How electron tunneling and uphill excitation energy transfer support photochemistry in Halomicronema hongdechloris.

Halomicronema hongdechloris, the first cyanobacterium reported to produce the red-shifted chlorophyll f (Chl f) upon acclimation to far-red light, demonstrates remarkable adaptability to diverse light conditions. The photosystem II (PS II) of this organism undergoes reversible changes in its Chl f content, ranging from practically zero under white-light culture conditions to a Chl f: Chl a ratio of up to 1:8 when exposed to far-red light (FRL) of 720-730 nm for several days. Our ps time- and wavelength-resolved fluorescence data obtained after excitation of living H. hongdechloris cells indicate that the Soret band of a far-red (FR) chlorophyll involved in charge separation absorbs around 470 nm. At 10 K, the fluorescence decay at 715-720 nm is still fast with a time constant of 165 ps indicating an efficient electron tunneling process. There is efficient excitation energy transfer (EET) from 715-720 nm to 745 nm with the latter resulting from FR Chl f, which mainly functions as light-harvesting pigment upon adaptation to FRL. From there, excitation energy reaches the primary donor in the reaction center of PS II with an energetic uphill EET mechanism inducing charge transfer. The fluorescence data are well explained with a secondary donor PD1 represented by a red-shifted Chl a molecule with characteristic fluorescence around 715 nm and a more red-shifted FR Chl f with fluorescence around 725 nm as primary donor at the ChlD1 or PD2 position.

The specificity of the bilin lyase CpcS for chromophore attachment to allophycocyanin in the chlorophyll f-containing cyanobacterium Halomicronima hongdechloris.

Phycobilisomes are light-harvesting antenna complexes of cyanobacteria and red algae that are comprised of chromoproteins called phycobiliproteins. PBS core structures are made up of allophycocyanin subunits. Halomicronema hongdechloris (H. hongdechloris) is one of the cyanobacteria that produce chlorophyll f (Chl f) under far-red light and is regulated by the Far-Red Light Photoacclimation gene cluster. There are five genes encoding APC in this specific gene cluster, and they are responsible for assembling the red-shifted PBS in H. hongdechloris grown under far-red light. In this study, the five apc genes located in the FaRLiP gene cluster were heterologously expressed in an Escherichia coli reconstitution system. The canonical APC-encoding genes were also constructed in the same system for comparison. Additionally, five annotated phycobiliprotein lyase-encoding genes (cpcS) from the H. hongdechloris genome were phylogenetically classified and experimentally tested for their catalytic properties including their contribution to the shifted absorption of PBS. Through analysis of recombinant proteins, we determined that the heterodimer of CpcS-I and CpcU are able to ligate a chromophore to the APC-α/APC-ß subunits. We discuss some hypotheses towards understanding the roles of the specialised APC and contributions of PBP lyases.

Genome and proteome of the chlorophyll f-producing cyanobacterium Halomicronema hongdechloris: adaptative proteomic shifts under different light conditions.

BACKGROUND: Halomicronema hongdechloris was the first cyanobacterium to be identified that produces chlorophyll (Chl) f. It contains Chl a and uses phycobiliproteins as its major light-harvesting components under white light conditions. However, under far-red light conditions H. hongdechloris produces Chl f and red-shifted phycobiliprotein complexes to absorb and use far-red light. In this study, we report the genomic sequence of H. hongdechloris and use quantitative proteomic approaches to confirm the deduced metabolic pathways as well as metabolic and photosynthetic changes in response to different photo-autotrophic conditions. RESULTS: The whole genome of H. hongdechloris was sequenced using three different technologies and assembled into a single circular scaffold with a genome size of 5,577,845 bp. The assembled genome has 54.6% GC content and encodes 5273 proteins covering 83.5% of the DNA sequence. Using Tandem Mass Tag labelling, the total proteome of H. hongdechloris grown under different light conditions was analyzed. A total of 1816 proteins were identified, with photosynthetic proteins accounting for 24% of the total mass spectral readings, of which 35% are phycobiliproteins. The proteomic data showed that essential cellular metabolic reactions remain unchanged under shifted light conditions. The largest differences in protein content between white and far-red light conditions reflect the changes to photosynthetic complexes, shifting from a standard phycobilisome and Chl a-based light harvesting system under white light, to modified, red-shifted phycobilisomes and Chl f-containing photosystems under far-red light conditions. CONCLUSION: We demonstrate that essential cellular metabolic reactions under different light conditions remain constant, including most of the enzymes in chlorophyll biosynthesis and photosynthetic carbon fixation. The changed light conditions cause significant changes in the make-up of photosynthetic protein complexes to improve photosynthetic light capture and reaction efficiencies. The integration of the global proteome with the genome sequence highlights that cyanobacterial adaptation strategies are focused on optimizing light capture and utilization, with minimal changes in other metabolic pathways. Our quantitative proteomic approach has enabled a deeper understanding of both the stability and the flexibility of cellular metabolic networks of H. hongdechloris in response to changes in its environment.

Photosynthesis supported by a chlorophyll f-dependent, entropy-driven uphill energy transfer in Halomicronema hongdechloris cells adapted to far-red light.

The phototrophic cyanobacterium Halomicronema hongdechloris shows far-red light-induced accumulation of chlorophyll (Chl) f, but the involvement of the pigment in photosynthetic energy harvesting by photosystem (PS) II is controversially discussed. While H. hongdechloris contains negligible amounts of Chl f in white-light culture conditions, the ratio of Chl f to Chl a is reversibly changed up to 1:8 under illumination with far-red light (720-730 nm). We performed UV-Vis absorption spectroscopy, time-integrated and time-resolved fluorescence spectroscopy for the calculation of decay-associated spectra (DAS) to determine excitation energy transfer (EET) processes between photosynthetic pigments in intact H. hongdechloris filaments. In cells grown under white light, highly efficient EET occurs from phycobilisomes (PBSs) to Chl a with an apparent time constant of about 100 ps. Charge separation occurs with a typical apparent time constant of 200-300 ps from Chl a. After 3-4 days of growth under far-red light, robust Chl f content was observed in H. hongdechloris and EET from PBSs reached Chl f efficiently within 200 ps. It is proposed based on mathematical modeling by rate equation systems for EET between the PBSs and PSII and subsequent electron transfer (ET) that charge separation occurs from Chl a and excitation energy is funneled from Chl f to Chl a via an energetically uphill EET driven by entropy, which is effective because the number of Chl a molecules coupled to Chl f is at least eight- to tenfold larger than the corresponding number of Chl f molecules. The long lifetime of Chl f molecules in contact to a tenfold larger pool of Chl a molecules allows Chl f to act as an intermediate energy storage level, from which the Gibbs free energy difference between Chl f and Chl a can be overcome by taking advantage from the favorable ratio of degeneracy coefficients, which formally represents a significant entropy gain in the Eyring formulation of the Arrhenius law. Direct evidence for energetically uphill EET and charge separation in PSII upon excitation of Chl f via anti-Stokes fluorescence in far-red light-adapted H. hongdechloris cells was obtained: Excitation by 720 nm laser light resulted in robust Chl a fluorescence at 680 nm that was distinctly temperature-dependent and, notably, increased upon DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) treatment in far-red light-adapted cells. Thus, rather than serving as an excitation energy trap, Chl f in far-red light-adapted H. hongdechloris cells is directly contributing to oxygenic photosynthesis at PSII.

Optimization and effects of different culture conditions on growth of Halomicronema hongdechloris - a filamentous cyanobacterium containing chlorophyll f.

A chlorophyll f containing cyanobacterium, Halomicronema hongdechloris (H. hongdechloris) was isolated from a stromatolite cyanobacterial community. The extremely slow growth rate of H. hongdechloris has hindered research on this newly isolated cyanobacterium and the investigation of chlorophyll f-photosynthesis. Therefore, optimizing H. hongdechloris culture conditions has become an essential requirement for future research. This work investigated the effects of various culture conditions, essential nutrients and light environments to determine the optimal growth conditions for H. hongdechloris and the biosynthetic rate of chlorophyll f. Based on the total chlorophyll concentration, an optimal growth rate of 0.22 ± 0.02 day(-1)(doubling time: 3.1 ± 0.3 days) was observed when cells were grown under continuous illumination with far-red light with an intensity of 20 µE at 32°C in modified K + ES seawater (pH 8.0) with additional nitrogen and phosphor supplements. High performance liquid chromatography on H. hongdechloris pigments confirmed that chlorophyll a is the major chlorophyll and chlorophyll f constitutes ~10% of the total chlorophyll from cells grown under far-red light. Fluorescence confocal image analysis demonstrated changes of photosynthetic membranes and the distribution of photopigments in response to different light conditions. The total photosynthetic oxygen evolution yield per cell showed no changes under different light conditions, which confirms the involvement of chlorophyll f in oxygenic photosynthesis. The implications of the presence of chlorophyll f in H. hongdechloris and its relationship with the ambient light environment are discussed.