Water oxidation by photosystem II (PSII) involves the release of O-2, electrons, and protons at the oxygen-evolving complex (OEC). These processes are facilitated by a hydrogen-bonded network of amino acid residues and waters surrounding the OEC. It is crucial to probe the proton-transfer pathways from the OEC as proton release helps to maintain the charge balance required for efficient water oxidation. In this study, we generate point mutations in the cyanobacterium Synechocystis sp. PCC 6803 at secondary-shell amino acid residues surrounding the OEC: D2-K317, D1-S169, CP43-R357, D1-D61, and D1-N181. We employ direct experimental methods to study the O-2 evolution rate under varying pH ranging from 3-8. The pH dependence follows a bell-shaped curve in both wild-type and mutated PSII from which we can derive the effective acidic pK(a). The effective acidic pK(a) provides insights into the protonation states of the amino acid residues participating in the proton-transfer process during the rate-determining step of water oxidation. The presence of an additional effective pK(a) in D1-S169A PSII and D2-K317A PSII indicates the possibility of multiple proton-transfer pathways during the ratedetermining step of water oxidation. We also studied the O-2 evolution rate in H2O and D2O with varying pL (L = H or D) to identify the amino acid residues participating in the proton-transfer process. We find that replacing the positively charged lysine with a neutral alanine in D2-K317A PSII and aspartate with alanine in D1-D6IA PSII significantly enhances the kinetic solvent isotope effect (KSIE), indicating that proton transfer becomes rate-limiting at the optimal pH in these mutated PSII. However, the KSIE remains unchanged for D1-N181A, D1-S169A, and CP43-R357K PSII. Thus, perturbing the channel defined by the D1-D61 and D2-K317 residues strongly hampers the proton-transfer mechanism, and in turn, the water oxidation reaction of PSII. Hence, our study provides a direct experimental probe to identify that the D1-D61 and D2-K317 residues participate in the proton-transfer process. These results, thereby, provide us a deeper understanding of the proton-transfer processes in the water oxidation mechanism.