Examination of the nature of different bond activations along the same catalytic path is of general interest in chemistry and biology. In this report, we compare the physical nature of two sequential H-transfers in the same enzymatic reaction. Thymidylate synthase (TSase) catalyzes a complex reaction that involves many chemical transformations including two different C-H bond cleavages, a rate-limiting C-H-C hydride transfer and a non-rate-limiting C-H-O proton transfer. Although the large kinetic complexity imposes difficulties in studying the proton transfer catalyzed by TSase, we are able to experimentally extract the intrinsic kinetic isotope effects (KIEs) on both steps. In contrast with the hydride transfer, the intrinsic KIEs of the proton transfer are temperature dependent. The results are interpreted within the framework of the Marcus-like model. This interpretation suggests that TSase optimizes the donor-acceptor geometries for the slower and overall rate-limiting hydride transfer but not for the faster proton transfer.