Systematic investigation on the energy transfer mechanisms occurring within light-harvesting complexes of the purple bacterium acidophilus Nery, E.¹*, Jones A.R.², Olaya-Castro A.³, and Jimenez J.I. ¹

¹ Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK. ² National Physical Laboratory, Teddington, UK. ³ Department of Physics and Astronomy, University College London, London, UK. *Corresponding author: [email protected]

Abstract: During the light dependent reactions of photosynthesis of purple , light harvesting (LH) complexes absorb and direct energy towards a reaction center with an efficiency of approximately 95% [1]. One of the hypotheses explaining this phenomenon argues that such high efficiency is achieved thanks to a quantum coherent state occurring between the bacteriochlorophyll molecules enclosed within these proteins. In the present study we will genetically modify the scaffold of LHs expressed by Rhodoblastus acidophilus for tuning the interactions occurring between their pigment molecules. The energy states of the proteins produced will then be calculated and compared with those corresponding to the wild type. The conclusions obtained in this project will constitute a foundation for experiments aiming at understanding whether quantum coherence plays a non-trivial role in photosynthesis. Moreover, it represents the first step in the development of high-performance technologies for sunlight harvesting.

Introduction Project Outline and Objectives How could quantum effects persist inside a living cell? It is thought that in this case, the protein scaffold would protect the Quantum Coherence in Photosynthesis “quantumness” and even enhance the coherent lifetimes of the system by tuning the interactions occurring between the pigment molecules [5]. We intend to reinforce this hypothesis by creating mutants of Rhodoblastus acidophilus expressing many LH2 variants and Done During the light-dependent reactions of photosynthesis, specialized membrane proteins containing pigment molecules work observing how the photosynthetic efficiency is affected. Moreover we will be calculating the energy states of some successful mutants by absorbing photons and funnelling the respective energy until a reaction centre (RC) where charge separation occurs. and analyse how it deviate from those obtained for the wild-type. Work in progress Traditional hypotheses postulate that the energy absorbed is transferred randomly by resonance between pigments until it Identifying active Light box Development of a eventually reaches the RC. The main critic against this hypothesis however, is that during this process the energy can easily be pucBA genes design mathematical model* To do lost to the environment, a fact that does not agree with the efficiency by which this phenomenon is known to occur in nature. Genome For trying to explain this paradox it was developed a new hypothesis based in quantum coherent effects. In this hypothesis, the sequencing and energy would be efficiently transferred until the RC by experimenting many pathways simultaneously (fig.1). annotation

Photosynthetic Development of Creation of a Calculation of efficiency analysis The traditional hypothesis: Random walk molecular tools deletion mutant energy states In this hypothesis the energy absorbed would be transferred randomly between pigment molecules until it finally reaches the RC. Literature Major checkpoint! review Creation of mutants Obtaining expressing LH2 absorption Comparison with the The Quantum walk *Developed by our collaborator at In this case the energy absorbed would lead to coupling variants spectra wild-type the University of Pisa, Benedetta between two or more pigment molecules, which means that Mennucci this energy would be delocalized within the protein. This coupling is responsible to what we call quantum coherence and would lead to the transfer of energy through different MAIN IMPACTS: 1. Understanding if and how quantum effects can persist within living cells and is evolutionary favoured; 2. Contribute to the development of high-performance technologies Fig.1. Quantum coherence in photosynthesis. Pictures extracted from the scientist paths simultaneously. for sunlight harvesting and other devices based on the transfer of energy. magazine (June, 2019).

Light-harvesting in Rhodoblastus acidophilus Work in Progress… References [1] Fleming, G. R., van Grondelle, R. (1997). Femtosecond spectroscopy of photosynthetic light-harvesting systems. Current opinion in structural biology, 7(5), 738-748. The absorption spectra of whole cells growing in our lab was obtained (fig. 3). 0.7

The light-harvesting complex 2 (LH2) of the photosynthetic Based in the literature, it could be identified spectral signatures of the light- 0.6 [2] Harel, E., Engel, G. S. (2012). Quantum coherence spectroscopy reveals complex purple bacterium Rhodoblastus acidophilus is composed by nine harvesting complexes being expressed under anaerobic conditions. Using this data, 0.5 dynamics in bacterial light-harvesting complex 2 (LH2). Proceedings of the National Academy of Sciences, 109(3), 706-711. protomers arranged symmetrically around a central axis. Each a light box that will be used during the next steps of the project could be designed. 0.4 0.3 protomer is composed by two subunits, the α and β-chains, which The variations in the absorption spectra obtained for the mutants that are being Absorbance [3] Scholes, G. D., Gould, I. R., Cogdell, R. J., Fleming, G. R. (1999). Ab initio molecular 0.2 orbital calculations of electronic couplings in the LH2 bacterial light-harvesting complex of enclose three bacteriochlorophylls (bchls) and one carotenoid created is the parameter that will allow the calculation of the energy states of their Rps. acidophila. The Journal of Physical Chemistry B, 103(13), 2543-2553. molecule. The strain possess many copies of the genes coding for LH2s which will be then compared with those obtained for the wild-type. 0.1 0 [4] Alden, R. G., Johnson, E., Nagarajan, V., Parson, W. W., Law, C. J., Cogdell, R. G. (1997). the LHs subunits (pucAB) which expression varies depending on the 650 700 750 800 850 900 950 1000 Wavelenght (nm) Calculations of Spectroscopic Properties of the LH2 Bacteriochlorophyll− Protein Antenna environmental conditions. Complex from Rhodopseudomonas Acidophila. The Journal of Physical Chemistry B, 101(23), 4667-4680. Fig.3. Absorption spectra obtained at the of whole cells growing anaerobically. The detail at the top right shows the The LH2 works by absorbing energy from sunlight and funnelling bacterium growing on Modified Pfenning agar under anaerobic conditions. [5] Ishizaki, A., Calhoun, T. R., Schlau-Cohen, G. S., Fleming, G. R. (2010). Quantum coherence and its interplay with protein environments in photosynthetic electronic energy it until another complex, the light harvesting complex 1 which transfer. Physical Chemistry Chemical Physics, 12(27), 7319-7337. contains the RC (LH1/RC). Even though strong coupling between its One of the biggest challenges faced during the accomplishment of this project is [6] Martínez‐García, E., de Lorenzo, V. (2011). Engineering multiple genomic deletions in pigments [2] and quantum signatures [3, 4] have already been Gram‐negative bacteria: analysis of the multi‐resistant antibiotic profile of Pseudomonas detected, the mechanism of energy transfer occurring within these the lack of molecular tools available for modifying bacterium in study. Based some putida KT2440. Environmental microbiology, 13(10), 2702-2716. systems still not well understood and is subject of intense debate. preliminary results (antibiotics resistance, growth rate and optimal growth conditions…) and what has been published regarding the genetic modification of similar purple bacteria, a protocol for transforming the by electroporation could be Project Funded finally developed. However, no mutants could be obtained until the present by: Fig.2. Light harvesting in Rhodoblastus acidophilus. Top left: LH2 of Rdb. Acidophilus observed from the top showing the ring of bchls absorbing at 800nm in red and at 850nm in blue Top right: Protomer composed by subunits α and β moment. The first system employed for deleting one of the pucBA genes was based enclosing the bchls and the carotenoid molecules (extracted from: O’Rilley PhD thesis, 2014). Bottom: Part of a in homologous recombination (pEMG/PSW-I system, ref. 6) and failed many times. photosynthetic unit (PSU) of Rdb. Acidophilus showing the energy pathway trough the light harvesting complexes until Fig.4. Design of the CRISPR interference system. Extracted from Qi et al., the RC (extracted from Hu et al., 1998). 2013 Our next trial will employ a variant of the dCas9 which lacks nuclease activity (fig. 4).