Cytochrome ba(3) from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba(3) oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169-16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved reaction between fully reduced cytochrome ba(3) and O(2) in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312 --> Ser variant in the ba(3) oxidase both reactions associated with proton uptake from solution, the P(R) --> F and F --> O transitions, were slowed compared to those of wild-type ba(3). The observed time constants were slowed approximately 3-fold (for P(R) --> F, from 60 to approximately 170 mus in the wild type) and approximately 30-fold (for F --> O, from 1.1 to approximately 40 ms). In the Thr-315 --> Val variant, the F --> O transition was approximately 5-fold slower (5 ms) than for the wild-type oxidase, whereas the P(R) --> F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 slower and decoupled from the optical P(R) --> F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O(2) reduction in the ba(3) oxidase.