Is there a weak H‐bond → LBHB transition on tetrahedral complex formation in serine proteases?

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102-His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme-inhibitor TC is a strong ionic asymmetric one-well hydrogen bond, in contrast to a previous suggestion that it is a weak H-bond in the former and a double-well LBHB in the latter.     

An effective enzyme interacting with poly (dT-dA).poly (dT-dA): a dynamic enhancer-repressor action.

The Green function technique is used to study the open hydrogen bond probability of poly(dT-dA).poly(dT-dA) when an effective enzyme is attached to the helix. The DNA interstrand hydrogen bond mean motion and probability of fluctuating to an open state depends on the internal vibrational frequency of the enzyme. An enzyme with internal frequency of 80 cm-1 reduces hydrogen bond motion and the resulting probability of hydrogen bond fluctuational opening. An enzyme with internal frequency of 72 cm-1 increases hydrogen bond motion and the probability of hydrogen bond breaking.     

Ab Initio Quantum Mechanics Analysis of Imidazole C-H…O Water Hydrogen Bonding and a Molecular Mechanics Forcefield Correction

Abstract

While it is well established that classical hydrogen bonds play an important role in enzyme structure, function and dynamics, the role of weaker, but ‘activated’ C-H donor hydrogen bonds is poorly understood. The most important such case involves histidine which often plays a direct role in enzyme catalysis and possesses the most acidic C-H donor group of the standard amino acids. In the present study, we obtained optimized geometries and hydrogen bond interaction energies for C-H…O hydrogen bonded complexes between methane, ethylene, benzene, acetylene, and imidazole with water at the MP2-FC/6-31++G(2d,2p) and MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) levels of theory. A strong linear relationship is obtained between the stability of the various hydrogen bonded complexes and both separation distances for H…0 and C—O. In general, these calculations indicate that C-H…0 interactions can be classified as hydrogen bonding interactions, albeit significantly weaker than the classical hydrogen bonds, but significantly stronger than just van der Waals interactions. For instance, while the electronic energy of stabilization at the MP2-FC/aug-cc-pVDZ//MP2-FC/6-31++G(2d,2p) level of theory of a water C-H…O water hydrogen bond is 4.36 kcal/mol more stable than the methane C-H…O water interaction, the water-water hydrogen bond is only 2.06 kcal/mol more stable than the imidazole C_e?H…O water hydrogen bond. Neglecting this latter hydrogen bonding interaction is obviously unacceptable. We next compare the potential energy surfaces for the imidazole C_e?H…O water and imidazole N_d?H…O hydrogen bonded complexes computed at the MP2/6-31++G(2d,2p) level of theory with the potential energy surface computed using the AMBER molecular mechanics program and forcefields. While the Weiner et al and Cornell et al AMBER forcefields reasonably account for the imidazole N-H…O water interaction, these forcefields do not adequately account for the imidazole C_e?H…O water hydrogen bond. A forcefield modification is offered that results in excellent agreement between the ab initio and molecular mechanics geometry and energy for this C-H…O hydrogen bonded complex.     

An Effective Enzyme Interacting with Poly (dT-dA)· Poly (dT-dA): A Dynamic Enhancer-Repressor Action

Abstract

The Green function technique is used to study the open hydrogen bond probability of poly(dT-dA)·poly(dT-dA) when an effective enzyme is attached to the helix. The DNA interstrand hydrogen bond mean motion and probability of fluctuating to an open state depends on the internal vibrational frequency of the enzyme. An enzyme with internal frequency of 80 cm ^?1 reduces hydrogen bond motion and the resulting probability of hydrogen bond fluctuational opening. An enzyme with internal frequency of 72 cm ^?1 increases hydrogen bond motion and the probability of hydrogen bond breaking.     

Critical hydrogen bonds and protonation states of pyridoxal 5'-phosphate revealed by NMR

In this contribution we review recent NMR studies of protonation and hydrogen bond states of pyridoxal 5'-phosphate (PLP) and PLP model Schiff bases in different environments, starting from aqueous solution, the organic solid state to polar organic solution and finally to enzyme environments. We have established hydrogen bond correlations that allow one to estimate hydrogen bond geometries from (15)N chemical shifts. It is shown that protonation of the pyridine ring of PLP in aspartate aminotransferase (AspAT) is achieved by (i) an intermolecular OHN hydrogen bond with an aspartate residue, assisted by the imidazole group of a histidine side chain and (ii) a local polarity as found for related model systems in a polar organic solvent exhibiting a dielectric constant of about 30. Model studies indicate that protonation of the pyridine ring of PLP leads to a dominance of the ketoenamine form, where the intramolecular OHN hydrogen bond of PLP exhibits a zwitterionic state. Thus, the PLP moiety in AspAT carries a net positive charge considered as a pre-requisite to initiate the enzyme reaction. However, it is shown that the ketoenamine form dominates in the absence of ring protonation when PLP is solvated by polar groups such as water. Finally, the differences between acid-base interactions in aqueous solution and in the interior of proteins are discussed. This article is part of a special issue entitled: Pyridoxal Phosphate Enzymology.     

Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques

We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5'-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide-NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic roles for two water bridged residues (Asp-98 and His-255) in the active site of copper-containing nitrite reductase

Two active site residues, Asp-98 and His-255, of copper-containing nitrite reductase (NIR) from Alcaligenes faecalis have been mutated to probe the catalytic mechanism. Three mutations at these two sites (D98N, H255D, and H255N) result in large reductions in activity relative to native NIR, suggesting that both residues are involved intimately in the reaction mechanism. Crystal structures of these mutants have been determined using data collected to better than 1. 9-A resolution. In the native structure, His-255 Nepsilon2 forms a hydrogen bond through a bridging water molecule to the side chain of Asp-98, which also forms a hydrogen bond to a water or nitrite oxygen ligated to the active site copper. In the D98N mutant, reorientation of the Asn-98 side chain results in the loss of the hydrogen bond to the copper ligand water, consistent with a negatively charged Asp-98 directing the binding and protonation of nitrite in the native enzyme. An additional solvent molecule is situated between residues 255 and the bridging water in the H255N and H255D mutants and likely inhibits nitrite binding. The interaction of His-255 with the bridging water appears to be necessary for catalysis and may donate a proton to reaction intermediates in addition to Asp-98.     

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.     

Refined crystal structure of carboxypeptidase A at 1.54 A resolution

The crystal structure of bovine carboxypeptidase A (Cox) has been refined at 1.54 A resolution using the restrained least-squares algorithm of Hendrickson & Konnert (1981). The crystallographic R factor (formula; see text) for structure factors calculated from the final model is 0.190. Bond lengths and bond angles in the carboxypeptidase A model have root-mean-square deviations from ideal values of 0.025 A and 3.6 degrees, respectively. Four examples of a reverse turn like structure (the "Asx" turn) requiring an aspartic acid or asparagine residue are observed in this structure. The Asx turn has the same number of atoms as a reverse turn, but only one peptide bond, and the hydrogen bond that closes the turn is between the Asx side-chain CO group and a main-chain NH group. The distributions of CO-N and NH-O hydrogen bond angles in the alpha-helices and beta-sheet structures of carboxypeptidase A are centered about 156 degrees. A total of 192 water molecules per molecule of enzyme are included in the final model. Unlike the hydrogen bonding geometry observed in the secondary structure of the enzyme, the CO-O(wat) hydrogen bond angle is distributed about 131 degrees, indicating the role of the lone pair electrons of the carbonyl oxygen in the hydrogen bond interaction. Twenty four solvent molecules are observed buried within the protein. Several of these waters are organized into hydrogen-bonded chains containing up to five waters. The average temperature factor for atoms in carboxypeptidase A is 8 A2, and varies from 5 A2 in the center of the protein, to over 30 A2 at the surface.     

α‐chymotrypsin in water–acetone and water–dimethyl sulfoxide mixtures: Effect of preferential solvation and hydration

We investigated water/organic solvent sorption and residual enzyme activity to simultaneously monitor preferential solvation/hydration of protein macromolecules in the entire range of water content at 25°C. We applied this approach to estimate protein destabilization/stabilization due to the preferential interactions of bovine pancreatic α‐chymotrypsin with water‐acetone (moderate‐strength H‐bond acceptor) and water‐DMSO (strong H‐bond acceptor) mixtures. There are three concentration regimes for the dried α‐chymotrypsin. α‐Chymotrypsin is preferentially hydrated at high water content. The residual enzyme activity values are close to 100%. At intermediate water content, the dehydrated α‐chymotrypsin has a higher affinity for acetone/DMSO than for water. Residual enzyme activity is minimal in this concentration range. The acetone/DMSO molecules are preferentially excluded from the protein surface at the lowest water content, resulting in preferential hydration. The residual catalytic activity in the water‐poor acetone is ～80%, compared with that observed after incubation in pure water. This effect is very small for the water‐poor DMSO. Two different schemes are operative for the hydrated enzyme. At high and intermediate water content, α‐chymotrypsin exhibits preferential hydration. However, at intermediate water content, in contrast to the dried enzyme, the initially hydrated α‐chymotrypsin possesses increased preferential hydration parameters. At low water content, no residual enzyme activity was observed. Preferential binding of DMSO/acetone to α‐chymotrypsin was detected. Our data clearly demonstrate that the hydrogen bond accepting ability of organic solvents and the protein hydration level constitute key factors in determining the stability of protein–water–organic solvent systems.     

Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase

The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA. The Ser-148----Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold. A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT. The three-dimensional structure of Ala-148 CAT at 2.34-A resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region. The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization. Ser-148 has also been replaced with cysteine and asparagine; the Ser-148----Cys mutation results in a 705-fold decrease in kcat and the Ser-148----Asn substitution in a 214-fold reduction in kcat. Removing the hydrogen bond donor (Ser-148----Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148----Cys or Asn).     

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Specificity and catalysis of uracil DNA glycosylase. A molecular dynamics study of reactant and product complexes with DNA.

The structure of uracil DNA glycosylase (UDG) in complex with a nonamer duplex DNA containing a uracil has been determined only in the product state. The reactant state was constructed by reattaching uracil to the deoxyribose, and both complexes were studied by molecular dynamics simulations. Significant changes in the positions of secondary structural elements in the enzyme are induced by the hydrolysis of the glycosidic bond. The simulations show that the specificity of the uracil pocket in the enzyme is largely retained in both complexes with the exception of Asn-204, which has been identified as a residue that contributes to discrimination between uracil and cytosine. The hydrogen bond between the amide group of Asn-204 and O(4) of uracil is disrupted by fluctuations of the side chain in the reactant state and is replaced by a hydrogen bond to water molecules trapped in the interior of the protein behind the uracil binding pocket. The role of two residues implicated by mutation experiments to be important in catalysis, His-268 and Asp-145, is clarified by the simulations. In the reactant state, His-268 is found 3.45 +/- 0.34 A from the uracil, allowing a water molecule to form a bridge to O(2). The environment in the enzyme raises the pK(a) value of His-268 to 7.1, establishing a protonated residue for assisting in the hydrolysis of the glycosidic bond. In agreement with the crystallographic structure, the DNA backbone retracts after the hydrolysis to allow His-268 to approach the O(2) of uracil with a concomitant release of the bridging water molecule and a reduction in the pK(a) to 5.5, which releases the proton to the product. The side chain of Asp-145 is fully solvated in the reactant state and H-bonded through a water molecule to the 3'-phosphate of uridine. Both the proximity of Asp-145 to the negatively charged phosphate and its pK(a) of 4.4 indicate that it cannot act as a general base catalyst. We propose a mechanism in which the bridging water between Asp-145 and the 3'-phosphate accepts a proton from another water to stabilize the bridge through a hydronium ion as well as to produce the hydroxide anion required for the hydrolytic step. The mechanism is consistent with known experimental data.     

The mechanism of glycerol conduction in aquaglyceroporins.

BACKGROUND: The E. coli glycerol facilitator, GlpF, selectively conducts glycerol and water, excluding ions and charged solutes. The detailed mechanism of the glycerol conduction and its relationship to the characteristic secondary structure of aquaporins and to the NPA motifs in the center of the channel are unknown. RESULTS: Molecular dynamics simulations of GlpF reveal spontaneous glycerol and water conduction driven, on a nanosecond timescale, by thermal fluctuations. The bidirectional conduction, guided and facilitated by the secondary structure, is characterized by breakage and formation of hydrogen bonds for which water and glycerol compete. The conduction involves only very minor changes in the protein structure, and cooperativity between the GlpF monomers is not evident. The two conserved NPA motifs are strictly linked together by several stable hydrogen bonds and their asparagine side chains form hydrogen bonds with the substrates passing the channel in single file. CONCLUSIONS: A complete conduction of glycerol through the GlpF was deduced from molecular dynamics simulations, and key residues facilitating the conduction were identified. The nonhelical parts of the two half-membrane-spanning segments expose carbonyl groups towards the channel interior, establishing a curve-linear pathway. The conformational stability of the NPA motifs is important in the conduction and critical for selectivity. Water and glycerol compete in a random manner for hydrogen bonding sites in the protein, and their translocations in single file are correlated. The suggested conduction mechanism should apply to the whole family.     

15N- and 13C-NMR investigations of glucose oxidase from Aspergillus niger

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N- and 13C-enriched FAD derivatives and investigated by 15N- and 13C-NMR spectroscopy. On the basis of the 15N-NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase. Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme-bound FAD were derived from the 13C-NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl-riboflavin in apolar solvents. In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein. As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzyme-bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution. Some of the 15N- and 13C-resonance positions of the enzyme-bound reduced cofactor are markedly pH-dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.     

Molecular basis for the reduced catalytic activity of the naturally occurring T560M mutant of human 12/15-lipoxygenase that has been implicated in coronary artery disease

Lipoxygenases have been implicated in cardiovascular disease. A rare single-nucleotide polymorphism causing T560M exchange has recently been described, and this mutation leads to a near null variant of the enzyme encoded for by the ALOX15 gene. When we inspected the three-dimensional structure of the rabbit ortholog, we localized Thr-560 outside the active site and identified a hydrogen bridge between its side chain and Gln-294. This interaction is part of a complex hydrogen bond network that appears to be conserved in other mammalian lipoxygenases. Gln-294 and Asn-287 are key amino acids in this network, and we hypothesized that disturbance of this hydrogen bond system causes the low activity of the T560M mutant. To test this hypothesis, we first mutated Thr-560 to amino acids not capable of forming side chain hydrogen bridges (T560M and T560A) and obtained enzyme variants with strongly reduced catalytic activity. In contrast, enzymatic activity was retained after T560S exchange. Enzyme variants with strongly reduced activity were also obtained when we mutated Gln-294 (binding partner of Thr-560) and Asn-287 (binding partner of Gln-294 and Met-418) to Leu. Basic kinetic characterization of the T560M mutant indicated that the enzyme lacks a kinetic lag phase but is rapidly inactivated. These data suggest that the low catalytic efficiency of the naturally occurring T560M mutant is caused by alterations of a hydrogen bond network interconnecting this residue with active site constituents. Disturbance of this bonding network increases the susceptibility of the enzyme for suicidal inactivation.     

Low-barrier hydrogen bond plays key role in active photosystem II--a new model for photosynthetic water oxidation

The function and mechanism of Tyr(Z) in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with Tyr(Z), and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of Tyr(Z) function in two aspects. First, the LBHB plays a key role to decrease the activation energy for Tyr(Z) oxidation and Tyr(Z)(.) reduction during photosynthetic water oxidation. Upon the oxidation of Tyr(Z), the hydrogen bond between Tyr(Z) and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon Tyr(Z)(.) reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after Tyr(Z) oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of Tyr(Z) oxidation and Tyr(Z)(.) reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between Tyr(Z) and Tyr(D) in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with Tyr(Z). This type of change arising from radiation damage has been confirmed in other enzyme systems.     

Hydrogen bonds from water molecules to aromatic acceptors in very high-resolution protein crystal structures

Short contacts of water molecules with the pi-faces of aromatic residues were studied in a set of 75 very high resolution (<1.1 A) protein X-ray crystal structures. For 18 water molecules found at distances to aromatic midpoints <3.5 A, it was attempted to assign the hydrogen bond configuration (without experimental knowledge of the H-atom positions) by inspection of the surrounding. For approximately one-quarter of the cases, evidence for an O-H...pi hydrogen bond was found, another one-quarter does not form such a hydrogen bond, and for the remaining half, no conclusive assignment could be made. The results confirm the relatively frequent occurrence of aromatic hydrogen bonding in biomolecular hydration, but also underline difficulties in hydrogen bond assignment without reliable knowledge of the H-atom positions.     

An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α‐helices compared to β‐sheets by ～0.2 kcal/mol. [2] Charge‐stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ～2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies.