Tésis en co-tutela con el Dr. Manuel Llansola-Portoles en Paris-Saclay
Role of excited states vibrational modes of chlorophylls in photosynthesis.
Abstract of the thesis project
Photosynthesis empowers the entire biosphere and is arguably the most important biological process on earth. The quantum efficiency of energy transfer in photosynthetic light-harvesting complexes can reach almost unity. This high efficiency is even more puzzling if we take into account that the high excitation energy transfer through hundreds of pigments in a disordered energetic landscape cannot be explained with the current models. We aim to create an interdisciplinary project in order to shed light on the mechanisms controlling energy transfer. We will combine biochemical techniques for protein purification, theoretical modeling, and biophysical techniques like 2DES and FSRS for evaluating excitation energy transfer, the configuration of excited states of the pigments, and intermolecular interactions. The goal of this project will be to create a robust model capable to describe the ultrafast excitation energy transfer in photosynthetic systems.
Energy harvesting, photosynthesis, control of excited states
The objective of this project is to provide a model capable to explain the ultrafast excitation energy transfer.
Photosynthesis is the process that converts the energy of solar photons into an electrochemical potential that is used to reduce CO2 to sugar. This process is of the utmost importance for our planet as it converts 90 TW of solar energy into biomass and oxygen. During the first steps of photosynthesis, two ultrafast processes occur in which the photon energy is converted into potential chemical energy: light-harvesting complexes first absorb solar photons, and then, a cascade of energy transfer leads the resulting excitation energy to a reaction center in about 20 picoseconds. All these steps occur in specialized proteins binding large amounts of photosynthetic cofactors (carotenoid and chlorophyll molecules). The primary steps of photosynthesis thus generally involve carotenoid and chlorophyll excited states. Amazingly, in optimal conditions, this process, which involves hundreds of excitation transfer steps, occurs at much faster timescales than predicted by theory with a quantum yield close to unity.
Ultrafast time-resolved absorption and 2D electronic spectroscopy (2DES) experiments have shown that the pigment-pigment excitation energy transfers and the first steps of charge separation are modulated by undulations (beats) over more than 2 ps. This was firstly interpreted as beats between coherently coupled states, suggesting that the excitation energy is probing more than a path at once. It was proposed that these quantum beats enter in resonance with vibrational modes of the cofactors, which help their survival over many picoseconds. However, the apparent matching between these beats and the vibrational modes of the cofactors is interpreted by other authors as arising from ‘vibrational assistance’. In this alternative model, the intersection between the excited state energy surface corresponds to the energy of a vibrational mode, and the oscillations observed originate from the movement of the wavepacket in the vibrational excited sub-state of the excited electronic level. The debate is still running, but not a single experiment could show that indeed the existence of quantum beats (or vibrational assistance) helps increase the rates of energy transfers.
There are two main challenges for validating excitation energy transfer models in photosynthetic systems. First, the current models are made adhoc for the few studied systems so it is necessary to probe them against perturbed systems. Second, no information exists on the structure of the involved excites states. In the absence of this information, it is impossible to corroborate the complex quantum modelling of these pigments, and thus to support any of the two current hypotheses (quantumness vs vibrational assistance).
This project aims to overcome these challenges by using as a protein model light harvesting complex II (LHCII). The LBMS group has shown recently that depending on the purification method, this protein can be in different light harvesting conformations, or even in dissipating energy mode. Thanks to Raman spectroscopy, it is known exactly the pigments with perturbed energy levels. Hence, this offers and unique opportunity to test different theories on the same protein-pigment complexes with identified energetic changes. The excitation energy transfer of the different LHCIIs will be characterized by ultrafast time-resolved absorption and 2D electronic spectroscopy (2DES). This data will already give us critical information about the validity of current models. However, it is necessary to have the structural information of excited states of the cofactors to determine the role and nature of vibrations and quantumness in the process. Presently, Femtosecond Stimulated Raman Spectroscopy (FSRS) is the only method that can provide the information to achieve the description of excited states structure, configuration, and intermolecular interactions with time resolutions in the 100s of femtoseconds. This method has not been yet applied in this field due to the complexity to implement in multi-chromophoric, fragile, photosynthetic complexes. The combination of the data obtained from FSRS, ultrafast TA, and 2DES with quantum modeling methods will allow us to obtain a precise picture of the organization of the wavefunctions in the excited states.
Pigment extraction and protein purification (LBMS-Paris Saclay/UNAM). Light-harvesting complex II (LHCII) is the most abundant antenna in oxygenic photosynthesis. It is found in a trimeric form with each monomer containing 14 chlorophylls (8 Chl-a, 6 chl-b) and 4 carotenoids (2 lutein, 1 violaxanthin, and 1 neoxanthin). First, we will extract the chlorophyll pigments to characterize the vibrational properties of each of them isolated. Then, we will isolate LHCII trimers following different routes that will yield two conformations of LHCII in harvesting mode, and one conformation in dissipation mode. The student will be trained on the necessary biochemical methods to extract and purify LHCII which LBMS is proficient and to extract and handle chlorophylls pigments by the UNAM.
Ultrafast time-resolved absorption (LBMS-Paris Saclay/UNAM). We will measure the excitation energy transfer in the different variations of LHCII. This will yield important information on changes in rates and pathways of the excitation energy. It will also show us the absorption region and time windows of the excited states, which are essential to perform efficiently FSRS. The student will be trained in performing these experiments and interpreting the results.
Femtosecond Stimulated Raman Spectroscopy (FSRS) (LBMS-Paris Saclay). We will characterize first the vibrational properties of the excited states of the isolated pigments (chlorophylls). There are a few reports characterizing vibrational properties of excited states of chlorophylls, but not all of the pigments present in LHCII. Then, we will access to vibrational properties of chlorophyll and carotenoid excited states in the different LHCII proteins. The student will be trained in performing these experiments and interpreting the results.
2D electronic spectroscopy (2DES) (UNAM). We will perform 2DES on the three different LHCII trimers to compare the changes in electronic coupling and energy transfer pathways in the tens of fs. The student will be trained in performing these experiments and interpreting the results.
Theoretical modeling (Vilnius University). The results obtained will be used to generate advanced models describing the ultrafast excitation energy transfer observed in the photosynthetic antenna. The student will be involved in the discussion with the Lithuanian group for the creation of this model.
The main outcome of this project would be a very significant advance in our understanding of the solar energy conversion during the early steps of photosynthesis.
Bibliography
Croce, R. and H. van Amerongen, Natural strategies for photosynthetic light harvesting. Nat. Chem. Biol., 2014. 10, 492-501 DOI: 10.1038/nchembio.1555.
Liu, Z., et al., Crystal structure of spinach major light-harvesting complex at 2.72 A resolution. Nature, 2004. 428, 287-292 DOI: 10.1038/nature02373.
Romero, E., et al., Quantum design of photosynthesis for bio-inspired solar-energy conversion. Nature, 2017. 543, 355-365 DOI: 10.1038/nature22012.
Zigmantas, D., et al., Ultrafast laser spectroscopy uncovers mechanisms of light energy conversion in photosynthesis and sustainable energy materials. Chemical Physics Reviews, 2022. 3, 041303 DOI: 10.1063/5.0092864.
Cao, J., et al., Quantum biology revisited. Science Advances, 2020. 6, eaaz4888 DOI: 10.1126/sciadv.aaz4888.
Pascal, A.A., et al., Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature, 2005. 436, 134-137.
Li, F., et al., A new, unquenched intermediate of LHCII. J. Biol. Chem., 2021. 296, 100322 DOI: 10.1016/j.jbc.2021.100322.
Llansola-Portoles, M.J., et al., Tuning antenna function through hydrogen bonds to chlorophyll a. Biochim. Biophys. Acta, Bioenerg., 2019. 1861, 148078 DOI: 10.1016/j.bbabio.2019.148078.