Ultrafast Triplet Generation and its Sensitization Drives Efficient

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Ultrafast Triplet Generation and its Sensitization Drives Efficient Photoisomerization of tetra-cis-lycopene to all-trans-lycopene Kanchustambham Vijayalakshmi, Ajay Jha, and Jyotishman Dasgupta J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b02086 • Publication Date (Web): 12 Jun 2015 Downloaded from http://pubs.acs.org on June 18, 2015

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The Journal of Physical Chemistry

Ultrafast Triplet Generation and its Sensitization Drives Efficient Photoisomerization of tetra-cislycopene to all-trans-lycopene Kanchustambham Vijayalakshmi, Ajay Jha and Jyotishman Dasgupta* *Email: [email protected] Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai-400005, INDIA

KEYWORDS: Carotenoid metabolism, photoisomerization, quantum yield, excited state dynamics, carotenoid isomerase.

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ABSTRACT Lycopene biosynthesis in photosynthetic organisms controls the metabolic flux of reaction center carotenoids like -carotene and lutein through the geometric four-step isomerization of 7, 9, 9’, 7’ tetra-cis-lycopene (prolycopene) to its all-trans form. In plants and cyanobacteria, a redoxcontrolled flavoenzyme carotenoid isomerase catalyzes the prolycopene isomerization although its functional loss inside the chloroplast can be rescued by light. In order to address the chloroplast-specificity and efficiency of the light-induced isomerization reaction, we need to critically understand the excited state dynamics of prolycopene and the nature of electronic states that lead to the isomerization. Using broadband femtosecond transient absorption spectroscopy, we observe ~610 fs rise of the long-lived triplet state from the photo-excited S2 with a quantum yield of ~0.19. The triplet state eventually triggers the first C=C bond isomerization at the symmetric 9 or 9’ position on the tetra-cis backbone to yield the tri-cis product with 15% quantum efficiency. However, direct sensitization of the photoreactive triplet state via mesotetraphenyl prophyrin sensitizer under steady state illumination leads to an efficient production of all-trans-lycopene with 58% quantum yield. Our work implies that chlorophyll-enriched chloroplasts should form an optimized photoreaction vessel for prolycopene isomerization, and synthetic utilization of such cis-carotenoids can result in an efficient triplet harvesting photonconversion devices.


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INTRODUCTION Carotenoids are conjugated tetraterpenes that function as visible light harvesting pigments as well as thermal sink for regulation of excess photons in photosynthesis.1-3 The specific physiological function of carotenoids is often dictated by the geometric cis/trans configuration of the conjugated polyene backbone especially in the reaction centers and light harvesting complexes.4-6 Due to the constant turnover of protein-pigment complexes during photosynthesis, it is imperative to understand the dynamic nature of carotenoid bioavailability and more significantly the biochemical control of various carotenoid isomers inside chloroplasts. The biosynthesis of primary carotenoids in photosynthetic organisms takes place via condensation of two C20 terpenoid molecules to furnish the first stable carotenoid 15-cisphytoene. The occurrence of cis-motif in phytoene and subsequent cis-intermediates in poly-cis pathway is well-documented for plants and cyanobacteria.7 The conversion of 15-cis-phytoene to all-trans-lycopene proceeds via two desaturation and two isomerization reactions accomplished by set of four enzymes.7-9 The final step in the lycopene formation involves isomerization of four cis C=C bonds present in the precursor molecule 7, 9, 9’, 7’ tetra-cis-lycopene also known as prolycopene. Prolycopene has two symmetric di-cis motifs in the polyene backbone (Scheme 1). Prolycopene conversion to all-trans-lycopene is catalyzed by the enzyme carotenoid isomerase (CRTISO) assisted by two electron reduction of bound co-factor flavin adenine dinucleotide.10-13 The efficiency of this redox-triggered enzymatic reaction is critical to the synthesis of downstream functional carotenoids e.g. xanthophylls and β-carotene.12-15


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Scheme 1: Cartoon representation of 7,9,9’,7’-tetra-cis-lycopene (prolycopene) isomerization to all-trans-lycopene catalyzed by the redox dependent flavoenzyme carotenoid isomerase (CRTISO) and the light induced isomerization reaction inside the chloroplast. It has been reported that light can rescue the impaired CRTISO activity only in the chloroplasts but not in chromoplasts where chlorophyll metabolism is typically ceased. Based on the steady state absorption spectrum it was previously shown that chlorophylls in chloroplast may drive photoisomerization of prolycopene in vivo in green cells of mutant C-6D of scenedesmus obliquus, green leaves of deletion mutant tangerinemic and the chloroplast of mutant strain (5/520) chlorella.12,16-18 It was concluded that stereo-configuration of poly-cis-isomers in the chloroplast is sensitive to both the quality of light and presence of oxygen. In addition, previous work has shown that prolycopene in solution undergoes stepwise isomerization to form all-trans-lycopene via several cis-intermediates in the presence and absence of photocatalyst like I2.19 However, the knowledge of involved excited states and the reaction efficiencies which detail the fundamental molecular mechanism of the photo-isomerization reaction is still unclear. Especially the reaction efficiency and its kinetic implications inside the chloroplasts remain unexplored. Unraveling all the biochemical pathways underlying the regulation of lycopene flux inside the chloroplasts will lead to deeper understanding of light usage in photosynthetic organisms during normal and stress conditions. Therefore it is imperative to temporally track and


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study the detailed excited state pathways that lead to lycopene formation subsequent to isomerization across four cis bonds. Ultrafast spectroscopy has been extensively used to interrogate the excited state dynamics of carotenoids. The assignment of various excited state spectral signatures has been an active area of research although the principal non-radiative transitions are now comprehensively benchmarked for model carotenoids.20-26 Previous work on cis-carotenoids has identified efficient formation of triplet states when bound to the light harvesting and reaction center complexes although the yield is usually small for either cis- or trans-carotenoids free in solution. 27-29

In certain cis carotenoids free in solution the triplet state has been identified as the reactive

surface for isomerization reaction.30-35 Herein, we use ultrafast broadband transient absorption spectroscopy in combination with steady state absorption and resonance Raman (RR) spectroscopy to elucidate the efficiency and mechanistic pathway for isomerization of four cisbonds at 7, 9, 7’, 9’ positions to yield all-trans-lycopene. Direct excitation at the intense S2 band of free prolycopene leads to extremely low quantum yield (1 ns (Figure S15a) although its decay kinetics is limited by our detection sensitivity (~0.1 mOD) and the extent of our pump-probe delays (2 ns). It is therefore evident from the time-resolved absorption of prolycopene in solution that a modest fraction of the excited population can yield triplet which is the reactive state. The quantum yield of the triplet state can be estimated by using the formation timescales of S1 and S* from the photo-excited S2 state. In order to confirm our state assignments and connect the individual states via a microscopic kinetic model we carried out detailed kinetic analysis. Kinetic analysis of the ESA dynamics. We determined the reaction kinetics at three distinct ESA features centered at 600 nm, 540 nm and 475 nm representing the S1, S* and ground state bleach regions of the spectrum, respectively. Figure 5a shows the kinetics at 600 nm was fitted to two-exponential model convoluted with IRF of ~100 fs. The rise time of 330 fs is the emergence of S1 state from the photoexcited S2 state while the decay time constant of ~6.4 ps is the timescale of its decay to the ground state. Figure 5b shows kinetics for the ESA at 540 nm which provides a glimpse of the kinetic scheme involving S1 and the postulated S*. The dynamics is fitted to three lifetime components: a rise time of about ~270 fs and two decay time constants of 5.2 ps and 18.9 ps, respectively. It is evident that the kinetics at 540 nm is dominated primarily by the S1 dynamics but an additional long kinetic component indicates the decay of the S* state. It has been shown previously that S* is an electronic state mediating the internal conversion from S2 to S1. However, S* was also primarily assigned to a state arising due to ground state heterogeneity in the molecule. Additionally, it has been identified as a precursor state to facilitate


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ultrafast singlet fission to the triplet manifold.33,61,63-65 In order to confirm the assignment of the S* state, we also fitted the data at 495 nm (Figure S14) which was away from the S1 although capturing the blue edge of the S* state. Fitting the data with four exponents provided a 280 fs rise time and three long decay times of: 15.8 ps, ~300 ps and a non-decaying triplet component. Although there may a strong overlap of the S* and triplet ESA features, we assign 280 fs to the rise of S* similar to the time constant obtained from decay kinetics at 540 nm. The time constants 15.8 ps and 300 ps were assigned to S* decay back to ground state through a putative Sr*.61

Figure 5: Multi-exponential single point kinetic analysis of prolycopene excited state dynamics at different wavelengths; (a) 600 nm; (b) 540 nm; and (c) 475 nm subsequent to 400 nm excitation. (d) Near-IR dynamics at 1040 nm subsequent to 480 nm excitation is plotted for S 2 lifetime The ground state bleach recovery dynamics were determined by fitting the kinetics at 475 nm to four exponents as shown in Figure 5c. The ultrafast component of 120 fs is assigned to the overlapping stimulated emission decay from the S2 state. The evidence for the stimulated emission can be seen in the very early time points (-100 fs to 50 fs) in the excited state evolution


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traces shown in Figure 4. The 4.2 ps dynamics is assigned to the major recovery channel of the ground state population through the combined relaxation pathways of the S1 and S* state. Through the kinetic fit at 600 nm and 495 nm, we estimate the S1 and S* lifetimes to be 6.4 ps and 15.8 ps respectively. Using these numbers we estimate the ground state recovery to occur with a 4.55 ps timescale which roughly matches the picosecond time constant obtained from the recovery dynamics at 475 nm. In addition, there is a long-lived positive ∆A signal at 475 nm that is seen to prevail till few nanoseconds although the intensity is affected by the S* decay of 300 ps. The 100 ps to 1 ns spectra obtained through femtosecond experiments are shown in Figure S15a in supporting information. The spectral evolution clearly enunciates that the non-decaying signal does not change its spectral shape between 500 ps to 1 ns. Therefore this signal is assigned to the long-lived prolycopene triplet state which ultimately should lead to the photoisomerization reaction. Under the assumption that the non-decaying triplet also originates from the S2 state, we carried out near-IR transient absorption measurements to measure the pure lifetime of the S 2 state. Fitting the transient data at ESA 1040 nm shown in Figure 5d, we obtained an ultrafast decay of the S2 state with a time constant ~120 ± 25 fs along with a second time constant of 5.6 ps assigned to S1 decay. 58 Using the intrinsic lifetime of the S2 state, we propose a ~610 fs rise time for the log-lived triplet state directly from S2 state ensuring almost 19 % triplet quantum yield (Scheme S1). The calculated quantum yield for the triplet state excellently correlates to the yield of the tri-cis-lycopene photoproduct as determined by the HPLC analysis for the unsensitized reaction. Our assignment of the kinetic pathway is consistent with the recently published report by Larsen and co-workers who propose similar scheme for β-carotene dynamics.61


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Global analysis of prolycopene excited state dynamics. The singular value decomposition was used to derive decay associated difference spectra (DADS).66 Four lifetime components 175 fs, 6.5 ps, 100 ps and non-decaying component were required to completely describe the time resolved spectra and the respective DADS is shown in Figure 6. The 175 fs spectrum shows pronounced negative feature between 460-600 nm along with an absorption extending up to 740 nm. This negative component matches well with the spectrum of S1 and S* states, thereby showing the kinetic partitioning of the S2 state towards the S1 and S* respectively. The region near 470 nm also shows a convolution of stimulated emission and possibly the triplet state that emerges directly from the S2 state. The second component (in red) shows a bleach signal and a broad intense absorption from 500-650 nm. The timescale of 6.5 ps matches with the S1 lifetime as measured from the single point kinetics at 600 nm. Since S1 does not have emission, the negative signal indicates that ground state recovery takes place with major amplitude of 6.5 ps. The third 105 ps component putatively assigned to S* state has a small contribution in overall transient signal intensity. However, our single wavelength kinetic fit shows a ~19 ps lifetime for the S* state. The spectrum of S* shows a vibronic like feature similar to that observed for βcarotene. The non-decaying component with absorption at 480 nm was assigned to the T1 to Tn transition in the triplet manifold. Based on the states and the associated spectra, we construct a model for the excited state evolution which is described in Scheme 2. We find that the S2 state branches to form all the new states that were detected in our time-resolved data. The formation of S1 and S* happens with similar time constants of 330 and 280 fs respectively although the triplet state forms on ~610 fs timescale. This microscopic kinetic scheme matches with known excited state dynamics of β-carotene although we cannot completely rule out the possibility that


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the triplet emerges from S*.33 The triplet surface therefore should provide the necessary reactive crossings for the efficient isomerization reaction.

Figure 6: Decay associated difference spectra (DADS) of excited state population for prolycopene in toluene measured upon 400 nm excitation. Sensitization of the triplet state for photoreaction. In order to test whether all the stepwise reactions proceed through respective triplet excited states, we carried out the isomerization reaction in presence of known triplet sensitizers.


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Figure 7: Triplet sensitized photoisomerization of prolycopene monitored using HPLC and compared to the chromatogram of all-trans-lycopene. Figure 7 shows the HPLC profiles for the isomerization reaction sensitized using mesotetraphenyl porphyrin (TPP). The reaction is triggered by exciting TPP at different wavelengths corresponding to its absorption spectrum and monitored after 5 minutes (Figure S6). For convenience, we only show the HPLC profile after the elution of TPP sensitizer at 5.4 minutes. It is evident that the lycopene yield increases substantially and the reaction speeds up rapidly when compared to unsensitized reaction described previously. Interestingly the profiles show pronounced wavelength dependence for lycopene formation with the highest yield observed at 520 nm and 595 nm corresponding to the porphyrin Q-bands. The quantum yield of the triplet sensitized photoisomerization of prolycopene is ~0.58 and the quantification is shown in Figure S12. We therefore conclude that the isomerization is vastly facilitated by the presence of a triplet sensitizer directly invoking the role of triplet states in the isomerization pathway (Figure S10S11). Our observation of high quantum efficiency under triplet sensitization clearly demonstrates the biological significance of light-dependent pathway during photosynthesis in chloroplasts.

Our results on prolycopene excited state evolution and isomerization dynamics is summarized in scheme 2 and the parameters obtained from the detailed kinetic analysis is provided in table ST1 in supporting information. Subsequent to S2 excitation of prolycopene, majority of the excited state population evolves rapidly to form singlet S1 and S* species via ultrafast internal conversion process with 330 fs and 270 fs time constants. The formed singlet states eventually relax to the ground state in ~5 ps from S1 respectively. However, a 610 fs time constant for the intersystem crossing signifies almost ~19% triplet quantum yield. In fact the reactive triplet state


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leads to the formation of the primary isomerization product tri-cis-lycopene with ~15% quantum yield under steady state illumination although the effective yield of the step-wise isomerization to all-trans-lycopene is only 0.003%. Upon sensitization with meso-tetraphenyl porphyrin the quantum yield for cis-trans isomerization across four double bonds increases to ~0.58. Our work demonstrates that although light-induced isomerization is efficient for flipping the symmetric 9 or 9’ ethylenic bond on the tetra-cis skeleton, the efficient four-step isomerization to all-trans product is only feasible under triplet sensitization. We believe that the ultrafast triplet generation in these classes of cis-carotenoids will find new applications for light energy utilization in photovoltaic devices.67-69 These results should also motivate new transient electronic and vibrational measurements to assess the nature of the reaction coordinate driving the singlet-totriplet state crossings.

Scheme 2: Kinetic model summarizing the excited state dynamics of prolycopene in toluene subsequent to 400 nm excitation. Arrows indicate the evolution of states and the time constant corresponding to the lifetimes of each state. Energy transfer from TPP to the prolycopene mediates the isomerization through reactive triplet manifold.


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In photosynthetic organisms the enzyme CRTISO controls the flux of all-trans-lycopene during the diurnal turnover although our results unequivocally suggest that direct prolycopene photoisomerization can be an efficient pathway in presence of triplet sensitizers. Chloroplasts naturally harbor chlorophyll molecules that can efficiently yield the necessary triplet states for triggering the photoisomerization chemistry, thereby forming an ideal reaction vessel. This alternate pathway for the conversion of prolycopene to all-trans-lycopene with high quantum yield should allow the photosynthetic organisms to counter high light stress, and carry out photoautotrophic growth. Our work therefore should initiate new in vivo experiments to precisely monitor the light-induced reaction inside the photosynthetic cells and evaluate the effect of oxygen production from photosystem II in CRTISO deletion mutant phenotype.

CONCLUSIONS

In conclusion, we demonstrate that light-induced cis-trans isomerization of prolycopene to alltrans-lycopene occurs through the triplet states. We provide the first spectroscopic evidence for an efficient sub-picosecond generation of prolycopene triplet state with quantum yield of ~ 19% in solution, and almost quantitative conversion from the reactive triplet state to its corresponding singly-isomerized tri-cis-lycopene photoproduct. In absence of triplet sensitizer, the isomerization of prolycopene to the all-trans product follows step-wise triplet generation mechanism which ultimately leads to poor quantum yield of ~0.003. Upon direct triplet sensitization, we show an efficient formation of all-trans-lycopene with quantum yield of ~0.58. Our work highlights the importance of the light-induced triplet isomerization pathway in carotenoid biosynthesis occurring inside chloroplasts during photosynthetic turnover.

We

envision that the ultrafast generation of triplet state in such cis-carotenoids should inspire its utilization in many photon-conversion technologies.


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ASSOCIATED CONTENT Supporting Information. Details of HPLC purification, characterization using NMR & MALDI, additional time-resolved data are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Phone: +91-22-22782383. Fax: +91-22-22804610. E-mail: [email protected] Notes: The authors declare no competing financial interest. ACKNOWLEDGMENT The authors thank Dr. Joseph Hirschberg, Dr. Varda Mann, Dr. Halim Jubran (Hebrew University) and Dr. Yaakov Tadmor (ARO, Israel) for providing tangerine fruits and watermelon seeds. For experimental help and discussions, Mr. Palas Roy, Dr. Asha Parmar, Ms. Ankita Das, Mr. Rahul Gera and Mr. Roshan Kulkarni, TIFR are acknowledged. We thank Prof. Sudipta Maiti and Dr. Ankona Datta, TIFR for HPLC and LC-MS instruments. Ms. Gitanjali Dhotre and Ms. Deepa S (TIFR) help with MALDI measurements is acknowledged. We thank Dr. Rajib Ghosh and Dr. Dipak Palit for flash photolysis measurements in BARC, Trombay. Ms. Geogy Abhraham, BARC is acknowledged for few Raman measurements. The national NMR facility at TIFR is acknowledged for 1H-NMR measurements. JD thanks the monetary support from the DAE and TIFR, Mumbai.


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ABBREVIATIONS CRTISO, carotenoid isomerase; IRF, instrument response function; ESA, excited state absorption;

GSB, ground state bleach; SVD, singular value decomposition; DAS, decay

associated spectra; RR, resonance Raman; TPP, meso-tetraphenyl porphyrin.


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Ultrafast Triplet Generation and its Sensitization Drives Efficient

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