Tautomerism of Histidine 64 Associated with Proton Transfer in Catalysis of Carbonic Anhydrase*

The imidazole 15N signals of histidine 64 (His64), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with 15N at ring-Nδ1 and Nϵ2, 13Cat ring-Cϵ1, 13C and 15N at all carbon and nitrogen, or 15N at the amide nitrogen and the labeled glycine with 13C at the carbonyl carbon. Using the pH dependence of ring-15N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (KT) of His64 is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His64 as both a general acid (a) and base (b): its ϵ2-nitrogen as (a) releases one proton into the bulk, whereas itsδ1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the δ1-nitrogen of His64. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His64 can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

ide (1). Isozymes of carbonic anhydrase regulate or function in such diverse physiological processes as pH regulation, ion transport, water-electrolyte balance, bicarbonate secretion-absorption, bone resorption, maintenance of intraocular pressure, renal acidification, and brain development (2). Nonfunctioning CA is implicated in such diseases as osteopetrosis syndrome, glaucoma, respiratory acidosis, epilepsy, and Ménière syndrome. Diseases due to CA deficiency include those affecting bones, the brain, and the kidneys. Consequently determining the detailed structure/function relationships or mechanisms responsible for its catalytic properties is mandatory for developing inhibitors or replacement therapies.
CA is present in at least three gene families (␣, ␤, and ␥), which has made it a popular model for the study of the evolution of gene families and protein folding, and for transgenic and gene target studies (2). Among the three families, the ␣ family is the best characterized, with 11 known isozymes identified in mammals. Earnhardt and co-workers have summarized maximal k cat and k cat /K m values for CO 2 hydration by isozyme I-VII (3). The human isozyme II (hCAII) has a remarkably high turnover rate or catalytic efficiency (k cat /K m ϭ 1.5 ϫ 10 8 M Ϫ1 s Ϫ1 ) that is very close to the frequency with which the enzyme and substrate molecules collide with each other in solution.
It is widely accepted that the hydration of CO 2 catalyzed by hCAII proceeds through several chemical steps as shown in Scheme 1 (1,4,5): the direct nucleophilic attack of the zinc-bound hydroxide ion on the carbonyl carbon of substrate CO 2 (structures 1-2), the formation of a zinc-bound bicarbonate intermediate (structures [2][3], the isomerization of the bicarbonate ion (structures [3][4], the exchange of the product bicarbonate ion with a H 2 O (structures 4 -5), and the regeneration of the zinc-bound hydroxide ion by the transfer of a proton to bulk solvent (structures [1][2][3][4][5]. The proton transfer step (structures 1-5) consists of two substeps: 1) an intra-molecular transfer of protons to another residue in the enzyme and 2) a release of protons to the outside of the enzyme with the aid of a base. The intra-molecular proton transfer is the rate-limiting step of the maximal turnover rate (10 6 s Ϫ1 ) at high concentrations of a base, whereas the proton release into the medium is rate-limiting at low buffer concentrations.
In this reaction mechanism, His 64 is thought to play an important role in shuttling protons between the inside and outside of the active site cleft (6 -9). As depicted in Fig. 1, the "in" (a) and "out" (b) conformations, representing the direction of the imidazole ring toward and away from the active site, were observed in pH-dependent x-ray crystallographic studies of hCAII (4, 5, 10 -12). The side chain imidazole ring takes the in conformation at pH 7.8, where His 64 should be electrically neutral because of the pK a value of 7 as determined by 1 H NMR (13). In this conformation, the ␦1-nitrogen of His 64 appears to be involved in a water bridge or solvent network connected to the zinc-bound hydroxide ion through a hydrogen bond (12,14,15). In contrast, the T200S mutant of this enzyme was found to have His 64 in the out conformation at pH 8.0, retaining the full enzymatic activity (16). Because the out conformation of the imidazole ring was also observed at pH 5.7 (10), a swinging movement between the in and out conformations was assumed in connection with the proton transfer between a water mole-cule near a zinc ion and a bulk water molecule (5): the productive proton, which is transferred to the ␦1-nitrogen via the water bridge, is released from its nitrogen to the bulk solution after swinging of the imidazole ring. This model is attractive because it appears to be able to account for a flow of water molecules in terms of space shared with the imidazole ring. However, there is no evidence supporting the notion that the two conformers are in the kinetically stable state at a given pH. In addition, molecular dynamics simulations show that His 64 vibrates rather than swings; it could be flexible enough to find the optimum geometry between active site solvent molecules and the bulk solvent (17)(18)(19).
Despite much effort, the proton-transfer mechanism involving the dynamic behavior of His 64 still remains controversial: the specific or reasonable manner in which His 64 participates in the proton-transfer needs to be explored. To address these issues, we labeled His 64 with 15 N nucleus to identify the tautomeric forms of the imidazole ring in connection with the chemical mechanism of proton transfer in hCAII. The goal of our study is to detail the mechanisms responsible for the catalytic properties of carbonic anhydrase.

MATERIALS AND METHODS
Isotope Labeling of hCAII-To detect imidazole 15 N signals and assign one of them to His 64 , selectively labeled enzymes were obtained from a double-auxotroph requiring glycine and histidine of bacterial cell Eschericha coli BL21(DE3) containing the pET-hCAII gene and pLys-S, grown in the presence of labeled histidines and/or glycine. The double auxotroph was prepared using two distinct procedures. First is the generalized transduction method using phage P1 vir (20). In this experiment, the glyA gene encoding the serine-glycine hydroxymethyl transferase in the chromosome of E. coli BL21(DE3) (21) was replaced with the deficient gene glyA6 in the chromosome of a glycine auxotroph E. coli IQ417 (22) via the P1 phage particle. The second procedure is the ampicillin treatment method for the isolation of histidine auxotrophic mutants (23). The cells treated with 0 -4 g/ml acridine mutagen ICR191 (6-chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxy-acridine dihydrochloride, Sigma) were grown in an M9 medium containing 50 g/ml ampicillin to enrich histidine auxotroph. The isolated double auxotroph requiring histidine and glycine, designated HS004, was cultured in an M9 medium containing 20 g/ml histidine and 80 g/ml glycine at 37°C. By using this auxotroph transformed by the pET-hCAII-gene plasmid, four types of selectively labeled enzymes ([ring- 15  Expression and Purification of Enzyme-The gene expression was induced by the addition of 1.2 mM isopropyl ␤-Dgalactopyranoside and 1.2 mM ZnSO 4 upon reaching the log  phase (A 600 ϭ 0.6) in the growth curve. The cells were collected 16 h later and the harvest was extracted in 50 mM Tris sulfate, 0.1% Triton X-100, pH 8.0, after sonication. The enzyme was purified by affinity column chromatography as described by Osborne and Tashian (25), followed by gel filtration with Sephadex G-75. The purified sample was stored as a lyophilized powder at Ϫ20°C. The zinc-free apoenzyme was prepared by treating the purified sample with pyridine-2,6-dicarboxylic acid (dipicolinic acid), according to Hunt et al. (26). Protein concentrations were determined by using the extinction coefficient ⑀ ϭ 54800 M Ϫ1 cm Ϫ1 at 280 nm for hCAII (27). The purity was confirmed by reverse-phase high performance liquid chromatography on a C4-column (YMC Co.). The molecular weight (29,000) of native enzyme was confirmed by the sedimentation equilibrium method with an Optima XL-A (28). The enzyme activity was confirmed by the hydrolysis rate of 1 mM 4-nitrophenyl acetate (29,30).
Determination of Acid Base and Tautomeric Equilibrium Constants of Histidine-The imidazolium cation exists in an acid-base equilibrium with two neutral species. These neutral forms of imidazole, the N ␦1 -H tautomer and the N ⑀2 -H tautomer, exist in tautomeric equilibrium. The acid-base equilibrium constants K 1 and K 2 are given by (39). The experimentally determined value K a is given by K a ϭ K 1 ϩ K 2 . The K T and pK a values of L-histidine are 0.25 and 6.2 in aqueous solution, respectively (40 -42). The 15 N chemical shifts at various pH values are derived from the Henderson-Hasselbalch equation as, where ␦ ϾN-H and ␦ ϾN are 15 N chemical shifts of pyrrole-like (ϾN-H) and pyridine-like (ϾN:) nitrogen types, respectively. ␦ ϾN and ␦ ϾN-H are assumed to be 249.5 and 167.5 ppm, respectively. These values were derived from small model compounds (43). The tautomeric equilibrium constant K T is given by the following equation.
The procedure cannot be verified as being highly accurate, but certainly it is more accurate than any other determination for solutions, including the use of C-N coupling constants (44). When there is the expected 82-ppm chemical shift difference, even a 2-3 ppm uncertainty in the limiting shift values of the numerator in Equation 3 will allow quite reasonable estimates (Ϯ5%) of K T . However, the same order of uncertainty in the denominator can cause a significant error, especially when the difference ␦ ϾN Ϫ ␦ ϾNH in Equation 2 is very small, or the tautomerization is in favor of the N ␦1 -H form.

RESULTS
Assignment of 1 H, 13 C, 15 N Signals of His 64 -There is no strategy for the simple direct assignment of the imidazole ring within histidine residues. By combining a unique method of amide assignment and the following techniques of intra-residual assignment, we carried out the unambiguous imidazole assignment of His 64 in hCAII. The double-labeling method (45) was applied to the amide assignment of His 64 , which was performed by using a selectively labeled enzyme, [␣-15 N]His/[1-13 C]Gly-hCAII. Among 12 histidine residues in hCAII, only His 64 is linked to Gly; the peptidyl bond between Gly 63 and His 64 is labeled by both 15 N and 13 C. Twelve singlets of histidine resonances in the decoupling spectrum shown in Fig. 2A change into 11 singlets and one doublet in the nondecoupled spectrum shown in Fig. 2B. This spectral change clearly demonstrates that the doublet is due to His 64 . This amide assignment was further confirmed in the two-dimensional HNCO spectrum of the same sample as shown in Fig. 3A. Fig. 3B shows the 13 C, 1 H plane of the three-dimensional HNCA spectrum of [U-13 C/ 15 N]His-hCAII at 15 N ϭ 117.2 ppm. The cross-peak between the amide proton and the C␣ carbon of His 64 was observed in the spectrum where C␣ ϭ 55.5 ppm. Fig.  3C shows the two-dimensional HCCH spectrum in which the resonances of C␤, H␣, and H␤ of His 64 were observed at 29.3, 5.80, and 4.05 ppm, respectively. Fig. 3D shows the two-dimensional (H␤)C␤(C␥C␦)H␦ spectrum to connect C␤ with H ␦2 . The H ␦2 resonance of His 64 was observed at 6.95 ppm. Fig. 3E shows the 15 N/ 1 H-HSQC spectrum of the [ring-15 N]His-hCAII at pH 5.2. In this spectrum, four correlation signals, N ␦1 -H ␦2 , N ␦1 -H ⑀1 , N ⑀2 -H ␦2 , and N⑀2-H ⑀1 , per histidine residue are observed. When both N ␦1 and N ⑀2 atoms are positively charged (designated as the ϩϾN-H nitrogen type) in the imidazolium cation, the 15 N signals of N ␦1 and N ⑀2 are observed around 176.5 ppm, with N ␦1 generally appearing at a ϳ2 ppm higher frequency than N ⑀2 (40 -43). The identification of N ␦1 and N ⑀2 nuclei can be confirmed at basic pH regions. By gradually changing the pH from 5.2 to basic, one of their signal intensities change characteristically; the N ␦1 -H ␦2 resonance weakens in intensity where the 3 J N␦1-H␦2 coupling is too small (Ϫ2 Hz) (46) to observe the resonance, as shown in Fig. 4. As a result, this weakening signal allows us to assign three other observable resonances, N ␦1 -H ⑀1 , N ⑀2 -H ⑀1 , and N ⑀2 -H ␦2 . Consequently, the H ⑀1 , N ␦1 , and N ⑀2 nuclei of His 64 were assigned to the 1 H and 15 N chemical shifts of 8.03, 177.8, and 175.4 ppm, respectively, at pH 5.2. Venters and co-workers (47) have reported the backbone resonance assignment of hCAII-substituted non-exchangeable protons for deuterium to detect the signals of this large-size protein without overlap, in which there is not enough available chemical shift data of the resonance to confirm our amide assignment of His 64 .
The Imidazole 15 N Signals and Proton-exchange Rate-The assigned imidazole 15 N signals of His 64 can serve as a good probe that provides both the pK a data and information concerning the tautomeric forms of this residue (K T ). Although the pK a data could be obtained using the 1 H signal of H ⑀1 (13), the 1 H signal cannot discriminate between two possible tautomers  of the imidazole ring. To determine the K T value, it is essential to observe the 15 N signals of N ␦1 and N ⑀2 simultaneously. The 15 N signals of the imidazole nitrogen nuclei characteristically reflect the charged states of the imidazole ring (43). In the cationic imidazolium form, both nuclei attached to protons showed a chemical shift at around 176.5 ppm. In the neutral form, N ␦1 -and N ⑀2 -15 N exhibit signals at 167.5 ppm when these nitrogen atoms are protonated and at 249.5 ppm when they are not protonated, allowing us to distinguish between two tauto-meric forms involving these nitrogen atoms. In favorable cases, these 15 N signals of N ␦1 and N ⑀2 are observed in a "fast exchange" regime, where their signals are averaged to give a single resonance. Note that the rate of proton-exchange between these two nitrogen atoms is more than 1.6 ϫ 10 4 s Ϫ1 . When the proton prefers one of the nitrogen nuclei, the weight averaging of the chemical shifts occurs in the N ␦1 and N ⑀2 signals.
For the observation of imidazole 15 N signals of His 64 in hCAII, two-dimensional 15 N/ 1 H-correlation spectroscopy was used, which detects the N ␦1 -H ⑀1 , N ␦1 -H ␦2 , N ⑀2 -H ⑀1 , and N ⑀2 -H ␦2 resonances described above. As shown in Fig. 4, the signals of His 64 were observed to be regarded as "fast" at pH 7.9. In this measurement, all other imidazole 15 N signals except the signal number 6 were also observed as the fast exchange. For signal number 6, considering the signals to be observed in the region of pH 5.2-6.7, one of the exchange rates may change from fast to "intermediate" with increasing pH. However, for this signal disappearance, this could not be concluded easily because the signal intensity is related not only to the exchange, but also to some other factors such as J-coupling constants dependent on pH.
The pH Dependence and Tautomeric Proportion of Histidine Residues-15 N chemical shifts were monitored as a function of pH to investigate the profile of acid-base and tautomeric equilibrium of the histidine residue. The pH-titration curves of N ␦1 and N ⑀2 for all 12 histidine residues are shown in Fig. 5. To simply illustrate the pH dependence of the 15 N chemical shift, the variation of the 15 N chemical shifts with pH are simulated by substituting the pK a value of L-histidine (6.2), the chemical shift value of the ϩϾN-H type, and the variable weight average of ϾN-H and ϾN: chemical shifts for Equations 1-3, as shown in Fig.  6. This figure allows us to facilitate the investigation of the tautomeric proportion in histidine residues under the fast exchange situation. Comparing Figs. 5 and 6, the approximate K T values of the histidine residues are quite obvious. In the case of pH-independent 15 N chemical shifts, their titration curves need not be compared with that of Fig. 6. In both cases, the K T values were calculated by Equations 2 and 3 using the basic 15 N-limitting shift. Table 2 summarizes the acidbase and tautomeric equilibrium constants of the histidine residues. According to their titration profiles, histidine residues of hCAII were classified into three groups, A, B, and C, as summarized in Table 2. Group A consists of seven histidine residues sensitive to the tested pH changes (Group A: the change between acid and base limiting shift values is Ͼ30 ppm for either N ␦1 or   Fig. 6. Those for histidine residues with pH-independent profiles were directly obtained from Equations 2 and 3. N ⑀2 ). These histidine residues would be distributed on the surface or in a solvent-accessible position in the molecule. For this study, one of them was unambiguously assigned to His 64 as described above. His 64 occurs in the equivalent proportion of the tautomer: K T ϭ 1.0. To our knowledge, no behavior similar to that of His 64 has been found in any other protein. The six other histidine signals are designated as 1-6. The K T constants are found to be in the range from 0.01 to 0.4. Curves 1 and 2 show that the hydrogen atoms are localized on N ⑀2 of their histidine residues, whereas curves 3-6 show normal tautomeric profiles, similar to that of L-histidine amino acid in aqueous solution. These histidine residues are thought to be on the surface of the molecule. Group B consists of two pH-insensitive histidines, designated as 7 and 8 (Group B: the change between acid and base limiting shift values is Ͻ0.1 ppm for both N ␦1 and N ⑀2 ). The N ␦1 signals of 7 and 8 appeared as ϾN-H type, and the N ⑀2 signal as the ϾN: type, thus indicating that these histidine residues exist as N ␦1 -H tautomers in all pH values tested. Group C consists of three slightly pH-sensitive histidines designated as curves 9 -11 (Group C: the change between acid and base limiting shift values is between 0.5 and 5 ppm for either N ␦1 or N ⑀2 ). The N ␦1 of 9 and 10, and N ⑀2 of 11 appear as ϾN-H types. This result shows that 9 and 10 histidines occur as N ␦1 -H tautomers; N ␦1 of 9 experiences a 7 ppm low field chemical shift change compared with typical pyrrole-like (ϾN-H) nitrogen and N ␦1 of 10 at 12.5 ppm. Number 11 of the histidine residue behaves like a N ⑀2 -H tautomer; N ⑀2 is a 9.5-ppm low field chemical shift change.
Identifications and Assignments of Zinc-bound and Buried Histidine Residues-Crystal structure shows two kinds of interior or not exposed histidine residues: zinc-bound histidines, His 94 , His 96 , and His 119 , and buried histidines, His 107 and His 122 (12). These residues except for His 122 are illustrated in Fig. 1. First, we distinguished the zinc-bound histidines from the buried histidines by comparing the C ⑀1 -H ⑀1 correlation signal of the holoenzyme with that of the apoenzymes. The pH titration experiment was carried out on [ring-C ⑀1 -13 C]His-hCAII using 13 C/ 1 H HSQC experiments. The H ⑀1 titration profiles are consistent with those from the 15 N/ 1 H experiments described above; the pK a values and chemical shift values of H ⑀1 were confirmed. Fig. 7, A and B, shows the spectra of holo-and apoenzymes labeled with [ring-C ⑀1 -13 C]His at pH 7.0. Comparing them, the His 64 signal and three other signals (numbers 9 -11) disappear from the spectrum of the apoenzyme. Instead of these signals, several other signals appear. This observation shows that signals 9 -11 were from three zinc-bound imidazoles of the histidine residues. This result is consistent with that of the above described 15 N experiment in which either a N ␦1 or N ⑀2 signal is observed in the region between 205 and 215 ppm, which is of the zinc-bound nitrogen type (48). Subsequently, we tentatively assigned signals 9 -11 to the zinc-bound histidine residues by using the crystal structure of enzyme. Among the three His residues coordinated with the zinc ion, His 119 is  a Numbers 1-6 histidine residues are located on the surface of molecule, which includes histidines 3, 4, 10, 15, 17, and 36. The number 1 or 2 may be from His 15 (see also "Discussion"). His 64 is assigned using unique NMR techniques. The residues in parentheses are tentatively assigned using crystal structure. b ND, not determined. unique in that its N ␦1 is coordinated with the zinc, whereas His 94 and His 96 are coordinated with the zinc via their N ⑀2 atoms, thus, number 11 would be assigned to the imidazole of His 119 . The H ⑀1 atom of His 119 exists in the plane of the indole ring of Trp 209 . The ring current effect of Trp 209 is expected to bring about the low field chemical shift change of the H ⑀1 . In fact, the H ⑀1 nucleus of number 11 was observed at 9.3 ppm. Numbers 9 and 10 are assigned to either the zinc-bound imidazole of His 94 or His 96 (these are designated as His 94/96 ). In the buried histidine residues, His 107 exists in the plane perpendicular to the indole ring of Trp 209 , in contrast to His 119 . The upfield chemical shift of the H ⑀1 observed in the spectra is 5.1 ppm of number 8, and thus, number 8 would be assigned to His 107 . The remaining signal of number 7 would be assigned to His 122 .
Direct Observation of Protons Fixed on Nitrogen within the Imidazole Group of Histidine Residues-Although, at a higher pH value than 2.0, an imidazole H N (H ␦1 or H ⑀2 ) signal is not observed because of the exchange of imidazole H N with the proton of bulk water, the imidazole H N shows its signal for a fixed or hydrogen-bonded proton in the downfield region around 13.5 ppm. Five signals were observed in this region of the 15 N/ 1 H HSQC spectrum for the 15 N-labeled enzyme, as shown in Fig. 8A. All five 15 N chemical shifts correspond with those of the above described ϾN-H type nitrogen of either Group B (N ␦1 of His 122 and N ␦1 of His 107 ) or C (N␦1 of His 94/96 and N ⑀2 of His 119 ), whereas no signal corresponds with the nitrogen of Group A (surface and His 64 ). The imidazole H N assignment is supported by an additional NOE cross-peak (49). The NOE cross-peaks for H ␦1 of His 107 , H ␦1 of His 94/96 , and H ⑀2 of His 119 were confirmed by using the NOESY as shown in Fig.   8B. For H ␦1 of His 122 , the NOE cross-peak was confirmed by using 15 N/ 1 H HMQC-NOESY as shown in Fig. 8C. The H N chemical shifts are added to Table 2. Scalar spin-spin coupling constants ( 1 J NH ) of the N-H bonds of the imidazole ring are summarized in Table 2. The values of 1 J NH provides a direct measure of covalent bond character; the observed values of 92-97 Hz indicate that these imino protons are fixed covalently about 90 -100% (50).
The H N chemical shifts were monitored as a function of pH to calculate pK a values. In the 15 N-labeled enzyme, all five H N signals were observed in the region of pH 5.7-8.8. All H N chemical shifts were slightly sensitive to pH change, as shown in Fig. 8D. For Group B, the H N signal of His 107 (H ␦1 ) shifts to a slightly lower field as the pH increases, which is different from the pH dependence of zinc-bound histidine residues in the direction of shift. The titration curve does not exhibit sigmoid behaviors and the difference between chemical shifts at acidic and basic is very small, 0.06 ppm. The H N signal of His 122 (H ␦1 ) shifts to a slightly higher field. In the H ␦1 of His 94/96 and H ⑀2 of His 119 of Group C, the titration curves exhibited the clearly sigmoid behaviors dependent on pH required to calculate pK a values and limiting shifts using ␦ obs of the proton instead of ␦ N obs in Equation 1. The pK a values of H ␦1 of His 94/96 (number 9), H ␦1 of His 94/96 (number 10), and H ⑀2 of His 119 are 7.3 Ϯ 0.04, 7.2 Ϯ 0.02, and 7.2 Ϯ 0.03, respectively. The pK a values of His 94 , His 96 , and His 119 probably reflect the titration behavior of other residues or groups because these residues are unattached to water molecules. Importantly, these pK a values are in good agreement with that of His 64 determined in our measurements. The coincidence implies that the titration behavior of His 64 is reflected on those of zinc-bound histidine residues. However, the possibility that the observed effect is due to the ionization of zinc-bound water could not be ruled out.

Implication of Tautomeric Equilibrium Constant of Histidine
Residues-We determined the tautomeric equilibrium constant (K T ) of the imidazole ring of His 64 to be 1.0, according to the unambiguous assignment of 15 N signals, the analysis of their pH dependences, and a comparison of experimental and simulated titration curves. This value was different from those of 11 other histidine residues in this enzyme, whereas its pK a value of 7.2-7.3 was indistinguishable from those of the others ( Table 2). The K T value of 1.0 indicates that two imidazole nitrogen atoms (N ␦1 and N ⑀2 ) can be equally involved in the catalytic reaction. It is therefore reasonable to assume that one of the imidazole nitrogen atoms acts as a general acid, whereas the other acts as a general base, as shown in Equation 4.

(Eq. 4)
Because the tautomeric equilibrium of an imidazole group is dominated by hydrogen bond interactions with the ␦1-nitrogen where an acid or base interacts strongly, the usual equilibrium condition gives a large deviation of the K T values from 1 (51). Four signals (numbers 9 -11, and His 64 ) disappeared compared with A. Instead of these signals, two sharp signals and several weak signals were observed. In these spectra, numbers 9 -11 were identified with zinc-bound histidine residues.
For example, the N ␦1 -H tautomer dominates in the imidazole group of cis-urocanic acid, as indicated by K T ϭ 5.2 (Equation 5), in which the intramolecular hydrogen bond can be formed, whereas the N ⑀2 -H tautomer is favorable in Equation 6 with the trans-configuration preventing the hydrogen bond though a carboxylate anion (K T ϭ 0.37).
(Eq. 5) (Eq. 6) These K T values suggest that the imidazole group intrinsically tends to be the N ⑀2 -H tautomer, unless a hydrogen bond interacts with the ␦1-nitrogen of the imidazole ring. In fact, the K T values for 6 histidine residues exposed to the solvent (Group A in Table 2) were shown to be less than 0.4, indicating the prevalence of the N ⑀2 -H tautomer.
(Eq. 7) As shown in Equation 7, the conformational flexibility along the C␤-C␥ bond of 3-(imidazol-4-yl)propionic acid permits the partial formation of a hydrogen bond. In this case, the N ⑀2 -H tautomer still dominates, as in Equation 6, but the equilibrium shifts in favor of the N ␦1 -H tautomer (K T ϭ 0.61). Based on this analogy, His 64 should have a structure-specific determinant to promote the partial formation of a hydrogen bond. As illustrated in Equation 8, we consider that a negative charge of the zinc-bound hydroxide ion is responsible for increasing the population of the N ␦1 -H tautomer, and a network of water molecules is responsible for attenuating the hydrogen bonding effect to a level comparable with that of the counterpart. Using this equation, we could consider that the tautomerization of His 64 would be coupled to the ionization of the zincbound solvent.
To our knowledge, no real compound model has been reported to explain the ⑀2-nitrogen of an imidazole group in hydrogen bond interactions. In this case, we assumed that a hydrogen bond partner in close proximity to the ⑀2-nitrogen affects the change in the K T value, in contrast to the ␦1-nitrogen case as described above, is expected to decrease to much less than 0.4. This implies that one of the K T values in Group A, Ͻ0.05 of signal number 1 or 0.1 of number 2, is from His 15 because the ⑀2-nitrogen of His 15 can form a hydrogen bond with oxygen of Lys-9 as a acceptor (distance: 3.19 Å), which may stabilize the N ⑀2 -H tautomeric form. For His 107 and His 122 in Group B, two hydrogen bond interactions are seen in the imidazole group, as shown in Fig. 8E, a and b, respectively. The conditions of His 107 and His 122 existing in a hydrogen bond network are apparently similar. Based on the structures, both histidine residues should take only the N ␦1 -H tautomer. This is also supported by our measurement for J values, in which the J N␦1-H␦1 value of His 107 is close to that of His 122 , indicating that hydrogen localization on the imidazole nitrogen of His 107 is essentially equal to that of His 122 . However, the apparent K T values, 7.6 for His 107 and Ͼ20 for His 122 , were calculated by Equations 2 and 3, although a small error contained in the difference ␦ ϾN Ϫ ␦ ϾNH in Equation 2 could make the comparison between their K T values difficult. For the difference of these histidine residues, it is possible to argue the difference of their strengths of hydrogen bonds in terms of chemical shift values. Comparing Fig. 8E, a and b, we note that the distance of hydrogen bond between N ␦1 of His 122 and the carbonyl oxygen of Ala 142 (3.12 Å) is appreciably longer than that between ␦1-nitrogen of His 107 and the carboxyl oxygen of Glu 117 (2.84 Å). Similarly, there is a slight increase in distance between the ␦1-nitrogen of His 122 and the hydroxyl oxygen of Tyr 51 (2.78 Å) compared with that between the ␦1-nitrogen of His 107 and the hydroxyl oxygen of Tyr 194 (2.66 Å). Such an increase in distances could lead to a weakening of hydrogen bond. In the N ␦1 -H tautomer illustrated in Fig. 8E, a, the chemical shift values of N ␦1 (177.5 ppm) and N ⑀2 (240.5 ppm) for His 107 agree well with the expected limiting shifts due to donation (ϩ10 ppm) and acceptance (Ϫ10 ppm) of hydrogen bonds, respectively (43,52). Using these limiting shifts, the K T value for His 107 , Ͼ20, is determined by the calculation using Equations 2 and 3, taking only N ␦1 -H tautomer. In contrast, the corresponding values of N ␦1 (167.8 ppm) and N ⑀2 (249.3 ppm) for His 122 do not accord with the above empirical rule, but appear to be independent of the hydrogen bonds with Tyr 51 and Ala 142 . That is, assume that neither ␦1nor ⑀2-nitrogen atoms of His 122 is firmly involved in the hydrogen bond interactions but the ⑀2-nitrogen is rather involved in hydrogen bond interaction with the hydroxyl oxygen of Tyr 51 because the slight or partial negative charge of the carbonyl oxygen of Ala 142 can likely balance with the amide of His 122 . For His 122 , thus, the limiting shifts without the hydrogen bond, 167.5 and 249.5 ppm, are used to determine its K T value, Ͼ20, taking only the N ␦1 -H tautomer. For Group C, because of zinc coordination and hydrogen bonding, His 94 , His 96 , and His 119 would exist only in one tautomeric form. Although their N H chemical shifts are ϳ10 ppm lower than a typical chemical shift, 167.5 ppm, the imidazole N-H spin-coupling constants range from 90 to 98 Hz. Therefore the H ␦1 protons of His 94 and His 96 and the H ⑀2 of His 119 are essentially 100% localized on these nitrogen atoms, based on their one-bond J coupling constants.
Catalytic Mechanism of Carbonic Anhydrase II-It has been accepted that protonation of the N ␦1 of His 64 results from the ionization of the water molecule to generate the hydroxide ion near the zinc ion, as shown in Equation 9 (5).
(Eq. 9) replacement of some water molecules between the zinc ion and His 64 with hydroxyl ions. The loss of their protons may decrease the effective transfer of the productive proton by tautomerization of His 64 .
In this study, our heteronuclear NMR approach to His 64 shows that both the N ␦1 -H and N ⑀2 -H tautomeric forms in equilibrium with an imidazolium ion are in the same population, providing information about the general acid-base function of the imidazole nitrogen. Here, we demonstrate a proton release model using the tautomeric information of His 64 , implying a new insight into the catalytic mechanism for the hydration or dehydration reaction in human carbonic anhydrase II, i.e. the split of the water bridge and the flow of water molecules.