The role of chloride in photosystem II (PSII) is unclear. Using structural information from PSII and a careful comparison with other chloride-activated enzymes, we proposed a role for chloride at the D2-K317 site in PSII [Pokhrel, R., et al. (2011) Biochemistry 50, 2725-2734]. To probe the role of chloride at this site, the D2-K317R, D2-K317A, D2-K317Q, and D2-K317E mutations were created in the cyanobacterium Synechocystis sp. PCC 6803. Purified PSII from the mutants was probed with Fourier transform infrared difference spectroscopy, demonstrating that compared to PSII from wild-type Synechocystis, PSII from all four mutants exhibit changes in the conformations of the polypeptide backbone and carboxylate groups. However, D2-K317R PSII exhibits minor changes, whereas D2-K317A, D2-K317Q, and D2-K317E PSII exhibit more substantial changes in polypeptide conformations. Steady-state oxygen-evolution measurements of purified PSII core complexes show that the oxygen-evolution activity of D2-K317A is independent of chloride. This is consistent with the loss of the chloride requirement when the charged K residue is replaced with an uncharged residue that no longer binds to an essential carboxylate (D1-D61) in the absence of chloride, analogous to observations in other chloride-activated enzymes. In contrast, the oxygen-evolution activity of D2-K317R is sensitive to the chloride concentration in the assay buffer; the effective KD for chloride binding is higher in D2-K317R than in wild-type PSII, possibly because of a less optimal binding site in the mutant. The S2 states of wild-type, D2-K317A, and D2-K317R PSII were probed using electron paramagnetic resonance spectroscopy. A g = 2 multiline signal, similar to the wild-type signal, was observed for D2-K317A and D2-K317R. However, a g = 4 signal was also observed for D2-K317R. Measurements of flash-dependent O2 yields showed that D2-K317A and D2-K317R have a higher miss factor than wild-type PSII. The oxygen-release kinetics of D2-K317A and D2-K317R were slower than those of the wild type, in the following order: D2-K317A < D2-K317R < wild type. These results collectively suggest that proton transfer is inefficient in D2-K317A and D2-K317R, thereby giving rise to a higher miss factor and slower oxygen-release kinetics.