In the recent X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is assigned as a ligand of the O2-evolving Mn4Ca cluster. In this communication, a preliminary characterization of the CP43-Glu354Gln mutant of the cyanobacterium Synechocystis sp. PCC 6803 is presented. The steady-state rate of O2 evolution in the mutant cells is only approximately 20% compared with the wild-type, but the kinetics of O2 release are essentially unchanged and the O2-flash yields show normal period-four oscillations, albeit with lower overall intensity. Purified PSII particles exhibit an essentially normal S2 state multiline electron paramagnetic resonance (EPR) signal, but exhibit a substantially altered S2-minus-S1 Fourier transform infrared (FTIR) difference spectrum. The intensities of the mutant EPR and FTIR difference spectra (above 75% compared with wild-type) are much greater than the O2 signals and suggest that CP43-Glu354Gln PSII reaction centres are heterogeneous, with a minority fraction able to evolve O2 with normal O2 release kinetics and a majority fraction unable to advance beyond the S2 or S3 states. The S2-minus-S1 FTIR difference spectrum of CP43-Glu354Gln PSII particles is altered in both the symmetric and asymmetric carboxylate stretching regions, implying either that CP43-Glu354 is exquisitely sensitive to the increased charge that develops on the Mn4Ca cluster during the S1-->S2 transition or that the CP43-Glu354Gln mutation changes the distribution of Mn(III) and Mn(IV) oxidation states within the Mn4Ca cluster in the S1 and/or S2 states.