stuart multipulse spectroscopy on the wild-type and ym210w bacterial reaction centre uncovers a new...

8/13/2019 Stuart Multipulse spectroscopy on the wild-type and YM210W Bacterial Reaction Centre uncovers a new intermedi

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2. Materials and methods

2.1. Sample preparation

Reaction centres were isolated from Rhodobacter sphaeroides

R26.1 according to procedures described in Ref. [21]. The

YM210W mutant of BRC, with a long-lived P* state, is constructed

using site-specific mutagenesis. The detergent purified membrane-bound, antenna deficient, reaction centres were dissolved in

10 mM Tris (pH 8.1) and circulated through a shaking 1-mm sam-

ple cell, to minimize the amount of RCs doubly excited. A 10 mM

ascorbate solution was added to the sample to re-reduce the spe-

cial pair[8,22,23]. Steady state spectra were recorded using a Per-

kin Elmer Lambda 40 UV/VIS spectrograph.

2.2. Femtosecond pumpdumpprobe spectroscopy

Transient laser spectra were recorded on a spectrometer, oper-

ating at a 1 kHz repetition rate. A seed-pulse from a diode-pumped

oscillator (Coherent Vitesse, 800 nm, 80 MHz) is amplified to 2.5 W

by using a Nd:YLF high-power pump-laser (Coherent Evolution-20,

527 nm). The Ti:Sapphire based amplifier (Coherent Legend-USP)

incorporates CPA, chirped pulse amplification, and a stretcher/

compressor combination to deliver sub-50 fs pulses, with a centre

wavelength of 800 nm and a bandwidth of 25 nm at the FWHM.

The pulse to pulse energy is 2.5 mJ (0.025 mJ). For control of the

time of arrival of the pump and dump pulse, two 60-cm long delay

stages (Newport IMS-6000) are placed in the beam paths. In the

single pass mode, this can delay the pulse up till 3.7 ns, reflecting

our time window.

The probe pulse, a white light continuum, is generated by focus-

ing a small fraction of the fundamental 800-nm pulses into a later-

ally rotating sapphire plate. These white light pulses are 300 fs

due to group velocity dispersion, introduced by the sapphire plate.

The pump and dump pulses are generated by a commercial OPA

(Coherent OperA), pumped by the Legend-USP. The pulses are

100 fs long and 10 nm wide at FWHM.Reflective optics is used to steer and overlap all pulses into a

rapidly shaking sample cuvette of 1 mm thickness. The probe pulse

was then spectrally dispersed by a spectrograph (Oriel) onto a

homebuilt photo-diode array detector. This 256-pixel array is read

out and the data is fed into a computer recording and calculating

the varying optical densities.

2.3. Experimental setup

In pumpdumpprobe spectroscopy, the molecule of subject is

excited by the dump pulse. While the system evolves into a differ-

ent state, a second pulse resonant with the stimulated emission of

the evolving excited state is applied, followed by the probe pulse.

In the case of BRC, the pump excites B to B*. This transfers its en-

ergy to P*, which is then dumped by a resonant dump pulse of

940 nm. A schematic PDP picture is shown in Fig. 1(seeSupple-

mentary material). In the wild-type experiment, dump pulses have

been applied at 1 and 2 ps delay after the pump pulse. This corre-

lates with the timescale on which the P

*

state evolves into chargeseparated species P+B or P+H. For the YM210W mutant, where P*

decays bi-exponentially with constants of 12 and 70 ps, dumping

pulses have been applied at 2, 4, 6, 8, and 10 ps delay. Varying

the dump-delay could show a gradual in or decrease in dump effi-

ciency, or could result in different intermediate states.

2.4. Data analysis

Global analysis [24] was simultaneously performed on the PP

and the PDP signals to see differences purely resulting from the

dump pulse. In practice, a direct comparison of the normal

pumpprobe measurements and the pumpdumpprobe mea-

surements is executed. Differences herein can only be introduced

by the dump pulse. New dynamics that arise from the dump pulse

can then be analysed and described. Secondly, target analysis of

the PP and PDP transient signals was performed. Several kinetic

models based on previously gathered data have been applied

[15,19]. The instrument response function is taken into account

using a GAUSSIAN[25]and dump-induced artefacts can be removed

by subtracting dumpprobe (DP) signals from the PDP.

3. Results

3.1. Steady state measurements

Absorption spectra of the two samples shown inFig. 2are in full

agreement with previously reported spectra. [10,19]. The three

band structure arises from the Qy transitions of the various pig-

ments present in the BRC. The blue-most band has been attributed

to the bacteriopheophytins HA and HB, the central band to the

accessory BA and BB bacteriochlorophyll band, and the red-most

band to the low-energy exciton transition of the special pair. The

slight difference in the relative amplitudes of the P, B and H bands

Fig. 1. The pump introduces an excited state (B *), its dynamics evolve unhindered

until the next pulse. The dump pulse will, when chosen to be resonant with the P *,

dump a percentage of this excited state population back to the ground-state. We

propose in this paper that the P*

state exists in an equilibrium with its chargetransfer (CT) state and when it is being dumped, this equilibrium is shortly shifted.

Fig. 2. The steady state spectrum of WT BRC (red) and the YM210W mutant (black).

(For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

T.A. Cohen Stuart, R. van Grondelle / Chemical Physics Letters 474 (2009) 352356 353


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of the WT and YM210W is a result of the mutation modifying the

electronic landscape around P, B and H[8].

3.2. Transient spectroscopy

PDP measurements were performed with excitation pulses cen-

tred at 795 nm, with a 100 nJ pulse to pulse energy. The de-excita-

tion or dump pulse is centred at 940 nm (5 nm width band-passfilter) and also has a 100 nJ energy. The pump and dump pulse

are parallel polarized to the magic angle, 54.7 relative to the

probe. The spotsize on the sample is a 100 lm. Steady state mea-

surements were taken before and after transient measurements

to ensure that no molecular breakdown is instigated by the excita-

tion and dump pulses. Using this power, non-linear effects are not

expected. The transient changes were recorded into four different

data sets. Recording PP, PDP and DP signals simultaneously, allows

a subtraction of the PDP signals from the PP signals. This results in

DDOD spectra that show dump-dependent dynamics. Two spectra

recorded for YM210W are depicted inFig. 3. The black spectrum

with a dump pulse present at 10 ps and the red spectrum without

this dump pulse. In this case the spectra are recorded just 1 ps after

the dump pulse. A dump pulse fired 10 ps after the pump is able to

remove up to 17% of the excited state if detected at 870 nm, the

maximum of the P* excited state. This is also clear from Fig. 3B,

where a time trace at 870 nm is shown during the measurement.

This efficiency appears reasonable, but it has not been studied in

detail. Note that in the spectrum shown inFig. 3A, before and after

the dump pulse, small but significant absorption changes can also

be observed in the region 740810 nm, most likely reflecting a

small amount of species being generated upon dumping. If we lookat the structure of the differences between dumped and not

dumped, we can more clearly observe the effect of the dump pulse

in this system. Time-gated DDOD spectra reveal interesting spec-

tral dynamics in the first ps following the dump pulse (see

Fig. 4). These early spectra are remarkably similar; irrespective of

the dump-delay chosen. In the initial spectra inFig. 4clear features

are observed around 810 nm which indicate the formation of P +, as

reflected by the BMband shift. Interesting is also the first spectrum

of the wild-type. The positive feature around 870 nm reflects the

loss of P* due to the dump. Since in the WT the amount of P* is

much lower, at 300 fs, this feature is overlapped by P* loss. This

behaviour is not seen in the YM210W mutant; however the larger

amount of P* still present at the dump moment may explain that

difference. At longer times, the DDOD spectra converge into a loss

of P+H. This is simply due to the fact that we have removed P* that

would have normally donated its energy to form P+H.

3.3. Global analysis

The species generated by the dump pulse in the mutant is

investigated by global analysis, using a well-established kinetic

model for BRC dynamics[15]with the possibility of an intermedi-

ate state being populated.

Besides the expected species that arise from this analysis (P*, B*,

P+H, etc.), a new state must be introduced in the YM210W mu-

tant. The model is shown in Fig. 5, with a preliminary SADS of

the intermediate species inFig. 6. Using this model, we find that

19% of the P* has been dumped and only a small part of that frac-

tion arrives at the intermediate state. The lifetimes found for thestates present are comparable to earlier literature, except for the

P+H lifetime. This lifetime is obscured by the dump-effect.

In Fig. 6 the intermediate species is compared to P+H. The

Fig. 3. Pumpdump experiment for the YM210W BRC. (A) The dump pulse arrives

10 ps after the pump and results in a 17% loss of the excited state population. (B)

The DDOD time trace at the excited state maximum (870 nm) shows the loss ofpopulation upon dumping.

Fig. 4. Time-gated DDOD spectra for the WT and YM210W as obtained by transient

multi-pulse spectroscopy. At T = 0 the dump pulse arrives. The reaction centre

shows a strong spectral feature around 800 nm reminiscent of the formation P+, andthe structure around 780 nm possibly represents involvement of a pheophytine.

354 T.A. Cohen Stuart, R. van Grondelle/ Chemical Physics Letters 474 (2009) 352356


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time-gated spectra fromFig. 4show a positive feature arising due

to the pump pulse. The spectrum that arises from the target anal-

ysis has its sign inverted compared to these time-gated spectra.

This is due to the fact that in our analysis method the dump pulse

is interpreted as removing population instead of generating it.

The species generated in the mutant very clearly shows the for-mation of P+ represented by the sharp band shift around 810 nm.

At 870 nm the strong P* loss is present and at lower wavelengths

some population present at 740 is reminiscent of a H band.

Although we have analysed the effect of the dump pulse applied

at 2, 4, 6, 8 and 10 ps, every experiment shows the same interme-

diate species arising shortly after the dump, in comparable quanti-

ties. This is against expectation, and puts forth a different picture

for the interaction between the excited state and the dump pulse.

The lifetime of this intermediate has been found to be comparablewith the P* lifetime (70 ps).

4. Discussion

The main goal of this study was to characterize the state P* and

to identify possible dynamics happening in the P* state while it

develops into P+B. In the wild-type BRC this process is very fast

and multi-exponential (3 and 12 ps). The precise mechanism of

charge separation has not been fully understood, in particular the

idea that an internal P+P charge transfer state exists before charge

separation takes place needs further investigation. Also the under-

lying cause for the multi-exponentiality is not well known. It has

been proposed that the charge separation may somehow be facili-

tated by a favourable protein conformation [26]. Then the fast

phase would reflect favourable conformations while the slow

phase is a manifestation of the unfavourable ones. In that case with

a second dump pulse it must be possible to interrogate the nature

of the excited state as a function of time after excitation and in par-

ticular to investigate the ability to produce useful product states

once P* has been formed. To try and identify these P* dynamics

we have performed multi-pulse spectroscopy. We have used WT-

BRCs that display fast charge separation and BRCs of the

YM210W mutant which is more than a factor of 10 slower. After

initiation of the photoreaction by a pump pulse of 795 nm, the evo-

lution of P* is manipulated by a 940 nm dump pulse. The time-

gated spectra show that both in WT and in YM210 BRCs the dump

pulse successfully removes about 20% of P* excited state popula-

tion and for a large part recovers to the ground state. In addition,in YM210W a new state is formed upon dumping P* that clearly

resembles a charge-separated state involving P+. Note that the

time-gatedDDOD spectra for the WT and the YM210W look very

similar, but we have not been able to retrieve a clear intermediate

for the WT. Apparently it is difficult to separate P+ formation from

P* dumping. In previous pumpdumpprobe studies, hot ground

states are regularly found[25], but in this case the target analysis

does not indicate formation of such a state. Also, although ex-

pected, varying the dump-time does not provide coherent informa-

tion on the charge-separation process. In the mutant, the state

appearing upon dumping shows the same signs of P+ around

805 nm, but now combined with a much larger bleaching of the

P-band at 870 nm. Because the mutant has a higher P* state stabil-

ity, this state is much easier to dump and thus a higher P* loss rel-ative to P+ is expected. TheYM210W has P+ present while P* is

developing into P+B, but significant bleaching of B is not observed.

A possible remaining candidate for the intermediate state is a P+/P

charge separated pair combined with P* loss. From our present data

however we cannot conclude that undoubtedly. In conclusion, by

pumpdumpprobe experiments on the BRC we have identified a

new charge-separated state, possibly P+/P that can only be pro-

duced following P* formation by a first pulse and dumping the ex-

cited state by a second laser pulse. We speculate that this state is

P+/P, the internal charge transfer state of the special pair. The life-

time connected to this intermediate seems to be of the order of the

slow phase of P*, namely around 70 ps. The fact that the slow P* and

P+/P lifetimes appear similar strengthens the belief that P* and P+/

P

co-exist. The dump pulse could shift this equilibrium shortlyand therefore reveal P+/P.

Fig. 5. The model used in global analysis of YM210W. The intermediate does not

contribute to the end product P+Q, and decays independently to the ground state.

The blue arrow depicts the pump pulse, and the red arrows depict the effect of the

dump pulse. Both the slow and the fast fraction of the P * population can be dumped

and they both produce the intermediate. From B* there is a 15% direct P+B

formation, 20% formation of fast P* and the rest, 65%, transfers into the slower P*.

(For interpretation of the references to colour in this figure legend, the reader is

referred to the web version of this article.)

Fig. 6. Species Associated Decay Species (SADS) as obtained from global analysis.

The red spectrum is the intermediate as generated by the dump in the YM210W

mutant. For comparison the steady state spectrum is shown in blue. The P+H

spectrum arising from the target analysis is also shown.

T.A. Cohen Stuart, R. van Grondelle / Chemical Physics Letters 474 (2009) 352356 355


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Acknowledgements

This research was supported by The Netherlands Organization

for Scientific Research through the Dutch Foundation for Earth

and Life Sciences (Investment Grant No. 834.01.002). T.A.C.S. re-

ceived support from NWO-CW (Grant No. 700.53.307). We grate-

fully acknowledge I.H.M. van Stokkum for extensive

computational data analysis, J. Thieme for technical support andM.R. Jones for supplying the samples.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in

the online version, atdoi:10.1016/j.cplett.2009.04.081.

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stuart multipulse spectroscopy on the wild-type and ym210w bacterial reaction centre uncovers a new...

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