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Picosecond kinetic absorption studies of an iron porphyrin and bacteriochlorophyll using a streak camera
Volume
84. number 2
PICOSECOND
CHEMLCAL
KINETIC ABSORPTION
PHYSICS
LE-ITERS
AND BACTERIOCHLOROPHYLL
Y. LIANG *, D.K. NEGUS,
R.M. HOCHSTRASSER.
Received
of Chemistry.
1981
STUDIES
OF AN IRON PORI’HYRIN
Department
1 December
Unrverstty of Pennsyhanra.
USING A STREAK CAMERA *
M GUNNER
Plmizdelpha,
and P L. DUTTON
Pennsylvama 19104
USA
8 June 1981, III final form 11 August 1981
The prcosecond absorptron krnetrcs of two novel systems were measured usmg a streak camera Qumone substrtuted bactenophotosynthetrc reactron centers showed dependence of electron transfer rate on qumone reductron potenhal. Addlnontiy,
tic
U-ansxnf
absorpuon
hfetlmc
of
an
Fe(lII)
porphyrm.
FeTPPCI,
was
measured
fa
be
31
ps_ T-be
apparatus
and its performance arc descrtbed
1. Introduction In thus paper
we descnbe
our adaptation
of a
streak camera system to the study of absorptton transients. This approach has the advantage of ultralugh tmme resolutron and accuracy obtamable on the basrs of smgle laser shot data [ I,?]. We present two novel apphcatrons of the new method. The first mvalves measurements of the rate of electron transfer m model reactron centers, and the other expenment concerns the decay kmetrcs of a short-hved transrent species that was recently discovered to anse rn optrtally pumped non tetraphenylporphynn [3]. Both of these expenments are Ideally surted to smgle-shot kmettc studtes of this sort, Conventronal prcosecond expenments
kinetics
on
these
systems
could consume
amed
at determintng
orders of magmtude
more tune than the present techmque. Tlus IS especrally true of the reachon center expenments whtch were requrred to be repeated for many drfferent samples [4]. The results reported for FeTPPCl yreld the first accurate measurement of the hfetrme of an iron porphyrm transient state. in recent years it has become evldent that many * This research
supported by the Nahonal Science Foundatron (CHE-7688428). Part of the mstrumentataon used m thrs research was provrdcd by the Regronal Laser Laboratory at the Umversrty of Pennsylvama. * IBM Pm-Doctoral Fellow.
236
was
molecular processes of great chenucal mterest occur on a prcosecond trmescale. These mclude molecular reonentatron and rsomensm, cage recombmatron dynanucs, vrbrattonal and electromc relaxation, electron transfer and energy transfer processes of many types m numerous systems. Clearly tt would be advantageous to have available methods that could be used to study such processes wrth sufficient accuracy that subtleties m the decay patterns could be exposed. The current situation is that picosecond expenments yreld data that frequently must be fitted by oversrmphfied model assumptions. Srgnal-to-norse ratros are slgmficantly higher for the hrgh-repetition cw modelocked system than for sohd-state mode-locked lasers. In the latter case, especrally for Nd - glass lasers, the shot-to-shot retability is very low and contributes substanttally to the noise level if the measurement requxes many laser shots. For thrs reason :t is demr-
able to destgn expenments so that as much informatton as possible is obtamable from a smgle firing of such lasers. The goal of smgle-shot expenments stmultaneously yreldmg both frequency and picosecond ttme evolution data can be acheved camera.
m principle using a streak
Such devices have been widely
used to study fluorescence decay curves, although frequency selection of the fluorescence and obtainment of sufficient signal on a smgle shot strh pose signrficant problems for most of these devrces [S-7]. A main difficulty is
Volume 84, number 2
CI-EMICAL PHYSICS LETTERS
that fast streak cameras often have low dynamic range which restricts their usefulness m generating kinetic data without extensive averaging.
2. Experimental In GUStechmque the tune resolution was determmed by the streak camera. A sample was excited to a non-equtibnum state by a picosecond laser pulse, then probed with a temporally long, spectrally narrow pulse. The probe pulse, generated by excitation of a dye was split m two and passed through the excited and an unexcited region of the sample. These two beams, contammg the transient information in a SIXI& shot, were then tune dispersed unth the streak
camera and dgM.ly recorded in an OMA. Thrs picoiecond transient absorptron spectrometer utilized smgle optical pulses generated in a modelocked Nd glass laser system. The oscillator catty was designed to ensure TEM,, mode structure_ A single 1.06 pm pulse was electro-optically switched from early in the train and amplified to a30 mJ. The second (530 nm) and tlurd (353 nm) harmonics of the laser fundamental weq!enerated in non&near crystals of KDP. The double-beam transient absorption configurahon 1s illustrated m fig. 1. It begms with excitation of a dye (OD per mm typically OS- 1 .O) by focusmg
the laser pulse with a 10 cm focal length lens. For the bacteriochlorophyll reaction center the dye was a mixed saturated solution of rhodamine B and cresyl violet perchlorate in ethanol excited by 530 nm light. The iron porphyrin was probed with fluorescence from diphenylstilbene in; dioxane excited with a 353 run laser pulse. The dye fluorescence was collimated with a 5 cm focal length quartz lens and subsequently split into two beams with a 60% transmitting, 4GG reflecting beam sphtter @St). The reflected beam was directed through a focusmg lens and the sample at a slight incident angle onto a pmhole in the image plate (I). The transnutted beam must first traverse a short delay line whoselength 1s determined by the position of a mirror (M,) allowmg both beams to arnve at the sample synchronously. It was then focused through the sample at normal incidence onto a second pinhole through the same quartz lens. The 530 run excitation pulse, typically 8 ps. was tuned with appropnate
having separations
the
equal to the round tnp time of
then focused onto a scatter plate over the third pinhole to pro-de mtensity and time cahbration for each laser shot. Wavelength selection of the probe light was accomplished close to the photocathode of the streak camera where spatial separation of the three images waz pomble. The two fluorescence beams, one contaming the transient information, were filtered using appropriate color and narrow bandpass interference filters. The streak camera, a GEAR PICO-V, is fitted with an S-25 photocathode in a proximity focused streak the etalon.
Fig. I. Schemahc representation of picosecond transient kmeuc spectrometer. The probe fluorescence pulse was generated by exatation of the dye (DC). The exatatzon pulse was brought to the sample (S) counterpropagating to the probe, via a small mirror &). Tune and intennty caliirat~on were provided for each shot by passmg a portion of the pulse through an etalon e). Wavelength selection of the probe fluoresa+nce was by narrow bandpass interference filters 0.
delay to arrive dunng
through the sample of the long fluorescence pulse. Tlus beam was aligned to be counterpropagatmg and collinear with the probe. It was focused with a 30 cm focal length lens onto the sample by way of an 8 X 8 mm mirror (h4,) behind the nnage plate. The mirror blocked ~10% of the available f/2 aperture between the image plate and the photocathode. The image plate conssted of three 650 pm lameter pinholes backed by a variable width slit whose center matched the center of each pmhole ensuring beam overlap and allowing for control of time resolution. The third pinhole transmitted a small portion of the excitatton pulse which was split off and passed through an arr gap Fatry--Wrot etalon. The etalon consisted of two SO/50 broadband reflector/transmitters. Its output was a decaying train of p& passage
This pulse tram was
237
Volume 84, number 2
CHEMICAL PHYSICS LETTERS
1 December 1981
tube. Dtfferent ramp voltage rates could be supplied to the tube for vanatton of the sweep speed. Dependmg 0;~ the destred sweep speed the camera could be tnggered by actlvatmg a rUgh band~vldth photodrode or a spark-gap cvlth the residual f -06 pm hght. Output of the streak camera was coupled throu@~ a high-quality demagmfymg lens to the face of a twodimensmnal optical multichannel analyzer (PAR 12 15) 16/54) and processed by a wnte-ut program. The t~vo=dirnensIon~ cspabtiltres allowed amultaneous recording of the several streak traces assocrated wv~theach laser shot.
The data collectton procedure uas destgned to tntntrmze any shot-to-shot fluctuations m the fluorescence profile. WI& the euxtatlon pulse blocked, a senes of probe fluorescence profdes were obtamed for both the “reference” and “excltabon” tracks. When ten profiles were collected whose mtenslttes matched withm the photon noise. they were averaged and stored m the computer. A stgnal-to-none ratio of 50 I was typical for these profdes. Subsequent unbloched shots bvere then compared by least-
squares attalysrs using the averaged “reference” track. This aHowed rejection of any shots showmg atyp!caI behavior. Finally, the fio@nShm of the ratio of the average blocked to unblocked ‘“excitation” track profiles was taken * . The result as shown m fig. 2 IS the effective &A as a function of time at the wavelength selected by the interference fdter. The resultmg averaged decay curve IS a convolutlo~ of functions corresponding to real agnal, the evcltatlon pulse width, and the resolution of the streak camera. The decay lunetlcs were deternuned by Iterative convolutions followed by least-squares analysis. The preclslon error bars quoted are the standard devratlons resultmg from the best fit. No attempt was made at resolving the nse time at the sweep speeds used for the reactlon center expenments. For the Ee(II[) TPPCl an excttatlon pulse with an apparent fwhm of 12 ps was necessary to fit the nsmg agnal (consatent urlth expectation for a I mm pa&length cell and counterpropagating beams)_ The reaction centers were Isolated from rhodo* ‘l-he logsnthm of the ratlo of the average blocked to unblocked “reference” track profdes yields the effectrve basehne whtch for these expenments indicated a sensmvtty bmxt of 50.02 OD uruts
238
FIG 2 Upper Wanslenf absorption kmettcs for two bactenochlorophyll rexnon CC~WI-S after 530 nm escatotson. The
bandpzz of the probe Pulse was lumted to 7.5 nm (fwhm) centered at 640 nm usmg a D~tnc mterferemx filter. The
dashed lme IS cxkulrrted
for mstant.ancous (on thrs tunesuie)
tramlent signal followed by decay of the form c-f/T 7 was deternuned by least-squares analysis to be 233 -c 25 ps for the nauve qumone and 110.5 % 36 ps for the 9.1 O-anthraqumone. These traces are an average of 10 laser shots Lower S&e-shot tramtent absorption kmettcs data for the nntive qumone reactfon center. budd-up
of the
pseudomonas
sphaerordes (blue green mutant; R26) dlmethylamme=N-oxlde (LDAO) and punfied on an ton exchange column (Whatman DE 52). Replacement qumones obtamed commercially
usmg lauryl
were punfied unth activated carbon and recrystallizatlon
From
ethanol
and
were
replaced
by
the method
Dutton et al. IS]. The reaction centers ClOOuM) were suspended III IO mM Tns HCl,pH = 8.0, The FeTPPCl crystals (a gtft of B.B. Wayland) were purified by analytical thin Iayer ~romatography usmg a 2 1 pen&me-ether developer. The fraction contammg the punfied FeTPPCl was then e&ted wrth spectrograde chloroform and evaporated to an OD per mm of 1.5 at the 417 nm Soret peak. The visible of
spectrum showed 0.62 OD at 530 nm, the pumping wavelength-
Volume 84, number 2
CHEMICAL
1 December 1981
LEl-lTRS
3.2. Iron tetmphenyiporphynn prcosecond &ransien&
and discussion
3. Results
PHYSICS
Photosynthetic reaction centers accomphsh the converQon of photon energy into electrochemically
TPPCI under picosecond [3]. The Soret band bleached by a 353 nm pulse from a Nd : glass laser, and a new absorption, peaking III the region 440460 nm, was &covered. The lifetime of this transient was estunated from point by point kinetic stules to be 50 + 20 ps, assuming its decay was exponential. We have suggested that this transient is a tnplet state of the porphyrin. The data of fig. 3 are consistent with the exponential decay of
stored energy. Visible excitation
the transient but indicate
In the present paper the capabilities of the streak camera spectrometer are exemplified through study of electron transfer m a photosynthetic reaction center system and through study of a short-hved transient m FeTPPCl.
3. I. Bactenophotosynthetzc reachon ten tern
results m electron
transfer and eventually a charge separation across the reaction center. Initially a fast electron transfer (GO
ps) takes place between two molecules of bacteriochlorophyll m dimer form (BChl),, and bacteriopheophytin (BPh). This IS followed either by the reverse reictlon, K = lo* s-l, or by a slower electron transfer from BPh- to ublqumone-10 (Q) with K’ = 4 X 109 s-l [9]. T& electron transfer step can be measured by momtoring
sorption
the transient
signal at 640 nm [9- 121. Recently,
have been accomplished
endogenous of diffenng
involvmg
ab-
studies
replacement
of the
qumone with a series of anthraqumones reduction potential Eli2 [S,l 11. Gunner
et al. [13] measured the EI12 dependence of @‘, the propotion of reaction centers which reach the stable (BChl);BPhQstate. It was found that G decreases with loweringE112, p erhaps as a result of a slowing of K’ with respect to K. If ths were so, K’ could be obtamed from 4, = K/(K’ + K). We therefore undertook the study of accurately measurmg K’ for a senes of twelve substituted qumones to venfy the vahdity of this partitioning approach. This would of course have been a monumental task utizmg conventional point by point picosecond techniques. The results for two of these qulnones ~th
are shown
The behavior
of Fe(III)
excitation was recently at 4 15 nm was mstautly
described
that the hfetime
is close to
31 ps for 530 run excitation. The choice of narrowband filters m this streak camera experiment was made with full knowledge of the spectrum of the transrent observed previously. What IS being measured is not a change in spectral shape, such as might accompany vibrational relaxation [ 151 but the disappearance of the transient population. In the present case we believe that the transient is generated by multiphoton pumpmg, the predominan t relaxation pathway of the inibally excited state being repopulation of the ground state m less than 150 fs. The 30 ps bottleneck reported here may correspond to a state for which the equdibnum configuration is signifrcantly different from that of the ground state so that spm-orbit couplmg at uon is not the only factor controlhng the lifetime.
I
#,
I
I
I
I,
I,
IRON 450””
020-
1
I
,-
tLU, TPPCX TRANSIENT_
m fig. 2
the balance bemg analyzed
elsewhere [ 141. These experiments with reaction centers where the native quinone had been replaced showed conclusvely that there was a change in electron transfer rate from BPh’ to Q which is a function of the half poten~KII of the quinone. For the native ubiquinone-10 = -600 mV versus saturated calomel elec~tJq2 trode the rate was found to be 4.29 X 109 s-1 _ However, for 9,IO-anthraquinone with Elj2 = -816 mV the rate was found to be slguificantly slower,K’ = 9.05 x 10s s-1.
TIME
(psec)
Fig. 3. Transent absorption kinetics of Fe(UI)TPPCl after 530 run exatation. l3e bandpass of the probe pulse was huted to 7.5 nm fwhm centered at 450 run. The smooth sohd line is calculated for mstantaneous build-up o the transient signal followed by decay of the form emrf 9 r was determined by least-squares analysis to be 31+ 9 ps. Thk trace LSan average of three laser shots.
239
Volume 84, number 2
CHEMICAL
PHYSICS
4. Conclusions We have documented a rehable double-beam streak camera techmque for measuring picosecond transient absorption lunetlcs. In domg so we have demonstrated its particular app~cab~~ to systems requuing many measurements and to systems requirmg high tLme resolution. We have shown by duect measurement that there IS a sigmficant change 111electron transfer rate for reaction centers containmg qumones of differing reduction potent&. We have also measured the hfeme of what IS proposed to be a tnplet state of an zron(lI1) po~hy~.
LETTER
1 December 1981
Trumpower (Acadenuc Press, New York, 1981). to be publi~ed. [51 G.R. Ffeming, I.M. Moms and G.W. Robmson, Chem. Phys. 17 (i976) 91. [61 F. Heisel, J.A. hflehe and B. S~pp, Chem. Phys. Letters 61 (1979) 115. 171 hf. Sum&m, N. Nakashtma, K, Yoshihara and S. Nagakura, Chem. Phys. Letters 51 (1977) 183. [81 P.L. Dutton, M.R. Gunner and R.C. Prmce, Proceedmgs of the 8th International Congress on PhotobIology, to be pu bhshed [PI T.L. Netzel, P.M. Rentzepls and J.S. Leigh, Science 182 (1973) 238 t101 3. Fajer, D.C. Brune, M-S. Davxs, A. Forman and L.D. Spauldmg, Proc. NatL AC&. Sa. US 72 (1975) 4956. t111 M.G. Rockfey, M-W.Wmdsor, R-3. Cogdell and W.W. Parsons, Proc. Nntl. Acad. Sex. US 72 t1975)
References
1-55 1.
L Dutton, T L Net&, J.S. Legh and P.M. Rentzepls. Science 188 (1975) 1301.
1121 K J Kaufmann.
P
t131 M.R. Gunner, D.M. Tlede, RC. Prmce and P.L. Dutton,
[ I J K. Yoshlhan, A Namxkr, M. Sum&am and N. Nakasfuma, J. Chern. PItys 71 f1979) 2892 [2] Y. Wang, hf K. Crawford, M-J. McAuhffe and K I3 Ewnthaf, Chem. Phys. Letters 74 (1980) 160. [ 31 P.A. Comehus, A.W. Steele, D. Chemoff and R M Hochstrasser, Chem. Phys Letters 82 (1981) 9. (41 RC Prmce, hf.R. Gunner and P-L. Dutton, ut- Function of qumones m energy conseivrng systems, ed. B.L.
240
m: Function
of qumones
tn energy conservmg
systems,
ed. B.L. Tmmpower (Acadermc Press, New York, I981), to be pubhshed. iI41 P.L. Dutton, I\f.R. Gunner, R.M. Hochstrasser, Y. fxng and D-K. Negus. m preparation. 1151 B.I. Greene, R.N. Hochstrasser and R.B. Wetsman, 3. Chem. Fhys. 70 (1979) 1247.
84. number 2
PICOSECOND
CHEMLCAL
KINETIC ABSORPTION
PHYSICS
LE-ITERS
AND BACTERIOCHLOROPHYLL
Y. LIANG *, D.K. NEGUS,
R.M. HOCHSTRASSER.
Received
of Chemistry.
1981
STUDIES
OF AN IRON PORI’HYRIN
Department
1 December
Unrverstty of Pennsyhanra.
USING A STREAK CAMERA *
M GUNNER
Plmizdelpha,
and P L. DUTTON
Pennsylvama 19104
USA
8 June 1981, III final form 11 August 1981
The prcosecond absorptron krnetrcs of two novel systems were measured usmg a streak camera Qumone substrtuted bactenophotosynthetrc reactron centers showed dependence of electron transfer rate on qumone reductron potenhal. Addlnontiy,
tic
U-ansxnf
absorpuon
hfetlmc
of
an
Fe(lII)
porphyrm.
FeTPPCI,
was
measured
fa
be
31
ps_ T-be
apparatus
and its performance arc descrtbed
1. Introduction In thus paper
we descnbe
our adaptation
of a
streak camera system to the study of absorptton transients. This approach has the advantage of ultralugh tmme resolutron and accuracy obtamable on the basrs of smgle laser shot data [ I,?]. We present two novel apphcatrons of the new method. The first mvalves measurements of the rate of electron transfer m model reactron centers, and the other expenment concerns the decay kmetrcs of a short-hved transrent species that was recently discovered to anse rn optrtally pumped non tetraphenylporphynn [3]. Both of these expenments are Ideally surted to smgle-shot kmettc studtes of this sort, Conventronal prcosecond expenments
kinetics
on
these
systems
could consume
amed
at determintng
orders of magmtude
more tune than the present techmque. Tlus IS especrally true of the reachon center expenments whtch were requrred to be repeated for many drfferent samples [4]. The results reported for FeTPPCl yreld the first accurate measurement of the hfetrme of an iron porphyrm transient state. in recent years it has become evldent that many * This research
supported by the Nahonal Science Foundatron (CHE-7688428). Part of the mstrumentataon used m thrs research was provrdcd by the Regronal Laser Laboratory at the Umversrty of Pennsylvama. * IBM Pm-Doctoral Fellow.
236
was
molecular processes of great chenucal mterest occur on a prcosecond trmescale. These mclude molecular reonentatron and rsomensm, cage recombmatron dynanucs, vrbrattonal and electromc relaxation, electron transfer and energy transfer processes of many types m numerous systems. Clearly tt would be advantageous to have available methods that could be used to study such processes wrth sufficient accuracy that subtleties m the decay patterns could be exposed. The current situation is that picosecond expenments yreld data that frequently must be fitted by oversrmphfied model assumptions. Srgnal-to-norse ratros are slgmficantly higher for the hrgh-repetition cw modelocked system than for sohd-state mode-locked lasers. In the latter case, especrally for Nd - glass lasers, the shot-to-shot retability is very low and contributes substanttally to the noise level if the measurement requxes many laser shots. For thrs reason :t is demr-
able to destgn expenments so that as much informatton as possible is obtamable from a smgle firing of such lasers. The goal of smgle-shot expenments stmultaneously yreldmg both frequency and picosecond ttme evolution data can be acheved camera.
m principle using a streak
Such devices have been widely
used to study fluorescence decay curves, although frequency selection of the fluorescence and obtainment of sufficient signal on a smgle shot strh pose signrficant problems for most of these devrces [S-7]. A main difficulty is
Volume 84, number 2
CI-EMICAL PHYSICS LETTERS
that fast streak cameras often have low dynamic range which restricts their usefulness m generating kinetic data without extensive averaging.
2. Experimental In GUStechmque the tune resolution was determmed by the streak camera. A sample was excited to a non-equtibnum state by a picosecond laser pulse, then probed with a temporally long, spectrally narrow pulse. The probe pulse, generated by excitation of a dye was split m two and passed through the excited and an unexcited region of the sample. These two beams, contammg the transient information in a SIXI& shot, were then tune dispersed unth the streak
camera and dgM.ly recorded in an OMA. Thrs picoiecond transient absorptron spectrometer utilized smgle optical pulses generated in a modelocked Nd glass laser system. The oscillator catty was designed to ensure TEM,, mode structure_ A single 1.06 pm pulse was electro-optically switched from early in the train and amplified to a30 mJ. The second (530 nm) and tlurd (353 nm) harmonics of the laser fundamental weq!enerated in non&near crystals of KDP. The double-beam transient absorption configurahon 1s illustrated m fig. 1. It begms with excitation of a dye (OD per mm typically OS- 1 .O) by focusmg
the laser pulse with a 10 cm focal length lens. For the bacteriochlorophyll reaction center the dye was a mixed saturated solution of rhodamine B and cresyl violet perchlorate in ethanol excited by 530 nm light. The iron porphyrin was probed with fluorescence from diphenylstilbene in; dioxane excited with a 353 run laser pulse. The dye fluorescence was collimated with a 5 cm focal length quartz lens and subsequently split into two beams with a 60% transmitting, 4GG reflecting beam sphtter @St). The reflected beam was directed through a focusmg lens and the sample at a slight incident angle onto a pmhole in the image plate (I). The transnutted beam must first traverse a short delay line whoselength 1s determined by the position of a mirror (M,) allowmg both beams to arnve at the sample synchronously. It was then focused through the sample at normal incidence onto a second pinhole through the same quartz lens. The 530 run excitation pulse, typically 8 ps. was tuned with appropnate
having separations
the
equal to the round tnp time of
then focused onto a scatter plate over the third pinhole to pro-de mtensity and time cahbration for each laser shot. Wavelength selection of the probe light was accomplished close to the photocathode of the streak camera where spatial separation of the three images waz pomble. The two fluorescence beams, one contaming the transient information, were filtered using appropriate color and narrow bandpass interference filters. The streak camera, a GEAR PICO-V, is fitted with an S-25 photocathode in a proximity focused streak the etalon.
Fig. I. Schemahc representation of picosecond transient kmeuc spectrometer. The probe fluorescence pulse was generated by exatation of the dye (DC). The exatatzon pulse was brought to the sample (S) counterpropagating to the probe, via a small mirror &). Tune and intennty caliirat~on were provided for each shot by passmg a portion of the pulse through an etalon e). Wavelength selection of the probe fluoresa+nce was by narrow bandpass interference filters 0.
delay to arrive dunng
through the sample of the long fluorescence pulse. Tlus beam was aligned to be counterpropagatmg and collinear with the probe. It was focused with a 30 cm focal length lens onto the sample by way of an 8 X 8 mm mirror (h4,) behind the nnage plate. The mirror blocked ~10% of the available f/2 aperture between the image plate and the photocathode. The image plate conssted of three 650 pm lameter pinholes backed by a variable width slit whose center matched the center of each pmhole ensuring beam overlap and allowing for control of time resolution. The third pinhole transmitted a small portion of the excitatton pulse which was split off and passed through an arr gap Fatry--Wrot etalon. The etalon consisted of two SO/50 broadband reflector/transmitters. Its output was a decaying train of p& passage
This pulse tram was
237
Volume 84, number 2
CHEMICAL PHYSICS LETTERS
1 December 1981
tube. Dtfferent ramp voltage rates could be supplied to the tube for vanatton of the sweep speed. Dependmg 0;~ the destred sweep speed the camera could be tnggered by actlvatmg a rUgh band~vldth photodrode or a spark-gap cvlth the residual f -06 pm hght. Output of the streak camera was coupled throu@~ a high-quality demagmfymg lens to the face of a twodimensmnal optical multichannel analyzer (PAR 12 15) 16/54) and processed by a wnte-ut program. The t~vo=dirnensIon~ cspabtiltres allowed amultaneous recording of the several streak traces assocrated wv~theach laser shot.
The data collectton procedure uas destgned to tntntrmze any shot-to-shot fluctuations m the fluorescence profile. WI& the euxtatlon pulse blocked, a senes of probe fluorescence profdes were obtamed for both the “reference” and “excltabon” tracks. When ten profiles were collected whose mtenslttes matched withm the photon noise. they were averaged and stored m the computer. A stgnal-to-none ratio of 50 I was typical for these profdes. Subsequent unbloched shots bvere then compared by least-
squares attalysrs using the averaged “reference” track. This aHowed rejection of any shots showmg atyp!caI behavior. Finally, the fio@nShm of the ratio of the average blocked to unblocked ‘“excitation” track profiles was taken * . The result as shown m fig. 2 IS the effective &A as a function of time at the wavelength selected by the interference fdter. The resultmg averaged decay curve IS a convolutlo~ of functions corresponding to real agnal, the evcltatlon pulse width, and the resolution of the streak camera. The decay lunetlcs were deternuned by Iterative convolutions followed by least-squares analysis. The preclslon error bars quoted are the standard devratlons resultmg from the best fit. No attempt was made at resolving the nse time at the sweep speeds used for the reactlon center expenments. For the Ee(II[) TPPCl an excttatlon pulse with an apparent fwhm of 12 ps was necessary to fit the nsmg agnal (consatent urlth expectation for a I mm pa&length cell and counterpropagating beams)_ The reaction centers were Isolated from rhodo* ‘l-he logsnthm of the ratlo of the average blocked to unblocked “reference” track profdes yields the effectrve basehne whtch for these expenments indicated a sensmvtty bmxt of 50.02 OD uruts
238
FIG 2 Upper Wanslenf absorption kmettcs for two bactenochlorophyll rexnon CC~WI-S after 530 nm escatotson. The
bandpzz of the probe Pulse was lumted to 7.5 nm (fwhm) centered at 640 nm usmg a D~tnc mterferemx filter. The
dashed lme IS cxkulrrted
for mstant.ancous (on thrs tunesuie)
tramlent signal followed by decay of the form c-f/T 7 was deternuned by least-squares analysis to be 233 -c 25 ps for the nauve qumone and 110.5 % 36 ps for the 9.1 O-anthraqumone. These traces are an average of 10 laser shots Lower S&e-shot tramtent absorption kmettcs data for the nntive qumone reactfon center. budd-up
of the
pseudomonas
sphaerordes (blue green mutant; R26) dlmethylamme=N-oxlde (LDAO) and punfied on an ton exchange column (Whatman DE 52). Replacement qumones obtamed commercially
usmg lauryl
were punfied unth activated carbon and recrystallizatlon
From
ethanol
and
were
replaced
by
the method
Dutton et al. IS]. The reaction centers ClOOuM) were suspended III IO mM Tns HCl,pH = 8.0, The FeTPPCl crystals (a gtft of B.B. Wayland) were purified by analytical thin Iayer ~romatography usmg a 2 1 pen&me-ether developer. The fraction contammg the punfied FeTPPCl was then e&ted wrth spectrograde chloroform and evaporated to an OD per mm of 1.5 at the 417 nm Soret peak. The visible of
spectrum showed 0.62 OD at 530 nm, the pumping wavelength-
Volume 84, number 2
CHEMICAL
1 December 1981
LEl-lTRS
3.2. Iron tetmphenyiporphynn prcosecond &ransien&
and discussion
3. Results
PHYSICS
Photosynthetic reaction centers accomphsh the converQon of photon energy into electrochemically
TPPCI under picosecond [3]. The Soret band bleached by a 353 nm pulse from a Nd : glass laser, and a new absorption, peaking III the region 440460 nm, was &covered. The lifetime of this transient was estunated from point by point kinetic stules to be 50 + 20 ps, assuming its decay was exponential. We have suggested that this transient is a tnplet state of the porphyrin. The data of fig. 3 are consistent with the exponential decay of
stored energy. Visible excitation
the transient but indicate
In the present paper the capabilities of the streak camera spectrometer are exemplified through study of electron transfer m a photosynthetic reaction center system and through study of a short-hved transient m FeTPPCl.
3. I. Bactenophotosynthetzc reachon ten tern
results m electron
transfer and eventually a charge separation across the reaction center. Initially a fast electron transfer (GO
ps) takes place between two molecules of bacteriochlorophyll m dimer form (BChl),, and bacteriopheophytin (BPh). This IS followed either by the reverse reictlon, K = lo* s-l, or by a slower electron transfer from BPh- to ublqumone-10 (Q) with K’ = 4 X 109 s-l [9]. T& electron transfer step can be measured by momtoring
sorption
the transient
signal at 640 nm [9- 121. Recently,
have been accomplished
endogenous of diffenng
involvmg
ab-
studies
replacement
of the
qumone with a series of anthraqumones reduction potential Eli2 [S,l 11. Gunner
et al. [13] measured the EI12 dependence of @‘, the propotion of reaction centers which reach the stable (BChl);BPhQstate. It was found that G decreases with loweringE112, p erhaps as a result of a slowing of K’ with respect to K. If ths were so, K’ could be obtamed from 4, = K/(K’ + K). We therefore undertook the study of accurately measurmg K’ for a senes of twelve substituted qumones to venfy the vahdity of this partitioning approach. This would of course have been a monumental task utizmg conventional point by point picosecond techniques. The results for two of these qulnones ~th
are shown
The behavior
of Fe(III)
excitation was recently at 4 15 nm was mstautly
described
that the hfetime
is close to
31 ps for 530 run excitation. The choice of narrowband filters m this streak camera experiment was made with full knowledge of the spectrum of the transrent observed previously. What IS being measured is not a change in spectral shape, such as might accompany vibrational relaxation [ 151 but the disappearance of the transient population. In the present case we believe that the transient is generated by multiphoton pumpmg, the predominan t relaxation pathway of the inibally excited state being repopulation of the ground state m less than 150 fs. The 30 ps bottleneck reported here may correspond to a state for which the equdibnum configuration is signifrcantly different from that of the ground state so that spm-orbit couplmg at uon is not the only factor controlhng the lifetime.
I
#,
I
I
I
I,
I,
IRON 450””
020-
1
I
,-
tLU, TPPCX TRANSIENT_
m fig. 2
the balance bemg analyzed
elsewhere [ 141. These experiments with reaction centers where the native quinone had been replaced showed conclusvely that there was a change in electron transfer rate from BPh’ to Q which is a function of the half poten~KII of the quinone. For the native ubiquinone-10 = -600 mV versus saturated calomel elec~tJq2 trode the rate was found to be 4.29 X 109 s-1 _ However, for 9,IO-anthraquinone with Elj2 = -816 mV the rate was found to be slguificantly slower,K’ = 9.05 x 10s s-1.
TIME
(psec)
Fig. 3. Transent absorption kinetics of Fe(UI)TPPCl after 530 run exatation. l3e bandpass of the probe pulse was huted to 7.5 nm fwhm centered at 450 run. The smooth sohd line is calculated for mstantaneous build-up o the transient signal followed by decay of the form emrf 9 r was determined by least-squares analysis to be 31+ 9 ps. Thk trace LSan average of three laser shots.
239
Volume 84, number 2
CHEMICAL
PHYSICS
4. Conclusions We have documented a rehable double-beam streak camera techmque for measuring picosecond transient absorption lunetlcs. In domg so we have demonstrated its particular app~cab~~ to systems requuing many measurements and to systems requirmg high tLme resolution. We have shown by duect measurement that there IS a sigmficant change 111electron transfer rate for reaction centers containmg qumones of differing reduction potent&. We have also measured the hfeme of what IS proposed to be a tnplet state of an zron(lI1) po~hy~.
LETTER
1 December 1981
Trumpower (Acadenuc Press, New York, 1981). to be publi~ed. [51 G.R. Ffeming, I.M. Moms and G.W. Robmson, Chem. Phys. 17 (i976) 91. [61 F. Heisel, J.A. hflehe and B. S~pp, Chem. Phys. Letters 61 (1979) 115. 171 hf. Sum&m, N. Nakashtma, K, Yoshihara and S. Nagakura, Chem. Phys. Letters 51 (1977) 183. [81 P.L. Dutton, M.R. Gunner and R.C. Prmce, Proceedmgs of the 8th International Congress on PhotobIology, to be pu bhshed [PI T.L. Netzel, P.M. Rentzepls and J.S. Leigh, Science 182 (1973) 238 t101 3. Fajer, D.C. Brune, M-S. Davxs, A. Forman and L.D. Spauldmg, Proc. NatL AC&. Sa. US 72 (1975) 4956. t111 M.G. Rockfey, M-W.Wmdsor, R-3. Cogdell and W.W. Parsons, Proc. Nntl. Acad. Sex. US 72 t1975)
References
1-55 1.
L Dutton, T L Net&, J.S. Legh and P.M. Rentzepls. Science 188 (1975) 1301.
1121 K J Kaufmann.
P
t131 M.R. Gunner, D.M. Tlede, RC. Prmce and P.L. Dutton,
[ I J K. Yoshlhan, A Namxkr, M. Sum&am and N. Nakasfuma, J. Chern. PItys 71 f1979) 2892 [2] Y. Wang, hf K. Crawford, M-J. McAuhffe and K I3 Ewnthaf, Chem. Phys. Letters 74 (1980) 160. [ 31 P.A. Comehus, A.W. Steele, D. Chemoff and R M Hochstrasser, Chem. Phys Letters 82 (1981) 9. (41 RC Prmce, hf.R. Gunner and P-L. Dutton, ut- Function of qumones m energy conseivrng systems, ed. B.L.
240
m: Function
of qumones
tn energy conservmg
systems,
ed. B.L. Tmmpower (Acadermc Press, New York, I981), to be pubhshed. iI41 P.L. Dutton, I\f.R. Gunner, R.M. Hochstrasser, Y. fxng and D-K. Negus. m preparation. 1151 B.I. Greene, R.N. Hochstrasser and R.B. Wetsman, 3. Chem. Fhys. 70 (1979) 1247.