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<p>In photosynthetic proteins, pigments at a higher energy level funnel excitation energy to pigments at a lower energy level. Specifically, in Photosystem II (PSII), energy is transferred downhill to the reaction center (RC), where water splitting occurs. However, the lowest-energy state in PSII is not the RC, but the F695 state, which can be observed using low-temperature spectroscopy. This lowest-energy state is typically assigned to a monomeric pigment, Chl B16 (ligated by His114), but this assignment has been called into question based on theoretical fits to low-temperature spectra. In this study, we set out to test concretely whether the F695 state is localized on Chl B16 using site-directed mutagenesis and 77 K fluorescence spectroscopy. To reduce spectral congestion for whole-cell PSII studies, we developed a background strain (PSI-kd/<InlineEquation ID="IEq1"> <EquationSource Format="TEX">\(\Delta\)</EquationSource> </InlineEquation>PBS) that combines a Photosystem I (PSI) knockdown with a Phycobilisome (PBS) knockout. In this background strain, we made site-directed mutations at site Thr5 in the PsbH subunit, which forms a hydrogen bond with the <InlineEquation ID="IEq2"> <EquationSource Format="TEX">\(13^{1}\)</EquationSource> </InlineEquation>-keto group of Chl B16. All mutants were capable of heterotrophic growth (without noticeable differences from wild-type), indicating the PSII function remains intact. As expected for Chl B16-localized fluorescence, the Thr5<InlineEquation ID="IEq3"> <EquationSource Format="TEX">\(\rightarrow\)</EquationSource> </InlineEquation>Arg mutation red-shifted the F695 state due to the strengthening of the hydrogen bond, while the Thr5<InlineEquation ID="IEq4"> <EquationSource Format="TEX">\(\rightarrow\)</EquationSource> </InlineEquation>Ala mutation exhibits a blue shift as the hydrogen bond is eliminated. Taken together, these findings provide strong confirmation that Chl B16 is responsible for the lowest-energy state.</p>

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Tuning the F695 fluorescent state in photosystem II using site-directed mutagenesis in Synechocystis sp. PCC 6803

  • 期刊论文

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  • 发表日期：

    2026年5月18日

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  • Amala Phadkule,
  • Amit Srivastava,
  • Alexandria Alailima Martin,
  • Lauren G. Dome,
  • Steven D. McKenzie,
  • Sujith Puthiyaveetil,
  • Mike Reppert

摘要

In photosynthetic proteins, pigments at a higher energy level funnel excitation energy to pigments at a lower energy level. Specifically, in Photosystem II (PSII), energy is transferred downhill to the reaction center (RC), where water splitting occurs. However, the lowest-energy state in PSII is not the RC, but the F695 state, which can be observed using low-temperature spectroscopy. This lowest-energy state is typically assigned to a monomeric pigment, Chl B16 (ligated by His114), but this assignment has been called into question based on theoretical fits to low-temperature spectra. In this study, we set out to test concretely whether the F695 state is localized on Chl B16 using site-directed mutagenesis and 77 K fluorescence spectroscopy. To reduce spectral congestion for whole-cell PSII studies, we developed a background strain (PSI-kd/ \(\Delta\) PBS) that combines a Photosystem I (PSI) knockdown with a Phycobilisome (PBS) knockout. In this background strain, we made site-directed mutations at site Thr5 in the PsbH subunit, which forms a hydrogen bond with the \(13^{1}\) -keto group of Chl B16. All mutants were capable of heterotrophic growth (without noticeable differences from wild-type), indicating the PSII function remains intact. As expected for Chl B16-localized fluorescence, the Thr5 \(\rightarrow\) Arg mutation red-shifted the F695 state due to the strengthening of the hydrogen bond, while the Thr5 \(\rightarrow\) Ala mutation exhibits a blue shift as the hydrogen bond is eliminated. Taken together, these findings provide strong confirmation that Chl B16 is responsible for the lowest-energy state.