Bicarbonate removal from the non-heme iron at the acceptor side of photosystem II (PSII) was recently shown to shift the midpoint potential of the primary quinone acceptor QA to a more positive potential and lowers the yield of singlet oxygen (1O2) production. The presence of QA- results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against 1O2 in vivo under photorespiratory conditions. Here we compared the properties of QA in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant hpr1-1, deficient in NADH-dependent, peroxisomal hydroxypyruvate reductase 1 (HPR1), which accumulates glycolate in leaves, to the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between QA and QB in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission indicating a shift of the midpoint potential of QA to a more positive value. The yield of 1O2 production was lowered in thylakoid membranes isolated from hpr1-1 compared to the wild type, consistent with a higher potential of QA/QA-. In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities consistent with diminished electron transfer out of photosystem II. This study indicates that replacement of bicarbonate at the non-heme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the non-heme iron by glycolate may represent a regulatory mechanism that protects PSII against photo-oxidative stress under low CO2 conditions.