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Low-temperature fluorescence kinetics of phytochrome New hints concerning the unknown mechanism of the early photocycle
JOURNAL OF
LUMINESCENCE ELSENIER
Journal of Luminescence 72-74 (1997) 603-604
Low-temperature fluorescence kinetics of phytochrome New hints concerning the unknown mechanism of the early photocycle M. Schulz-Evers”> *, K. Teuchner a, G. Hermannb, W. RiidigerC a Max-Born-Institut fiir Nichtlineare Optik und Kurzzeitspektroskopie, Rudower Chausee 6, D-12489 Berlin, Germany b Institut fiir Biochemie und Biophysik der Universitiit Jena, Philosophenweg 12, D-07743 Jena, Germany c Botanisches Institut der Ludwigs-Maximilians-Universitiit Miinchen, Menzinger Str. 67, D-80638 Miinchen, Germany
Abstract The relaxation rate of primary excited native (124 kDa) phytochrome from oat, is measured by time-resolved fluorescence techniques at temperatures between 77 and 300K in a buffer/glycerol mixture. The known multiexponential fluorescence decay of the red absorbing form P, is found at physiological temperatures while it can be characterized by a single exponential below 160 K with a lifetime of (1.3 & 0.2) ns at 77 K. This is consistent with the dynamics of the initially excited P, and an excited intermediate of the chromophore during the Z-E photoisomerization. Keywords: Phytochrome;
Primary photocycle;
Time-resolved
fluorescence;
Temperature
dependence
1. Introduction
2. Material and methods
The plant chromoprotein phytochrome, exists in two stable forms: P, (A,,, = 665 nm) and PF~ (A,,, = 730nm), both connected by a photocycle involving different intermediates [ 11. At ambient temperatures the P, fluorescence exhibits two photochromic exponentials [2, 41. Different models are currently discussed for the pathway from P, to the first longer lived intermediate lumi-R: (a) the fluorescence decay may be caused by a conformational heterogeneity of the chromophore and/or potein [2], (b) the fluorescence decay can be explained by the dynamics of two excited states which are in thermal equilibrium with each other [3, 41. At ambient temperatures both models are not distinguishable by fluorescence decay measurements, which we did extend down to 77 K.
Native oat phytochrome (124kDa) was isolated according to Ref. [5] with slight modifications. The phytochrome samples were used with a potassiurnphosphate buffer pH 7.6 and 66% v/v glycerol. Fluorescence emission was measured perpendicular to the excitation beam (,I = 630 nm, FWHM = 25 ps; Nd :YAG laser-pumped parametric generator; photon flux density 1O24cm-* s-’ ) with a single-shot streak camera (Hamamatsu C 1587, 2 ps time resolution). The sample with optical density of 0.3/cm was placed in a temperature controlled cryostat, which was moved after every laser shot. To increase the signal-to-noise ratio, up to 100 streak profiles were added. A back-transformation of photoproducts was driven by pulses with 700 nm 10 ns after the primary excitation. The applied numerical package CALE is described in Ref. [6].
* Corresponding author. [email protected].
Fax:
+49-30-6392-1309;
e-mail:
0022-23 13/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SOO22-2313(96)00386-9
M. Schulz-Even
604
et al. /Journal
of Luminescence 72-74 (1997) 603-604
3. Results and discussion Deconvolution analysis was applied with a sum of exponentials. At room temperature the two photochromic decay components were found to be zi=15f5ps(ai=0.6)andz2=60f7ps(a~=0.3). The longer but weaker components, fitted by an effective component of z3 = 350~s (a3 = O.l), are usually attributed to impurities [4] and omitted in the following. A reduction of temperature leads to the lengthening of the emission decays and only one single exponential can be found for T < 160 K. At 77 K the fluorescence lifetime equals (1.3 f 0.2) ns. The measured fluorescence decays were fitted to the energy level scheme (cf. Fig. 1) by target analysis. PT represents the initially excited fluorescing state of phytochrome, whereas X* denotes an excited, state preceding formation of lumi-R. For this model biexponential emission decay is expected. Fitted parameters are given in Table 1. Variation of y3 over a wide range has no significant influence on the fitted rate constants ~5, k+, k_ and thus was kept fixed. A detailed discussion will be given elsewhere [7]. Analysis for two fluorescable P, species reveals that heterogeneity does not exist: one single exponential for T < 160 K supports only one species. In contrast the equilibrium model can explain the temperature-dependent effect. When lowering the temperature the fluorescence
Table 1 Determined rate constants. The relative errors of k+/k-, are 15%, those of ys are 30% T WI
73 (fix)
300 260 160
10’0 lo9 IO’
(s-’
1
k+/k-
k+
4.8 6.1 18.9
4.8 x 10’0 1.2 x 10’0 1.1 x109
(s-‘1
and k+
YS (~~‘1
4 x 10’0 10’0 Not deducible
decay becomes longer and below 180 K lumi-R can be stabilized [ 11, the photocycle P, H Pk reduces to photochromic equilibrium P, H lumi-R. For T < 160 K the repopulation of Pr* (rate constant k- ) is too small to overcome the energy barrier between level 5 and 3 and does not influence the excited state P, significantly. Thus, the investigations are consistent with the dynamics of two excited states which are in thermal equilibrium with each other and exclude the species model.
Acknowledgements This work has been Forschungsgemeinschaft Ru 108/29-2).
supported (projects
by the Deutsche Te 188/l -3 and
References
X” t”““‘~“““““‘l h
1.0 i 0.8
P;"
.g 0.6 E al 0.4 E 0.2
0.0
2.0
4.0
6.0
8.0
time [ns] Fig. 1. Fluorescence decay of Pr at T = 77 K and result deconvolution analysis. Inset: energy level scheme.
[l] P. Eilfeld and W. Riidiger, Z. Naturforsch. C 40 (1985) 109-l 14. [2] K. Schaffner, S.E. Braslavsky and A.R. Holzwarth, Adv. Photochem. 5 (1990) 229-277. [3] K. Teuchner, D. Leupold and W. Riidiger, Projects Te 188/ l-l and Ru 108/29-l of the Deutsche Forschungsgesellschaft (working hypothesis), (1991), unpublished. [4] A.R. Holzwarth, E. Venuti, S.E. Braslavski and K. Schaffner, Biochim. Biophys. Acta 1140 (1992) 59-68. [5] R. Grimm and W. Riidiger, Z. Naturforsch. C 41 (1986) 988-992. [6] H. Stiel, K. Teuchner, D. Leupold, S. Oberllnder, J. Ehlert and R. Jahnke, Intell. Instr. Comp. 9 (1992) 79-88. [7] K. Teuchner, M. Schulz-Evers, D. Leupold, G. Hermann, W. Riidiger, Chem. Phys. Lett. (1996), submitted.
LUMINESCENCE ELSENIER
Journal of Luminescence 72-74 (1997) 603-604
Low-temperature fluorescence kinetics of phytochrome New hints concerning the unknown mechanism of the early photocycle M. Schulz-Evers”> *, K. Teuchner a, G. Hermannb, W. RiidigerC a Max-Born-Institut fiir Nichtlineare Optik und Kurzzeitspektroskopie, Rudower Chausee 6, D-12489 Berlin, Germany b Institut fiir Biochemie und Biophysik der Universitiit Jena, Philosophenweg 12, D-07743 Jena, Germany c Botanisches Institut der Ludwigs-Maximilians-Universitiit Miinchen, Menzinger Str. 67, D-80638 Miinchen, Germany
Abstract The relaxation rate of primary excited native (124 kDa) phytochrome from oat, is measured by time-resolved fluorescence techniques at temperatures between 77 and 300K in a buffer/glycerol mixture. The known multiexponential fluorescence decay of the red absorbing form P, is found at physiological temperatures while it can be characterized by a single exponential below 160 K with a lifetime of (1.3 & 0.2) ns at 77 K. This is consistent with the dynamics of the initially excited P, and an excited intermediate of the chromophore during the Z-E photoisomerization. Keywords: Phytochrome;
Primary photocycle;
Time-resolved
fluorescence;
Temperature
dependence
1. Introduction
2. Material and methods
The plant chromoprotein phytochrome, exists in two stable forms: P, (A,,, = 665 nm) and PF~ (A,,, = 730nm), both connected by a photocycle involving different intermediates [ 11. At ambient temperatures the P, fluorescence exhibits two photochromic exponentials [2, 41. Different models are currently discussed for the pathway from P, to the first longer lived intermediate lumi-R: (a) the fluorescence decay may be caused by a conformational heterogeneity of the chromophore and/or potein [2], (b) the fluorescence decay can be explained by the dynamics of two excited states which are in thermal equilibrium with each other [3, 41. At ambient temperatures both models are not distinguishable by fluorescence decay measurements, which we did extend down to 77 K.
Native oat phytochrome (124kDa) was isolated according to Ref. [5] with slight modifications. The phytochrome samples were used with a potassiurnphosphate buffer pH 7.6 and 66% v/v glycerol. Fluorescence emission was measured perpendicular to the excitation beam (,I = 630 nm, FWHM = 25 ps; Nd :YAG laser-pumped parametric generator; photon flux density 1O24cm-* s-’ ) with a single-shot streak camera (Hamamatsu C 1587, 2 ps time resolution). The sample with optical density of 0.3/cm was placed in a temperature controlled cryostat, which was moved after every laser shot. To increase the signal-to-noise ratio, up to 100 streak profiles were added. A back-transformation of photoproducts was driven by pulses with 700 nm 10 ns after the primary excitation. The applied numerical package CALE is described in Ref. [6].
* Corresponding author. [email protected].
Fax:
+49-30-6392-1309;
e-mail:
0022-23 13/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SOO22-2313(96)00386-9
M. Schulz-Even
604
et al. /Journal
of Luminescence 72-74 (1997) 603-604
3. Results and discussion Deconvolution analysis was applied with a sum of exponentials. At room temperature the two photochromic decay components were found to be zi=15f5ps(ai=0.6)andz2=60f7ps(a~=0.3). The longer but weaker components, fitted by an effective component of z3 = 350~s (a3 = O.l), are usually attributed to impurities [4] and omitted in the following. A reduction of temperature leads to the lengthening of the emission decays and only one single exponential can be found for T < 160 K. At 77 K the fluorescence lifetime equals (1.3 f 0.2) ns. The measured fluorescence decays were fitted to the energy level scheme (cf. Fig. 1) by target analysis. PT represents the initially excited fluorescing state of phytochrome, whereas X* denotes an excited, state preceding formation of lumi-R. For this model biexponential emission decay is expected. Fitted parameters are given in Table 1. Variation of y3 over a wide range has no significant influence on the fitted rate constants ~5, k+, k_ and thus was kept fixed. A detailed discussion will be given elsewhere [7]. Analysis for two fluorescable P, species reveals that heterogeneity does not exist: one single exponential for T < 160 K supports only one species. In contrast the equilibrium model can explain the temperature-dependent effect. When lowering the temperature the fluorescence
Table 1 Determined rate constants. The relative errors of k+/k-, are 15%, those of ys are 30% T WI
73 (fix)
300 260 160
10’0 lo9 IO’
(s-’
1
k+/k-
k+
4.8 6.1 18.9
4.8 x 10’0 1.2 x 10’0 1.1 x109
(s-‘1
and k+
YS (~~‘1
4 x 10’0 10’0 Not deducible
decay becomes longer and below 180 K lumi-R can be stabilized [ 11, the photocycle P, H Pk reduces to photochromic equilibrium P, H lumi-R. For T < 160 K the repopulation of Pr* (rate constant k- ) is too small to overcome the energy barrier between level 5 and 3 and does not influence the excited state P, significantly. Thus, the investigations are consistent with the dynamics of two excited states which are in thermal equilibrium with each other and exclude the species model.
Acknowledgements This work has been Forschungsgemeinschaft Ru 108/29-2).
supported (projects
by the Deutsche Te 188/l -3 and
References
X” t”““‘~“““““‘l h
1.0 i 0.8
P;"
.g 0.6 E al 0.4 E 0.2
0.0
2.0
4.0
6.0
8.0
time [ns] Fig. 1. Fluorescence decay of Pr at T = 77 K and result deconvolution analysis. Inset: energy level scheme.
[l] P. Eilfeld and W. Riidiger, Z. Naturforsch. C 40 (1985) 109-l 14. [2] K. Schaffner, S.E. Braslavsky and A.R. Holzwarth, Adv. Photochem. 5 (1990) 229-277. [3] K. Teuchner, D. Leupold and W. Riidiger, Projects Te 188/ l-l and Ru 108/29-l of the Deutsche Forschungsgesellschaft (working hypothesis), (1991), unpublished. [4] A.R. Holzwarth, E. Venuti, S.E. Braslavski and K. Schaffner, Biochim. Biophys. Acta 1140 (1992) 59-68. [5] R. Grimm and W. Riidiger, Z. Naturforsch. C 41 (1986) 988-992. [6] H. Stiel, K. Teuchner, D. Leupold, S. Oberllnder, J. Ehlert and R. Jahnke, Intell. Instr. Comp. 9 (1992) 79-88. [7] K. Teuchner, M. Schulz-Evers, D. Leupold, G. Hermann, W. Riidiger, Chem. Phys. Lett. (1996), submitted.