The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA(3) gene compared to that deduced from the psbA(1) gene points a difference of 21 residues. In this work. PSII isolated from a wild type T. elongatus strain expressing PsbA(1) or from a strain in which both the psbA(1) and psbA(2) genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo(D1) is increased by 17 mV from -522 mV in PsbA1-PSII to -505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S(2)QA(-center dot). charge recombination and the C equivalent to N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q(B) pocket. The temperature dependences of the S(2)Q(A)(-center dot) charge recombination show that the strength of the H-bond to Pheo(D1) is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q(A) (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P-680(+center dot) and Y-z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P-680/P-680(+center dot) couple (and hence that of P-1(680)center dot/P-680(+)center dot) is tuned as well when shifting from PsbAl to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes. (C) 2010 Elsevier B.V. All rights reserved.