Human carbonic anhydrase II (HCA II) uses a Zn-bound OH − /H 2 O mechanism to catalyze the reversible hydration of CO 2 . This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pK a compared with free tyrosine. To further probe the pK a of this residue, NMR spectroscopic measurements of [ 13 C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pK a of Tyr7 and how pH and Tyr proximity to Zn affect hydrogenbonding interactions.proton transfer | neutron crystallography | perturbed pKa | solution NMR C arbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the interconversion of CO 2 and HCO 3 − . In humans, CAs are involved in a great variety of physiological processes, from respiration, ureagenesis, gluconeogenesis, and cerebrospinal fluid production to general acid/base homeostasis (1). The most abundant and well-characterized human CA isoform is human carbonic anhydrase II (HCA II), which displays a bellshaped pH dependence on catalytic activity, with a maximal k cat of 10 6 per second at pH 7.2. In the hydration direction, the first step of catalysis is a Zn-OH − -mediated nucleophilic attack on CO 2 to form HCO 3 − and one H + , leaving one water molecule bound to the zinc. The second, rate-limiting step occurs via proton transfer (PT) that removes one H + from the metal-bound water to regenerate the catalytic Zn-OH − (2). This PT step is thought to adopt classic Grotthuss-type hopping and it is mediated by a well-ordered network of hydrogen-bonded (H-bonded) water molecules that spans the ∼8-Å distance between the Znwater and proton-shuttling residue, His64 (Figs. 1-3). In previous neutron structures, the Zn-bound solvent has been observed to be an H 2 O (or exchanged D 2 O) and this may imply that H 2 O is the favored species over OH − in the absence of substrate (3). In chemical crystallography extended X-ray absorption fine structure can be used to determine Zn:Zn-solvent distances as a way to discriminate between H 2 O or OH − . For Zn-OH − the distance should be ∼1.80 Å and for Zn-H 2 O it should be closer to 2.0 Å (4). Despite the availability of ultra-high-resolution X-ray crystal structures of holo HCA II (no substrate present) the range of Zn:Zn-solvent distances observed has led to inconclusive results. Also, within the coordinate error it is unfeasible to determine the Zn-bound solvent species using distance with any certainty (5, 6).His64 is thought to be one component in the delivery of excess H + to the bulk solvent, resetting the a...