich B. te´r. 1., Hungary b Kyoto University, Graduate School of Science, Department of Chemistry, Kyoto 6068502, Japan

Received 17 September 2008; accepted 23 September 2008

Available online 7 October 2008

Edited by Richard Cogdell

There are several evidences that RCs are in different confor- Abstract Spectrally silent conformation change after photoexcitation of photosynthetic reaction centers isolated from mation states in dark and light, e.g. [2–6]. Kinetic components Rhodobacter sphaeroides R-26 was observed by the optical in transient absorption were observed at specific wavelength heterodyne transient grating technique. The signal showed spec- [7,8], in capacitive potentiometry [9] and time resolved FTIR trally silent structural change in photosynthetic reaction centers spectroscopy [10,11] which are generally assigned to conforma- followed by the primary P+BPh charge separation and this tional transients related to QA QB to QAQB electron transfer. change remains even after the charge recombination. Without Recent crystallographic experiments did not show large qui- bound quinone to the RC, the conformation change relaxes with none displacements at the QB site on the time scale of the sec- about 28 ls lifetime. The presence of quinone at the primary qui- ondary electron transfer [12,13] in agreement with FTIR none (QA) site may suppress this conformation change. How- studies [14,15]. It seems reasonable to conclude that even larger ever, a weak relaxation with 30–40 ls lifetime is still observed quinone movement is not necessarily accompanied with con- under the presence of QA, which increases up to 40 ls as a function of the occupancy of the secondary quinone (QB) site. siderable structural change [13]. 2008 Federation of European Biochemical Societies. Pub- Transient grating technique was successfully used for many lished by Elsevier B.V. All rights reserved. applications including thermodynamics and kinetics of CO binding of myoglobin [16,17], the photocycle of the photoac- Keywords: Transient grating; Heterodyne detection; Reaction tive yellow protein (PYP, [18]), determining diffusion coeffi- center cient of proteins and DNA [19] and conformational changes of photosensor proteins [20–23]. In a recent publication we have shown that this method can be successfully applied to investigation of light induced charge transfer processes and accompanied protein relaxation movements in bacterial reac- 1. Introduction tion centers [24]. Here, we give further evidences.

There are several types of photosynthetic reaction centers 1.1. Principle of the OHD-TG measurement (RCs) in living organisms (PS-I and PS-II of plants and cyano- The principle of the optical heterodyne detection (OHD) of bacteria, RCs of purple and green bacteria; see e.g. [1]) which the transient grating (TG) signal was described previously [25– are developed to convert light energy into chemical potential in 28]. After photoexcitation of a sample with a grating pulsed a series of (photo)physical and (photo)chemical reactions. light, a signal field (ES(t)) is created by the diffraction of a The basic processes of the photosynthetic energy conversion probe light. If it is interfered with a local oscillator (LO) field in all types of the RCs include (a) electron excitation of chlo- (ELO) the observed light intensity is rine pigments by light, (b) charge separation and stabilization 2 reactions between redox active cofactors bound to the protein, IðtÞ¼ajELO þ EsðtÞj ILO þ dnðtÞ cos D/IexIpr; ð1Þ (c) rearrangement of the dielectric medium and hydrogen bond interactions (including protonation and deprotonation of spe- where a is an instrumental constant, dn(t) is the refractive in- cific amino acids), and (d) conformational movements within dex change, D/ phase difference between the local oscillator the protein [including transition of (sub)states between dark (LO) and probe light fields and Iex, ILO, and Ipr are the inten- and light adapted forms]. sities of the excitation, LO, and probe light, respectively. Here we assume that the refractive index change is the dominant source of the signal, and the signal field is weak compared to ELO (|ELO|>>|ES|). Since the local oscillator light intensity is a constant, I provides constant background. The second *Corresponding author. Fax: + 36 62 544121. LO E-mail address: [email protected] (L. Nagy). term of Eq. (1) represents the OHD-TG signal. The OHD- TG signal intensity depends on the relative phase difference be- Abbreviations: BPh, bacteriopheophytin; DEAE, diethylaminoethyl; tween the LO and signal fields, D/. The maximum intensity of LDAO, N,N-dimethyldodecylamine-N-oxide; LO, local oscillator; the thermal grating component of a reference sample is OHD, optical heterodyne detection; P, primary donor; QA, primary achieved at D/ =0. quinone; QB, secondary quinone; R., Rhodobacter; RC, photosynthetic reaction center; TG, transient grating; TrL, transient lens; UQ-10, Two main factors contribute to the refractive index change: ubiquinone-10 the thermal effect [thermal grating, dnth(t)] and a change in

0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.09.048 3658 L. Nagy et al. / FEBS Letters 582 (2008) 3657–3662 chemical species by the reaction [species grating, dnspe(t)]. The 2.2. Transient grating measurements species grating component consists of the population and vol- An excitation laser beam (from the second harmonics of a Nd:YAG ume terms. Hence, we can monitor the temporal changes of the laser, k = 532 nm, s = 10 ns) and a cw IR beam (YAG, 1064 nm) were split by a transmission grating (optical mask) and the first order dif- energy, absorption change, and molecular volume by analyz- fracted beams were combined again on the sample using a concave ing the OHD-TG signal. mirror. In order to avoid overexcitation of the sample, the energy of When the energy relaxation is faster than the diffusion pro- the excitation beam was reduced by neutral density filters (typically be- cess, the temporal profile of dn (t) is determined by the ther- low 100 lJ). One of the cw IR beams was used for the probe beam of th the TG signal. The other beam intensity was attenuated about 1/100 by mal diffusion, and is given by a neutral density filter for the use of the local LO light and of the dn ðtÞ¼dn0 expðD q2tÞ; ð2Þ OHD-TG signal. The filter was slightly adjusted tilting by a computer th th th and used for a fine adjustment of the phase of the LO field to the probe where D is the diffusion constant, q is the grating wavenum- field by changing the optical path length inside. The LO light intensity th was detected by a photodiode. The LO light intensity could be changed ber. not only by the interference between the signal and the LO light, but The kinetics of the species grating signal intensity, dnspe(t), is also by the transient lens (TrL) signal created by the pump beam given by the difference of the refractive index changes due to [31]. The OHD-TG signal was obtained by calculating the difference between minimum and maximum LO light intensities matching two the reactant (dnr) and product (dnp). If the back reaction from the product to the reactant is comparable to the diffusion pro- opposite phases after filter adjustments. The spacing of the grating fringe, equivalently, the grating wave- cess it is given by number, was measured by the decay rate constant of the thermal grat- k ing signal from a calorimetric standard sample (bromocresol purple). dn t dn0 dn0 k The signal was averaged and stored by a digital oscilloscope (Tektron- speð Þ¼ p 2 r expfð k þðDr DpÞq ics, TDS-520) [17]. In some experiments, absorption change was mon- dn0ðD D Þq2 itored by a He–Ne laser (590 nm) after the flash excitation. The D q2t r r p D q2t ; experimental set up was described in our earlier work [24]. þ p Þg þ 2 expf r g ð3Þ k þðDr DpÞq where Dr and Dp are diffusion coefficients of the reactant and the product, respectively. 3. Results and discussion

After photoexcitation of the sample solutions, the detected 2. Materials and methods probe light intensity increased abruptly then decreased as a function of time. The two components in the signal (TG and 2.1. Sample preparations TrL) can be separated based on the phase sensitive nature of Rhodobacter sphaeroides R-26 cells were grown photoheterotrophi- the OHD-TG signal. In contrast to TrL, the amplitude and cally under anaerobic conditions. RCs were prepared by detergent (LDAO, N,N-dimethyldodecylamine-N-oxide) solubilization followed the sign of the OHD-TG signal depends on the phase differ- by ammonium sulfate precipitation and diethylaminoethyl (DEAE) ence between the LO and the signal (see D/ in Eq. (1)). The Sephacell anion exchange chromatography [29]. The primary (QA) OHD-TG signal changes the sign by phase difference of and the secondary (QB) quinones were extracted out according to 180. Hence, subtraction of the D/ = 180 signal from the Okamura et al. [30]. RCs with different amount of bound quinones D/ =0 signal provides OHD-TG without TrL contribution. were prepared by addition of ubiquinone-10 (UQ-10) to the solution. The quinone/RC ratio was checked by kinetic absorption change mea- The curves TG+ and TG in Fig. 1 show the probe light inten- surement as described by Tandori et al. [29]. sities measured at D/ = 180 and D/ =0, respectively in QB

Fig. 1. The intensity of different components of the measured grating signal at different LO light conditions for QB reconstituted RCs. TG+ and TG indicate the original data measured with two opposite phases of the LO light (D/ = 180 and D/ =0, respectively). TrL and OHD-TG indicate the transient lens and grating components (gray curves), respectively, calculated from TG+ and TG as described in the Section 1.1. The best fitted curves are shown by the black lines. The best fitting parameters for the OHD-TG and TrL components are I = 0.97exp(t/0.15 s) + 0.03exp(t/0.055 s) for the OHD-TG component and I = 0.97exp(t/1.5 s) 0.03exp(t/0.11 s) for the TrL component. Here the amplitudes are normalized to 1 for better comparison. L. Nagy et al. / FEBS Letters 582 (2008) 3657–3662 3659

Fig. 2. The intensity of the OHD-TG signal of RCs at different (bound quinone)/RC ratios as indicated. The signal of the reference sample is also shown. Measurement was done by the experimental setup outlined in Ohmori et al. [24] and the OHD-TG signal was calculated as described in Fig. 1. RCs were suspended in 10 mM Tris, pH 8.0, 0.01% LDAO, 100 mM NaCl. The optical density of the sample was 1 at the excitation wavelength of 532 nm. The frequency of the excitation was 0.2 Hz for each sample, except for that of the reference (10 Hz). reconstituted RCs. The OHD-TG signal was obtained by sub- (D = 3.8 Æ 1011 m2/s and d = 11.4 nm), and other data ob- traction of these signals. Once, we obtained the OHD-TG sig- tained e.g. by AFM [32] or dynamic light scattering [33] exper- nal, the TrL component may be calculated from the OHD-TG iments. and the observed signals. Since the time constant of the fast component did not de- The OHD-TG signals measured for the calorimetric refer- pend on the grating wavenumber, this phase is not due to ence and for RCs at different ratio of bound quinone to the the thermal diffusion or protein diffusion processes, but it reaction center ([Q]/[RC]) are shown in Fig. 2. For the refer- should represent reaction dynamics of the photoexcited RC. ence solution, the TG signal demonstrated monoexponential The thermal grating signal intensity was too weak to be ob- decay of about 35 ls lifetime. Apparently, this is the thermal served under these experimental conditions. The thermal grat- 2 grating signal and the decay rate was determined by Dthq ing could be seen only when the RC was overexcited by high (see Eq. (2)). For photosynthetic RCs typically two main excitation rate and the absorbed laser energy was released as phases appeared after the abrupt initial increase of the refrac- heat (Fig. 3). After the photoexcitation, the P+BPh charge tive index. The fast phase (lifetime s1) decayed in the few pair is formed in about 3 ps after the flash excitation and de- 10s ls time range and the slow phase (lifetime s2) in the milli- cays via charge recombination in about 10 ns (see [34]). The second–second time scale: observed TG signal of 28 ls lifetime does not appear in the transient absorption measurement. Hence, this component is I ðtÞ¼afA expðt=s ÞþA expðt=s Þg ð4Þ TG 1 1 2 2 spectrally silent and represents conformation change induced + In RCs of 8% Q/RC we obtained s1 = 28.1 ls, s2 = 110 ms and by PBPh fi P BPh charge separation. Interestingly, the the ratio of the amplitudes A1:A2 = 94.2:5.8. With increasing relaxation rate constant of this conformation change is small, [Q]/[RC], the fast component decreased and that of the slow and the protein reserves its state even after the charge recom- component increased. Hence, it is reasonable to consider that bination when the chromophores relaxed to their initial the fast component represents the dynamics of RC without Q. (ground) states. As the amplitude of this component is large First, the slow component is discussed. We reported previ- for RC without Q sample, the 28 ls-signal should be due to ously [24] that the lifetime of the milliseconds–seconds dynam- structural rearrangement of the protein in the absence of the ics depended on the grating wavenumber that indicated that primary and secondary quinones. ‘‘Spectrally silent’’ compo- this kinetics represented the protein diffusion process. The sin- nents are well known in many proteins [16,18,23], and there gle exponential behavior of this component showed that the are only few appropriate methods suitable to investigate them; diffusion coefficient of the reactant was almost the same as that transient grating is one of these techniques. of the charge separated state. The rate constant of the grating The decrease of the relative amplitude of the 28 ls decay signal was expressed as Dq2 + k, where k was the rate constant component by addition of quinone to the solution suggests 1 1 of the charge recombination: (100 ms) and (1 s) for that the presence of quinone at the QA site suppresses this con- + + P QA fi PQA and P QAQB fi PQAQB, respectively. From formational relaxation, or the conformation change is fixed these data, we calculated the diffusion coefficient until the charge recombination occurs with the lifetimes of 11 2 D = 4.5 Æ 10 m /s and the hydrodynamic diameter 100 ms for QA sample and of 1 s for QAQB sample. d = 10.2 nm (estimated from the Einstein–Stokes relationship) However, we should note that a weak 30 ls-component was for the QA reconstituted RC/LDAO micelles. These values observed even for QA and QAQB samples. This component agreed fairly well with results of our earlier publication might be observed by other spectroscopic techniques. In fact, 3660 L. Nagy et al. / FEBS Letters 582 (2008) 3657–3662

12 QB+trb

10 QB high energy 8 0.8

(rel.) Ref: TG

I 6 : 8.7 μτ s

0.32 2 NoQ : 26.0 μτ s 0 1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 Time (s)

Fig. 3. The intensity of the OHD-TG signal of RCs at different (bound quinone)/RC ratios as indicated. Measurement was done as described in Fig. 2 but the grating conditions were changed. This is indicated by the lifetime of the reference signal. The OHD-TG signal was calculated as described in Fig. 1. ‘‘QB high energy’’ indicates reconstituted QB sample, but the excitation repetition rate increased to 10 Hz and the energy of the laser beam was oversaturating. several experimental techniques demonstrated two or more ki- Only the observed slow phase is speeded up which is under- netic components in the 20–40 ls and 200–500 ls range which standable, since the life time of the absorption change is mod- + + were suggested to be related to the P QA QB to P QAQB ified from sslow = 1.5 s to sfast = 0.11 s after the treatment with electron transfer [7–9]. this chemical. This fact indicates that ‘‘empty’’ quinone site is Additional increase in the lifetime of this decay component responsible for this kinetic component in the microsecond time can be seen, although its amplitude is small, if quinone is titrated scale, regardless the QB site is occupied by the redox active into the QB site (Fig. 4 and Table 1). Since this component is re- UQ-10 or non-redox active terbutryn. lated to the presence of the secondary quinone, QB, it can be as- Fig. 5 shows that the primary quinone is sitting in a loop signed to the interquinone electron transfer. To our surprise the formed by random structures at the end of D and E transmem- amplitude of this component is smallest for the fully reconsti- brane and mde cytoplasmic helices. The strong packing of the tuted 2Q/RC sample and we did not see other kinetic compo- intramembrane helix structure does not support large confor- nents, e.g. the components of few hundreds of microseconds, mation movements, but the empty random loop can be very described by the ‘‘conformational gating’’ model [3]. flexible. We also indicate the amino acid residue TrpM252 Interestingly, the presence of non-redox active inhibitor at which is in close contact with the BPh in the A electron trans- the QB site, like terbutryn, does not affect the 28 ls component. port branch and QA and supports tunneling of the electron to

40 s)

μ 30 (

τ 16

12 20 8 A(rel.) 10 4

0 0 0.5 1 1.5 2 0 Q/RC 0 0.5 1 1.5 2 2.5 Q/RC

Fig. 4. The lifetime of the submillisecond component of the transient grating signal as a function of the Q/RC ratio. Filled circles and squares represent the data calculated from ﬁrst (Fig. 2) and second (Fig. 3) grating conditions, respectively. Measurement was done as described in Fig. 1. The insert shows the relative amplitude of the TG component as a function of the Q/RC ratio. L. Nagy et al. / FEBS Letters 582 (2008) 3657–3662 3661

Table 1 Summary of kinetic parameters of the measured transient grating signal presented in Fig 2 and hydrodynamic data calculated for isolated RCs. A1 (%), A2 (%) and s1 (ls), s2 (ms) represent the amplitudes and life times of the main phases of the kinetic components of the ITG signal, respectively. sTrL (ms) is the life time of the thermal lens component of the signal. D is the diﬀusion coeﬃcient, and d is the hydrodynamic diameter of the RC micellar system. 11 2 Q/RC A1 (%) s1 (ls) A2 (%) s2 (ms) sTrL (ms) D 10 (m /s) d (nm) 0a 100 34.7 0ab 100 15.0 0a 100 8.7

0.06 94.2 28.1 5.8 110 0.32 1.2 28.7 28.8 52.4 115 0.54 48.5 29.0 51.5 49.3 119 (0.5)b (50.0)b (28.0)b (50.0)b (54.3)b (108)b 4.5c 10.2c 0.68 37.1 29.6 62.4 63.4 106 (3.8)b (11.4)b 0.81 23.7 32.4 75.4 64.8 122 0.91 17.9 31.7 84.8 54.6 111 1.00 6.7 40.8 92.6 52.6 123

1.2 6.7 39.5 92.6 70.9 182.3 1.3 8.2 2.3 92.6 71.7 285.8 2.0 1.8 46.1 98.2 158.1 1490 2.8 15.4 aCalorimetric reference sample at diﬀerent grating conditions. bOhmori et al. (2008). cCalculated from the average of samples Q/RC < 1 as described in the text.

the primary acceptor. In the absence of QA and its interactions cept a large conformational movement in the empty QA pock- with the surrounding amino acid residues it is reasonable to ac- et. Also, the contribution of relaxation of water molecules (internal and external ones) should be taken into account. Although the lifetime of this dynamics is in the range of the triplet formation, the probability of this process is too small in our experimental conditions (no secondary donor is present, primary quinone is oxidized, low probability for double excitation, oxygen concentration is saturating). The discussion of the contribution of the triplet states to the TG signal would be important, but it needs more investigation and cannot be the scope of this paper. The absence of the few hundreds of microsecond component is rather surprising because it is well stated by other measurements in the literature [3,7–9]. Considering the extre- mely high sensitivity of the OHD-TG method, we can argue for no large displacement of the secondary quinone induced by the charge movements and the other volume changes due to charge relaxation processes should be very small. Re- cently Koepke et al. [35] studied crystal structures with QB in proximal and distal positions and found that the quinone movement between the two positions was not accompanied with large conformational rearrangement. This result supports our observation.

4. Conclusions

The photoreaction of photosynthetic reaction center was investigated by the OHD-TG technique. Here, for the first time in the literature, we demonstrated that the PBPh fi P+BPh charge separation induced considerable structural change in the protein that was relaxed much slower (28 ls) than the P+BPh fi PBPh charge recombination (10 ns). The conformation change was suppressed by the presence of quinone at Fig. 5. The position of the primary quinone, QA, at the loop formed by helical and random structures of the protein close to the membrane the QA site. surface. The BPh in the active ‘‘A’’ electron transport branch and the characteristic amino acids around the QA (HisM219, AlaM260, Acknowledgments: This work was supported by the grant of Japan TrpM252) are also indicated. Picture was drawn by HyperChem using Society for Promotion of Science, by the Hungarian Technology and the crystal structure 1PCR [36] downloaded from the Protein Data Science Foundation (JP-12/2006) and NKTH-OTKA (K 67850) and Bank, Brookhaven. also by the MEXT Japan to MT (Nos. 15076204, 18205002). 3662 L. Nagy et al. / FEBS Letters 582 (2008) 3657–3662

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