1：Experimental Design and Method Selection:

The study involved constructing site-directed mutants (D1/T179H and D1/T179V) in Thermosynechococcus elongatus using mutagenesis kits and characterizing them with biophysical techniques including EPR, FTIR, fluorescence and absorption spectroscopies at room temperature and 77 K, time-resolved UV–vis absorption spectroscopy, polarography, and thermoluminescence to analyze electron transfer and photodamage mechanisms.

2：Sample Selection and Data Sources:

PSII samples were purified from mutant and wild-type T. elongatus strains. Mutants were selected using antibiotic resistance and confirmed by genomic DNA analysis and sequencing.

3：List of Experimental Equipment and Materials:

Equipment included a Gene Pulser (BioRad) for transformation, spectrophotometers (Hitachi U-3900H, U-3900), EPR spectrometer (Bruker Elexsys 500), FTIR spectrophotometer (Bruker VERTEX 80), oxygen electrode (Hansatech), thermoluminescence apparatus, and time-resolved absorption spectrophotometer. Materials included primers, restriction enzymes, chloramphenicol, gentamycin, kanamycin, DCMU, PPBQ, histidine, and various buffers.

4：Experimental Procedures and Operational Workflow:

Mutants were constructed via site-directed mutagenesis, transformed, and selected. PSII was purified and characterized for oxygen evolution, singlet oxygen production, EPR signals, thermoluminescence, absorption changes, and fluorescence spectra under controlled conditions (e.g., dark adaptation, specific temperatures).

5：Data Analysis Methods:

Data were analyzed using spectral comparisons, oscillation patterns in flash-induced changes, and normalization techniques. Statistical comparisons were made between mutants and wild-type based on spectral shifts, recombination rates, and bleaching patterns.