Structure and function of photochemical reaction centers

This is a research proposal that I submitted as part of an application for a position as an assistant professor at IST Austria. I was not shortlisted: evidently, they did not understand my awesomeness.

Structure and function of photochemical reaction centers

Research proposal for the position of Assistant Professor at IST Austria

Dr. Tanai Cardona

Department of Life Sciences, Imperial College London

London, UK

Why photosynthesis research at IST Austria

Life on earth is sustained by photosynthesis. Civilization depends on it because photosynthesis is the source of our food and gave rise to the fuels we burn to power our society. Scientists world-wide are trying to improve photo­synthesis to enhance crop productivity and thus feed a rapidly growing global population^1,2. At the same time, scientists are looking for ways to engineer photosynthetic organisms to produce clean energy alternatives to fossil fuels or high-value products, like plastics or pharmaceuticals³. Intensive research is also carried out to develop synthetic compounds that mimic natural photosynthesis to generate fuels directly from sunlight and water⁴, or to sequester carbon as a promising strategy to combat climate change⁵. As a result, the study of photosynthesis now is more relevant than ever before, as it is at the heart of many solutions to overcome current global challenges. However, our understanding of photosynthesis is enormously biased (Figure 1); our knowledge is based on just a handful of model plants and a few strains of bacteria. These are a very tiny fraction of the entire biodiversity of photosynthetic systems that abound in the planet and that still remains largely unexplored. It is this potential for breakthrough and discovery that excites me, it is the main reason I decided to pursue a career doing photosynthe­sis research when I was still an undergrad student, and it is the reason why I want to lead my own research group today.

Figure 1. This graph shows how little we know about photosynthetic systems. In the past 5 years there have been more than 20000 publications on Arabidopsis alone, a model plant system. Arabidopsis and Spinacia are plants; Synechocystis, Thermosynechococcus, and Gloeobacter, are model strains of Cyanobacteria. Rhodobacter and Blastochlororis are strains of Proteobacteria (purple bacteria). Together these strains make for 98.5% of all the publications accessible with PubMed by searches using the genus name of “the most popular” model organisms of each group. Chloroflexus and Roseiflexus are strains of Chloroflexi (green nonsulfur bacteria); Chlorobaculum and Chlorobium are strains of Chlorobi (green sulfur bacteria); Heliobacterium and Heliobacillus are strains of Firmicutes (heliobacteria); Chloracidobacterium and Gemmatimonas are newly discovered types of phototrophs belonging to the phylum Acidobacteria and Gemmatimonadetes, respectively. The data was retrieved September 2015.

Photosynthesis is possible thanks to photochemical reaction centers (Figure 2). These are molecular machines that transform the energy of light into chemical energy. In other words, nanoscaled photovoltaic solar panels found within the photosynthetic cell. They are fascinating because of their complexity, variety of forms, specialized chemistry, and incredible technological potential. As a result, I plan to build a multidisciplinary research group that combines biochemistry, biophysics, structural, evolutionary, and synthetic biology to study and exploit photochemical reaction centers and photosynthesis. In particular, focusing on those photosynthetic systems that are still very poorly understood. I propose here three research modules that I will develop at IST Austria:

1.       Functional and structural characterization of  photochemical reaction centers

2.       Design, remodeling, and enhancement of photosynthetic systems

3.       Reconstructing the origin and evolution of photosynthesis

Figure 2. Photochemical reaction centers from Cyanobacteria. Both are transmembrane multiprotein complexes carrying hundreds of cofactors, included chlorophylls, carotenoids, lipids, hemes, FeS clusters, among others. Type II reaction centers are distinguished because they lack FeS clusters. In this system after light absorption and charge separation, electrons are shuttled to quinones molecule (e.g. menaquinone, ubiquinone, plastoquinone). After it is reduced the quinone is released to the membrane to be oxidized by a Cytochrome bc₁ or b₆f complex. This contributes to the formation of a proton gradient and ATP synthesis. Among Type II reaction centers, Photosystem II is unique because of the water oxidizing complex, were oxygen evolution occurs. Type I reaction centers are characterized for having FeS clusters as terminal electron acceptors. After charge separation, the FeS clusters are oxidized by a ferredoxin, which then can go on to power metabolism (e.g. carbon or nitrogen fixation).

Photochemical reaction centers are thought to have originated only once in the domain Bacteria. This is because currently there are no described strains in the domain Archaea with photosynthesis based on protein complexes containing chlorophyll or bacteriochlorophyll^6,7. At the other end of the tree of life, eukaryotic algae and plants obtained photosynthesis via the endosymbiosis of a cyanobacterium. Within Bacteria, there are currently seven phyla known to have strains with reaction centers, these are: Cyanobacteria, Chloroflexi, Firmicutes, Chlorobi, Proteobacteria, and those recently found in Acidobacteria⁸ and Gemmatimonadetes⁹. Just a while ago, it was suggested that the phylum Actinobacteria might have been ancestrally capable of photosynthesis¹⁰, as some strains in this phylum seem to have a vestigial chlorophyll synthesis pathway. So today, 95% of research in photosynthesis is done in the Cyanobacteria/plant system, and less than 5% in the remaining types of phototrophic systems, as determined by the number of publications in the last five years (Figure 1). From this 5%, 3.5% is research performed in a single strain of Alphaproteobacteria, Rhodobacter spheroides, probably best known for the 1988 Nobel Prize in Chemistry. Therefore, we know virtually nothing about most of the diversity of photosynthetic organisms currently inhabiting Earth.

Even though the less studied groups of phototrophs represent, at best, less than 1.5% of the total photosyn­thesis research carried out in the world, there is a lot to be learnt from them. For example, quantum coherence in living systems was first discovered in the FMO light harvesting complex of the Chlorobi¹¹, and potential applications of this quality may provide some insight to develop more efficient solar cells¹². Strains of the phylum Chloroflexi have a unique car­bon fixation pathway, unlike those in plants and Cyanobacteria, which is thought to be more energy efficient under specific conditions¹³. Efforts to engineer this pathway in a model cyanobacterium have been attempted recently¹⁴. Heliobacteria are ubiquitous and powerful nitrogen fixers, commonly found in rice paddies around the world¹⁵. They are some of the fastest growing phototrophic bacteria in nature; however, their ecological importance has not been determined and their biotechnological potential has not even been acknowledged in the literature. In conclusion, the entire diversity of photosynthetic bacteria represents a new frontier of research, that if pursued, will certainly have a far-reaching societal and technological impact.

Table 1. Phototrophic groups and some facts regarding crystal structures of reaction centers (RC).

PHYLUM	RC	STRUC-TURESa	SOURCES	BEST	INTERESTING TRAITS
Cyanobacteria and plastids	I	10	T. elongatus S. sp. 6803 Pea	2.5 Å16	Photosystem I comes in monomers, trimers, and tetramers
	II	15	T. elongatus T. vulcanus	1.9 Å17	The Mn4CaO5 cluster and water oxidation
Proteobacteria Purples	II	~100	R. sphaeroides B. viridis T. tepidum	1.8 Å18	Nobel Prize
Firmicutes Heliobacteria	I	0	―	―	It is made of a single subunit
Chlorobi Green sulfur	I	0	―	―	Chlorosomes and the Fenna-Matthews-Olson complex
Acidobacteria Chloracidobacterium	I	0	―	―	12 membrane-bound carotenoid protein subunits surround the complex
Chloroflexi Green non-sulfur	II	0	―	―	Three-pheophytin system
Gemmatimonadetes Gemmatinonas	II	0	―	―	Obtained RC via horizontal gene transfer recently
Actinobacteria Rubrobacter	―	―	―	―	Only has a vestigial chlorophyll synthesis pathway

^aCrystal structures with resolution better than 4.0 Å. Less than 1% of all protein structures in the PDB (Protein Data Bank) are from phototrophic organisms.

Functional and structural characterization of photochemical reaction centers

As mentioned above, reaction centers are distributed in at least seven groups of distantly related bacteria. They come in two forms distinguished by the primary photochemical steps and known as Type I and Type II reaction centers (Figure 2). Today, there are crystal structures available for reaction centers in Cyanobcateria, plants, and in Proteo­bacteria, but none in the other phototrophic systems (Table 1). I will therefore focus on strains that are very poorly understood; namely, Heliobacterium modesticaldum (Heliobacteria), Roseiflexus castenholzii (Chlor­oflexi), and Chlorobium tepidum (Chlorobi). Nonetheless, I already have in my laboratory 8 additional strains selected because of their remarkable reaction centers that I could bring with me to Austria.

I have selected those three targets because their function and structure still remains to be elucidated. Moreover, they contain interesting evolutionary information. For example, the Type I reaction center from Heliobacteria and the Chlorobi are the simplest in nature and might be structurally similar to the earliest evolving systems. However, their fundamental chemistry still remains a puzzle because the role of quinones in electron transfer has not been conclusively demonstrated. It is possible that under certain conditions these Type I reaction centers may behave like Type II instead. Demonstrating that these simple reaction centers could have a dual function would be a fantastic discovery that could change the way we think about photosynthesis. On the other hand, the reaction center from Roseiflexus has a unique protein domain not seen in any other proteins that could give clues on how water oxidation catalysis evolved in Cyanobacteria⁶.

This proposal is an expansion of an independent re­search program I started as a Research Fellow about two years ago here at Imperial. Experimentally the reaction centers will be studied in vivo, in isolated membranes, and in the purified enzyme. For example, excitation energy and electron transfer under different conditions will be measured; as well as any alterations to the energetics of cofactors, protein composition, or oligomeric forms. Changes to the photosynthetic machinery under stress conditions (e.g. high light intensity, iron or nitrogen starva­tion) will be monitored too. The results will be compared to those in Cyanobacteria for which extensive data is available. The key objective is to have a clear picture of the function and dynamics of the reaction center in the target strains, a picture that is not yet available to a satisfactory level of detail, if at all. Simultaneously, crystal trials will be initiated as purified enzymes become available.
I have experience purifying the reaction center from H. modesticaldum, Photosystem II, and Photosystem I. I also have experience with various spectroscopic methods such as, absorption, fluorescence, and electron paramagnetic resonance (EPR) spectroscopy; in addition to gel-based and gel-free proteomic approaches and in the application of electrochemical methods to reaction centers. These techniques will be applied judiciously in order to study function as deemed necessary.

Currently, I am optimizing crystallization conditions for the reaction center from H. modesticaldum; pre­liminary data suggests promising conditions. These conditions and those available for Photosystem I¹⁶ could be used as a starting base for the structure of Chlorobium. An attempt at crystallizing the reaction center from Chlor­o­flexus aurantiacus was published 20 years ago, but a structure was never re­leased¹⁹. This protocol could be further improved for the structure of Roseiflexus. Alternatively, modifications to available meth­ods to crystallize Photosystem II¹⁷ or the proteobacterial reaction center¹⁸ could be tried as well. Another possibility is to obtain structural models using electron microscopy. The complete characterization and structural determination of one or two of the reaction center shall make for a very exciting PhD project or postdoctoral position, which should pro­vide extensive results for multiple high-impact publications.

Design, remodeling, and enhancement of photosynthetic systems

We require a new source of energy. Biofuels from photosynthetic organisms have been considered to be part of the solution to the energy crisis, but one of the grand challenges is that overall, the efficiency of photosynthesis is low³. This is because the solar to biomass energy conversion efficiency is around 1% or less (in real life, not under optimal laboratory conditions). In other words, the ratio of en­ergy returned in the biofuel relative to the energy invested to produce it is currently quite unfavorable, even in the best case scenarios. As a result, it has been hypothesized that the natural limits of photosynthesis could be enhanced or overcome^20,21, but no experimental validation of such hypotheses has been provided yet. All of the approaches proposed to improve photosynthesis in living system require genetic engineering. For example, it has been suggested that a reaction center could be engineered to absorb light in the far-red region beyond PAR (photosyn­thetically active radiation), and this could potentially double its photosynthetic efficiency. Such approach requires: 1) the expression of new pigment synthesis pathways in parallel to the native ones, 2) the expression of a new reaction center from a distinct organism into the host strain, or 3) both 1) and 2) at the same time. However, we still do not completely understand pigment synthesis and reaction center biogenesis. Although great advances have been made in the past decades, still some of the steps, enzymes in the pathway, and assembly factors, have not been identified or are very poorly characterized^22,23.

            I propose here a novel strategy―not yet discussed in the literature―to get great insight into how correctly engineer a photosynthetic system and consequently, how to improve it. The first stage of this module is to engineer photosynthesis in a heterotrophic bacterium; or in other words, to reverse engineer photosynthesis from scratch. My group will transfer a photosynthetic gene cluster from a phototrophic gammaproteobacterium to Escherichia coli, which is also a gammaproteobacterium. Genetically, they should be somewhat alike. Similar approaches have been attempted before to engineer N₂-fixation in E. coli successfully²⁴. To do this, a nitrogenase gene cluster from Klebsiella oxytoca containing about 20 genes was refactored and then transferred into E. coli. K. oxytoca is also a gammaproteobacterium. The photosynthetic gene cluster varies in size from organism to organism ranging from 15 to 25 genes. Therefore, this technology could be used as a starting foundation. We will take it several steps further.

In addition, photosynthetic gammaproteobacteria are known to be good at horizontally transferring genes (HGT) in nature: for exam­ple, Cyanobacteria of the marine Synechococcus/Prochlorococcus clade have obtained numerous photosynthetic genes from Gammaproteobacteria, including circadian clock components²⁵, carboxysome components and Ru­bisco²⁶, summed to chlorophyll synthesis genes²⁷, among many others²⁸. Gammaproteobacteria have also been shown to donate a photosynthetic gene cluster to strains of the rare phylum Gemmatimondetes, and these have been demonstrated to be able to express functional reaction centers⁹. Evidence for HGT of photosynthesis genes from Gammaproteobacteria to strains of Firmicutes of the genus Alkalibacterium has also been provided²⁹, but this result still awaits further experimental validation. HGT events between organisms of different phylum should be much more difficult to occur than within more closely related bacteria. Furthermore, several phototrophic gammaproteobacte­rial genomes are publicly available and some strains are amenable to cultivation and genetic engineering. I will start with the photosynthesis gene cluster of Thiocapsa roseopercisina; an anoxygenic photosynthetic gammapro­teobacterium, which has been of particular interests because of its O₂-tolerant hydrogenase³⁰.

            If functional reaction center can be engineer as a proof-of-concept in a non-phototrophic bacterium, the possibilities to follow this up are limitless. First, selected genes in the cluster could be removed or new ones added, in order to find the minimum necessary genetic requirements for phototrophy and photoautotrophy. Combination of genes from different organisms could be mixed into novel gene clusters to test whether functionality or activity yields could be improved or not. In addition, the gene cluster could be inserted into strains of E. coli that have already been engineered to produce diverse biofuels or compounds of interest. Like this, it could be possible to test whether production of the desired compound can be enhanced by the acquisition of phototrophy. A step farther would consist in expressing a photosynthetic gene cluster in a yeast model. However, the ultimate goal is to transfer a photosynthetic gene cluster encoding the capacity for oxygenic photosynthesis from Cyanobacteria. A gene cluster for oxygenic photosynthesis does not exist in nature, so it would be 100% artificially designed. In this case, the engineered strain would use water and light as the main energy source and thus would be completely photoautrotrophic. This project, though risky, will provide invaluable insight into the nature of photosynthesis and teach us immeasurably on the creation of novel life forms.

           

Reconstructing the origin and evolution of photosynthetic systems

Another one of my personal scientific interests is evolution. How photosynthesis originated and diversified remains one of the greatest puzzles in the history of life. I have set myself the personal goal to reconstruct the most detailed evolutionary scenario yet for the origin and diversification of photosynthesis. I have made good progress towards this with my publications in the past four years^6,7,31. Earlier this year, I published a major reassessment of the evolution of reaction centers⁶. In addition, I led and published an exhaustive phylogenetic study of the D1 protein of Photosystem II, which provided for the first time, a clear picture of how the water oxidizing complex of oxygenic photosynthesis evolved and the dramatic transitions Photosystem II underwent in its path to acquiring water oxidation catalysis³¹. My work demonstrated how the structural and functional data available for Photosystem II can be used to gain evolutionary information at an unprecedented level of detail, if integrated with powerful phylogenetic analysis. I am currently performing molecular clock analysis of reaction center proteins to time the origin of photosynthesis and to date important evolutionary events, such as the origin of the water oxidizing complex of Photosystem II. As structural and functional information from the targeted strains become available in my group, these will be used to create even more precise molecular evolutionary models.

          At IST Austria I also plan to extend these evolutionary studies to the evolution of several major cofactor synthesis pathways relevant to photosynthesis: namely, the chlorophyll, heme, quinone, and carotenoid biosynthesis pathways. Understanding the evolution and extent of the current diversity of cofactor biosynthetic pathway could come in handy when redesigning and refactoring the photosynthetic gene clusters. It could inform us on what genes or strains could be most promising. This would be harder to achieve if a good understanding of the diversity and evolution of phototrophy is lacking.

Although my research program at Imperial is set within an evolutionary context, I intend to develop and lead a more comprehensive and multidisciplinary research profile at the next stage of my career. I aim to branch from purely fundamental research into more applied biotechnological fields.

References

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2        Long, S.P., Marshall-Colon, A. & Zhu, X. G. Meeting the global food demand of the future by engineering crop photosynthesis and yield potential. Cell (2015) 161, 56-66.

3        Cotton, C. A., Douglas J. S., De Causmaecker, S., Brinkert, K., Cardona T., et al. Photosynthetic constraints on fuel from microbes. Front Bioeng Biotechnol (2015) 3, 36, doi: 10.3389/fbioe.2015.00036.

4        Faunce, T. et al. Artificial photosynthesis as a frontier technology for energy sustainability. Energ Environ Sci (2013) 6, 1074-1076.

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6        Cardona, T. A fresh look at the evolution and diversification of photochemical reaction centers. Photosynth res (2015) 126, 111-134.

7        Cardona, T., Sedoud, A., Cox, N. & Rutherford, A. W. Charge separation in Photosystem II: A comparative and evolutionary overview. BBA-Bioenergetics (2012) 1817, 26-43,.

8        Bryant, D. A. et al. Candidatus Chloracidobacterium thermophilum: An aerobic phototrophic acidobacterium. Science (2007) 317, 523-526.

9        Zeng, Y. H., Feng, F. Y., Medova, H., Dean, J. & Koblizek, M. Functional Type 2 photosynthetic reaction centers found in the rare bacterial phylum Gemmatimonadetes. PNAS (2014) 111, 7795-7800.

10      Gupta, R. S. & Khadka, B. Evidence for the presence of key chlorophyll-biosynthesis-related proteins in the genus Rubrobacter (Phylum Actinobacteria) and its implications for the evolution and origin of photosynthesis. Photosynth res, doi:10.1007/s11120-015-0177-y (2015).

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17      Umena, Y., Kawakami, K., Shen, J. R. & Kamiya, N. Crystal structure of oxygen-evolving Photosystem II at a resolution of 1.9 Å. Nature (2011) 473, 55-60.

18      Xu, Q. et al. X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb. sphaeroides; altered proton transfer pathways. Structure (2004) 12, 703-715.

19      Feick, R., Ertlmaier, A. & Ermler, U. Crystallization and X-ray analysis of the reaction center from the thermophilic green bacterium Chloroflexus aurantiacus. FEBS lett (1996) 396, 161-164.

20      Blankenship, R. E. et al. Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement. Science (2011) 332, 805-809.

21      Ort, D. R. et al. Redesigning photosynthesis to sustainably meet global food and bioenergy demand. PNAS (2015) 112, 8529-8536.

22      Komenda, J., Sobotka, R. & Nixon, P. J. Assembling and maintaining the Photosystem II complex in chloroplasts and cyanobacteria. Curr Opin Plant Biol (2012) 15, 245-251.

23      Chen, M. Chlorophyll modifications and their spectral extension in oxygenic photosynthesis. Annu Rev Biochem (2014) 83, 317-340.

24      Smanski, M. J. et al. Functional optimization of gene clusters by combinatorial design and assembly. Nat Biotechnol (2014) 32, 1241-U1104.

25      Dvornyk, V. Subfamilies of cpmA, a gene involved in circadian output, have different evolutionary histories in cyanobacteria. Microbiol-Sgm (2006) 152, 75-84.

26      Marin, B., Nowack, E. C. M., Glockner, G. & Melkonian, M. The ancestor of the Paulinella chromatophore obtained a carboxysomal operon by horizontal gene transfer from a Nitrococcus-like gammaproteobacterium. BMC Evol Biol (2007) 7, doi: 10.1186/1471-2148-7-85.

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28      Zhaxybayeva, O., Doolittle, W. F., Papke, R. T. & Gogarten, J. P. Intertwined evolutionary histories of marine Synechococcus and Prochlorococcus marinus. Genome Biol Evol (2009) 1, 325-339.

29      Perreault, N. N. et al. Heterotrophic and autotrophic microbial populations in cold perennial springs of the high arctic. Appl Environ Microb (2008) 74, 6898-6907.

30      Maroti, G. et al. Discovery of [NiFe]-hydrogenase genes in metagenomic DNA: Cloning and heterologous expression in Thiocapsa roseopersicina. Appl Environ Microb (2009) 75, 5821-5830.

31      Cardona, T., Murray, J. W. & Rutherford, A. W. Origin and evolution of water oxidation before the last common ancestor of the cyanobacteria. Mol Biol Evol (2015) 32, 1310-1328