Recent advances in the structural diversity of reaction centers.

Photosynthetic reaction centers (RC) catalyze the conversion of light to chemical energy that supports life on Earth, but they exhibit substantial diversity among different phyla. This is exemplified in a recent structure of the RC from an anoxygenic green sulfur bacterium (GsbRC) which has characteristics that may challenge the canonical view of RC classification. The GsbRC structure is analyzed and compared with other RCs, and the observations reveal important but unstudied research directions that are vital for disentangling RC evolution and diversity. Namely, (1) common themes of electron donation implicate a Ca2+ site whose role is unknown; (2) a previously unidentified lipid molecule with unclear functional significance is involved in the axial ligation of a cofactor in the electron transfer chain; (3) the GsbRC features surprising structural similarities with the distantly-related photosystem II; and (4) a structural basis for energy quenching in the GsbRC can be gleaned that exemplifies the importance of how exposure to oxygen has shaped the evolution of RCs. The analysis highlights these novel avenues of research that are critical for revealing evolutionary relationships that underpin the great diversity observed in extant RCs.

On the origin of oxygenic photosynthesis and Cyanobacteria.

Oxygenic phototrophs have played a fundamental role in Earth's history by enabling the rise of atmospheric oxygen (O2 ) and paving the way for animal evolution. Understanding the origins of oxygenic photosynthesis and Cyanobacteria is key when piecing together the events around Earth's oxygenation. It is likely that photosynthesis evolved within bacterial lineages that are not extant, so it can be challenging when studying the early history of photosynthesis. Recent genomic and molecular evolution studies have transformed our understanding about the evolution of photosynthetic reaction centres and the evolution of Cyanobacteria. The evidence reviewed here highlights some of the most recent advances on the origin of photosynthesis both at the genomic and gene family levels.

Thylakoid membrane function in heterocysts.

Multicellular cyanobacteria form different cell types in response to environmental stimuli. Under nitrogen limiting conditions a fraction of the vegetative cells in the filament differentiate into heterocysts. Heterocysts are specialized in atmospheric nitrogen fixation and differentiation involves drastic morphological changes on the cellular level, such as reorganization of the thylakoid membranes and differential expression of thylakoid membrane proteins. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase by developing an extra polysaccharide layer that limits air diffusion into the heterocyst and by upregulating heterocyst-specific respiratory enzymes. In this review article, we summarize what is known about the thylakoid membrane in heterocysts and compare its function with that of the vegetative cells. We emphasize the role of photosynthetic electron transport in providing the required amounts of ATP and reductants to the nitrogenase enzyme. In the light of recent high-throughput proteomic and transcriptomic data, as well as recently discovered electron transfer pathways in cyanobacteria, our aim is to broaden current views of the bioenergetics of heterocysts. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Origin and Evolution of Water Oxidation before the Last Common Ancestor of the Cyanobacteria.

Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages toward the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria.

A fresh look at the evolution and diversification of photochemical reaction centers.

In this review, I reexamine the origin and diversification of photochemical reaction centers based on the known phylogenetic relations of the core subunits, and with the aid of sequence and structural alignments. I show, for example, that the protein folds at the C-terminus of the D1 and D2 subunits of Photosystem II, which are essential for the coordination of the water-oxidizing complex, were already in place in the most ancestral Type II reaction center subunit. I then evaluate the evolution of reaction centers in the context of the rise and expansion of the different groups of bacteria based on recent large-scale phylogenetic analyses. I find that the Heliobacteriaceae family of Firmicutes appears to be the earliest branching of the known groups of phototrophic bacteria; however, the origin of photochemical reaction centers and chlorophyll synthesis cannot be placed in this group. Moreover, it becomes evident that the Acidobacteria and the Proteobacteria shared a more recent common phototrophic ancestor, and this is also likely for the Chloroflexi and the Cyanobacteria. Finally, I argue that the discrepancies among the phylogenies of the reaction center proteins, chlorophyll synthesis enzymes, and the species tree of bacteria are best explained if both types of photochemical reaction centers evolved before the diversification of the known phyla of phototrophic bacteria. The primordial phototrophic ancestor must have had both Type I and Type II reaction centers.

Charge separation in photosystem II: a comparative and evolutionary overview.

Our current understanding of the PSII reaction centre owes a great deal to comparisons to the simpler and better understood, purple bacterial reaction centre. Here we provide an overview of the similarities with a focus on charge separation and the electron acceptors. We go on to discuss some of the main differences between the two kinds of reaction centres that have been highlighted by the improving knowledge of PSII. We attempt to relate these differences to functional requirements of water splitting. Some are directly associated with that function, e.g. high oxidation potentials, while others are associated with regulation and protection against photodamage. The protective and regulatory functions are associated with the harsh chemistry performed during its normal function but also with requirements of the enzyme while it is undergoing assembly and repair. Key aspects of PSII reaction centre evolution are also addressed. This article is part of a Special Issue entitled: Photosystem II.

Covalent immobilization of oriented photosystem II on a nanostructured electrode for solar water oxidation.

Photosystem II (PSII) offers a biological and sustainable route of photochemical water oxidation to O2 and can provide protons and electrons for the generation of solar fuels, such as H2. We present a rational strategy to electrostatically improve the orientation of PSII from a thermophilic cyanobacterium, Thermosynechococcus elongatus , on a nanostructured indium tin oxide (ITO) electrode and to covalently immobilize PSII on the electrode. The ITO electrode was modified with a self-assembled monolayer (SAM) of phosphonic acid ITO linkers with a dangling carboxylate moiety. The negatively charged carboxylate attracts the positive dipole on the electron acceptor side of PSII via Coulomb interactions. Covalent attachment of PSII in its electrostatically improved orientation to the SAM-modified ITO electrode was accomplished via an amide bond to further enhance red-light-driven, direct electron transfer and stability of the PSII hybrid photoelectrode.

Molecular diversity and evolution of far-red light-acclimated photosystem I.

The need to acclimate to different environmental conditions is central to the evolution of cyanobacteria. Far-red light (FRL) photoacclimation, or FaRLiP, is an acclimation mechanism that enables certain cyanobacteria to use FRL to drive photosynthesis. During this process, a well-defined gene cluster is upregulated, resulting in changes to the photosystems that allow them to absorb FRL to perform photochemistry. Because FaRLiP is widespread, and because it exemplifies cyanobacterial adaptation mechanisms in nature, it is of interest to understand its molecular evolution. Here, we performed a phylogenetic analysis of the photosystem I subunits encoded in the FaRLiP gene cluster and analyzed the available structural data to predict ancestral characteristics of FRL-absorbing photosystem I. The analysis suggests that FRL-specific photosystem I subunits arose relatively late during the evolution of cyanobacteria when compared with some of the FRL-specific subunits of photosystem II, and that the order Nodosilineales, which include strains like Halomicronema hongdechloris and Synechococcus sp. PCC 7335, could have obtained FaRLiP via horizontal gene transfer. We show that the ancestral form of FRL-absorbing photosystem I contained three chlorophyll f-binding sites in the PsaB2 subunit, and a rotated chlorophyll a molecule in the A0B site of the electron transfer chain. Along with our previous study of photosystem II expressed during FaRLiP, these studies describe the molecular evolution of the photosystem complexes encoded by the FaRLiP gene cluster.

The Evolution and Evolvability of Photosystem II.

Photosystem II is the water-oxidizing and O2-evolving enzyme of photosynthesis. How and when this remarkable enzyme arose are fundamental questions in the history of life that have remained difficult to answer. Here, recent advances in our understanding of the origin and evolution of photosystem II are reviewed and discussed in detail. The evolution of photosystem II indicates that water oxidation originated early in the history of life, long before the diversification of cyanobacteria and other major groups of prokaryotes, challenging and transforming current paradigms on the evolution of photosynthesis. We show that photosystem II has remained virtually unchanged for billions of years, and yet the nonstop duplication process of the D1 subunit of photosystem II, which controls photochemistry and catalysis, has enabled the enzyme to become adaptable to variable environmental conditions and even to innovate enzymatic functions beyond water oxidation. We suggest that this evolvability can be harnessed to develop novel light-powered enzymes with the capacity to carry out complex multistep oxidative transformations for sustainable biocatalysis.

Photoelectrochemical water oxidation with photosystem II integrated in a mesoporous indium-tin oxide electrode.

We report on a hybrid photoanode for water oxidation consisting of a cyanobacterial photosystem II (PSII) from Thermosynechococcus elongatus on a mesoporous indium-tin oxide (mesoITO) electrode. The three-dimensional metal oxide environment allows for high protein coverage (26 times an ideal monolayer coverage) and direct (mediator-free) electron transfer from PSII to mesoITO. The oxidation of water occurs with 1.6 ± 0.3 µA cm(-2) and a corresponding turnover frequency of approximately 0.18 ± 0.04 (mol O(2)) (mol PSII)(-1) s(-1) during red light irradiation. Mechanistic studies are consistent with interfacial electron transfer occurring not only from the terminal quinone Q(B), but also from the quinone Q(A) through an unnatural electron transfer pathway to the ITO surface.

Molecular Evolution of Far-Red Light-Acclimated Photosystem II.

Cyanobacteria are major contributors to global carbon fixation and primarily use visible light (400-700 nm) to drive oxygenic photosynthesis. When shifted into environments where visible light is attenuated, a small, but highly diverse and widespread number of cyanobacteria can express modified pigments and paralogous versions of photosystem subunits and phycobiliproteins that confer far-red light (FRL) absorbance (700-800 nm), a process termed far-red light photoacclimation, or FaRLiP. During FaRLiP, alternate photosystem II (PSII) subunits enable the complex to bind chlorophylls d and f, which absorb at lower energy than chlorophyll a but still support water oxidation. How the FaRLiP response arose remains poorly studied. Here, we report ancestral sequence reconstruction and structure-based molecular evolutionary studies of the FRL-specific subunits of FRL-PSII. We show that the duplications leading to the origin of two PsbA (D1) paralogs required to make chlorophyll f and to bind chlorophyll d in water-splitting FRL-PSII are likely the first to have occurred prior to the diversification of extant cyanobacteria. These duplications were followed by those leading to alternative PsbC (CP43) and PsbD (D2) subunits, occurring early during the diversification of cyanobacteria, and culminating with those leading to PsbB (CP47) and PsbH paralogs coincident with the radiation of the major groups. We show that the origin of FRL-PSII required the accumulation of a relatively small number of amino acid changes and that the ancestral FRL-PSII likely contained a chlorophyll d molecule in the electron transfer chain, two chlorophyll f molecules in the antenna subunits at equivalent positions, and three chlorophyll a molecules whose site energies were altered. The results suggest a minimal model for engineering far-red light absorbance into plant PSII for biotechnological applications.

Excitation energy transfer to Photosystem I in filaments and heterocysts of Nostoc punctiforme.

Cyanobacteria adapt to varying light conditions by controlling the amount of excitation energy to the photosystems. On the minute time scale this leads to redirection of the excitation energy, usually referred to as state transitions, which involves movement of the phycobilisomes. We have studied short-term light adaptation in isolated heterocysts and intact filaments from the cyanobacterium Nostoc punctiforme ATCC 29133. In N. punctiforme vegetative cells differentiate into heterocysts where nitrogen fixation takes place. Photosystem II is inactivated in the heterocysts, and the abundancy of Photosystem I is increased relative to the vegetative cells. To study light-induced changes in energy transfer to Photosystem I, pre-illumination was made to dark adapted isolated heterocysts. Illumination wavelengths were chosen to excite Photosystem I (708nm) or phycobilisomes (560nm) specifically. In heterocysts that were pre-illuminated at 708nm, fluorescence from the phycobilisome terminal emitter was observed in the 77K emission spectrum. However, illumination with 560nm light caused quenching of the emission from the terminal emitter, with a simultaneous increase in the emission at 750nm, indicating that the 560nm pre-illumination caused trimerization of Photosystem I. Excitation spectra showed that 560nm pre-illumination led to an increase in excitation transfer from the phycobilisomes to trimeric Photosystem I. Illumination at 708nm did not lead to increased energy transfer from the phycobilisome to Photosystem I compared to dark adapted samples. The measurements were repeated using intact filaments containing vegetative cells, and found to give very similar results as the heterocysts. This demonstrates that molecular events leading to increased excitation energy transfer to Photosystem I, including trimerization, are independent of Photosystem II activity.

Complete Genome Sequencing of a Novel Gloeobacter Species from a Waterfall Cave in Mexico.

Only two complete genomes of the cyanobacterial genus Gloeobacter from two very different regions of the world currently exist. Here, we present the complete genome sequence of a third member of the genus isolated from a waterfall cave in Mexico. Analysis of the average nucleotide identities (ANIs) between published Gloeobacter genomes revealed that the complete genome of this new member is only 92.7% similar to Gloeobacter violaceus and therefore we determined it to be a new species. We propose to name this new species Gloeobacter morelensis after the location in Mexico where it was isolated. The complete genome consists of one circular chromosome (4,921,229 bp), one linear plasmid (172,328 bp), and one circular plasmid (8,839 bp). Its genome is the largest of all completely sequenced genomes of Gloeobacter species. Pangenomic comparisons revealed that G. morelensis encodes 759 genes not shared with other Gloeobacter species. Despite being more closely related to G. violaceus, it features an extremely divergent psbA gene encoding an atypical D1 core subunit of Photosystem II previously only found within the genome of Gloeobacter kilaueensis. In addition, we detected evidence of concerted evolution of psbA genes encoding identical D1 in all three Gloeobacter genomes, a characteristic that seems widespread in cyanobacteria and may therefore be traced back to their last common ancestor.

Time-resolved comparative molecular evolution of oxygenic photosynthesis.

Oxygenic photosynthesis starts with the oxidation of water to O2, a light-driven reaction catalysed by photosystem II. Cyanobacteria are the only prokaryotes capable of water oxidation and therefore, it is assumed that the origin of oxygenic photosynthesis is a late innovation relative to the origin of life and bioenergetics. However, when exactly water oxidation originated remains an unanswered question. Here we use phylogenetic analysis to study a gene duplication event that is unique to photosystem II: the duplication that led to the evolution of the core antenna subunits CP43 and CP47. We compare the changes in the rates of evolution of this duplication with those of some of the oldest well-described events in the history of life: namely, the duplication leading to the Alpha and Beta subunits of the catalytic head of ATP synthase, and the divergence of archaeal and bacterial RNA polymerases and ribosomes. We also compare it with more recent events such as the duplication of Cyanobacteria-specific FtsH metalloprotease subunits and the radiation leading to Margulisbacteria, Sericytochromatia, Vampirovibrionia, and other clades containing anoxygenic phototrophs. We demonstrate that the ancestral core duplication of photosystem II exhibits patterns in the rates of protein evolution through geological time that are nearly identical to those of the ATP synthase, RNA polymerase, or the ribosome. Furthermore, we use ancestral sequence reconstruction in combination with comparative structural biology of photosystem subunits, to provide additional evidence supporting the premise that water oxidation had originated before the ancestral core duplications. Our work suggests that photosynthetic water oxidation originated closer to the origin of life and bioenergetics than can be documented based on phylogenetic or phylogenomic species trees alone.

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme.

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.

Global distribution of a chlorophyll f cyanobacterial marker.

Some cyanobacteria use light outside the visible spectrum for oxygenic photosynthesis. The far-red light (FRL) region is made accessible through a complex acclimation process that involves the formation of new phycobilisomes and photosystems containing chlorophyll f. Diverse cyanobacteria ranging from unicellular to branched-filamentous forms show this response. These organisms have been isolated from shaded environments such as microbial mats, soil, rock, and stromatolites. However, the full spread of chlorophyll f-containing species in nature is still unknown. Currently, discovering new chlorophyll f cyanobacteria involves lengthy incubation times under selective far-red light. We have used a marker gene to detect chlorophyll f organisms in environmental samples and metagenomic data. This marker, apcE2, encodes a phycobilisome linker associated with FRL-photosynthesis. By focusing on a far-red motif within the sequence, degenerate PCR and BLAST searches can effectively discriminate against the normal chlorophyll a-associated apcE. Even short recovered sequences carry enough information for phylogenetic placement. Markers of chlorophyll f photosynthesis were found in metagenomic datasets from diverse environments around the globe, including cyanobacterial symbionts, hypersaline lakes, corals, and the Arctic/Antarctic regions. This additional information enabled higher phylogenetic resolution supporting the hypothesis that vertical descent, as opposed to horizontal gene transfer, is largely responsible for this phenotype's distribution.

Thinking twice about the evolution of photosynthesis.

Sam Granick opened his seminal 1957 paper titled 'Speculations on the origins and evolution of photosynthesis' with the assertion that there is a constant urge in human beings to seek beginnings (I concur). This urge has led to an incessant stream of speculative ideas and debates on the evolution of photosynthesis that started in the first half of the twentieth century and shows no signs of abating. Some of these speculative ideas have become commonplace, are taken as fact, but find little support. Here, I review and scrutinize three widely accepted ideas that underpin the current study of the evolution of photosynthesis: first, that the photochemical reaction centres used in anoxygenic photosynthesis are more primitive than those in oxygenic photosynthesis; second, that the probability of acquiring photosynthesis via horizontal gene transfer is greater than the probability of losing photosynthesis; and third, and most important, that the origin of anoxygenic photosynthesis pre-dates the origin of oxygenic photosynthesis. I shall attempt to demonstrate that these three ideas are often grounded in incorrect assumptions built on more assumptions with no experimental or observational support. I hope that this brief review will not only serve as a cautionary tale but also that it will open new avenues of research aimed at disentangling the complex evolution of photosynthesis and its impact on the early history of life and the planet.

Evolution of Photochemical Reaction Centres: More Twists?

One of the earliest events in the molecular evolution of photosynthesis is the structural and functional specialisation of type I (ferredoxin-reducing) and type II (quinone-reducing) reaction centres. In this opinion article we point out that the homodimeric type I reaction centre of heliobacteria has a calcium-binding site with striking structural similarities to the Mn4CaO5 cluster of photosystem II. These similarities indicate that most of the structural elements required to evolve water oxidation chemistry were present in the earliest reaction centres. We suggest that the divergence of type I and type II reaction centres was made possible by a drastic structural shift linked to a change in redox properties that coincided with or facilitated the origin of photosynthetic water oxidation.

Evolutionary Implications of Anoxygenic Phototrophy in the Bacterial Phylum Candidatus Eremiobacterota (WPS-2).

Genome-resolved environmental metagenomic sequencing has uncovered substantial previously unrecognized microbial diversity relevant for understanding the ecology and evolution of the biosphere, providing a more nuanced view of the distribution and ecological significance of traits including phototrophy across diverse niches. Recently, the capacity for bacteriochlorophyll-based anoxygenic photosynthesis has been proposed in the uncultured bacterial WPS-2 phylum (recently proposed as Candidatus Eremiobacterota) that are in close association with boreal moss. Here, we use phylogenomic analysis to investigate the diversity and evolution of phototrophic WPS-2. We demonstrate that phototrophic WPS-2 show significant genetic and metabolic divergence from other phototrophic and non-phototrophic lineages. The genomes of these organisms encode a new family of anoxygenic Type II photochemical reaction centers and other phototrophy-related proteins that are both phylogenetically and structurally distinct from those found in previously described phototrophs. We propose the name Candidatus Baltobacterales for the order-level aerobic WPS-2 clade which contains phototrophic lineages, from the Greek for "bog" or "swamp," in reference to the typical habitat of phototrophic members of this clade.