Low-barrier H-bond Research Articles

Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin.

Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.

Exploring the Deprotonation Process during Incorporation of a Ligand Water Molecule at the Dangling Mn Site in Photosystem II.

The Mn4CaO5 cluster, featuring four ligand water molecules (W1 to W4), serves as the water-splitting site in photosystem II (PSII). X-ray free electron laser (XFEL) structures exhibit an additional oxygen site (O6) adjacent to the O5 site in the fourth lowest oxidation state, S3, forming Mn4CaO6. Here, we investigate the mechanism of the second water ligand molecule at the dangling Mn (W2) as a potential incorporating species, using a quantum mechanical/molecular mechanical (QM/MM) approach. Previous QM/MM calculations demonstrated that W1 releases two protons through a low-barrier H-bond toward D1-Asp61 and subsequently releases an electron during the S2 to S3 transition, resulting in O•- at W1 and protonated D1-Asp61. During the process of Mn4CaO6 formation, O•-, rather than H2O or OH-, best reproduced the O5···O6 distance. Although the catalytic cluster with O•- at O6 is more stable than that with O•- at W1 in S3, it does not occur spontaneously due to the significantly uphill deprotonation process. Assuming O•- at W2 incorporates into the O6 site, an exergonic conversion from Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV) (equivalent to the open-cubane S2 valence state) to Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III) (equivalent to the closed-cubane S2 valence state) occurs. These findings provide energetic insights into the deprotonation and structural conversion events required for the Mn4CaO6 formation.

Interplay of two low-barrier hydrogen bonds in long-distance proton-coupled electron transfer for water oxidation.

D1-Tyr161 (TyrZ) forms a low-barrier H-bond with D1-His190 and functions as a redox-active group in photosystem II. When oxidized to the radical form (TyrZ-O•), it accepts an electron from the oxygen-evolving Mn4CaO5 cluster, facilitating an increase in the oxidation state (Sn; n = 0-3). In this study, we investigated the mechanism of how TyrZ-O• drives proton-coupled electron transfer during the S2 to S3 transition using a quantum mechanical/molecular mechanical approach. In response to TyrZ-O• formation and subsequent loss of the low-barrier H-bond, the ligand water molecule at the Ca2+ site (W4) reorients away from TyrZ and donates an H-bond to D1-Glu189 at Mn4 of Mn4CaO5 together with an adjacent water molecule. The H-bond donation to the Mn4CaO5 cluster triggers the release of the proton from the lowest pKa site (W1 at Mn4) along the W1…D1-Asp61 low-barrier H-bond, leading to protonation of D1-Asp61. The interplay of the two low-barrier H-bonds, involving the Ca2+ interface and forming the extended Grotthuss-like network [TyrZ…D1-His190]-[Mn4CaO5]-[W1…D1-Asp61], rather than the direct electrostatic interaction, is likely a basis of the apparent long-distance interaction (11.4 Å) between TyrZ-O• formation and D1-Asp61 protonation.

Structural and energetic insights into Mn-to-Fe substitution in the oxygen-evolving complex

Manganese (Mn) serves as the catalytic center for water splitting in photosystem II (PSII), despite the abundance of iron (Fe) on earth. As a first step toward why Mn and not Fe is employed by Nature in the water oxidation catalyst, we investigated the Fe4CaO5 cluster in the PSII protein environment using a quantum mechanical/molecular mechanical (QM/MM) approach, assuming an equivalence between Mn(III/IV) and Fe(II/III). Substituting Mn with Fe resulted in the protonation of μ-oxo bridges at sites O2 and O3 by Arg357 and D1-His337, respectively. While the Mn4CaO5 cluster exhibits distinct open- and closed-cubane S2 conformations, the Fe4CaO5 cluster lacks this variability due to an equal spin distribution over sites Fe1 and Fe4. The absence of a low-barrier H-bond between a ligand water molecule (W1) and D1-Asp61 in the Fe4CaO5 cluster may underlie its incapability for ligand water deprotonation, highlighting the relevance of Mn in natural water splitting.

Mechanism of Asparagine-Mediated Proton Transfer in Photosynthetic Reaction Centers.

In photosynthetic reaction centers from purple bacteria (PbRCs), light-induced charge separation leads to the reduction of the terminal electron acceptor quinone, QB. The reduction of QB to QB•- is followed by protonation via Asp-L213 and Ser-L223 in PbRC from Rhodobacter sphaeroides. However, Asp-L213 is replaced with nontitratable Asn-L222 and Asn-L213 in PbRCs from Thermochromatium tepidum and Blastochloris viridis, respectively. Here, we investigated the energetics of proton transfer along the asparagine-involved H-bond network using a quantum mechanical/molecular mechanical approach. The potential energy profile for the H-bond between H3O+ and the carbonyl O site of Asn-L222 shows that the proton is predominantly localized at the Asn-L222 moiety in the T. tepidum PbRC protein environment, easily forming the enol species. The release of the proton from the amide -NH2 site toward Ser-L232 via tautomerization suffers from the energy barrier. Upon reorientation of Asn-L222, the enol -OH site forms a short low-barrier H-bond with Ser-L232, facilitating protonation of QB•- in a Grotthuss-like mechanism. This is a basis of how asparagine or glutamine side chains function as acceptors/donors in proton transfer pathways.

Release of a Proton and Formation of a Low-Barrier Hydrogen Bond between Tyrosine D and D2-His189 in Photosystem II.

In photosystem II (PSII), the second-lowest oxidation state (S1) of the oxygen-evolving Mn4CaO5 cluster is the most stable, as the radical form of the redox-active D2-Tyr160 is considered to be a candidate that accepts an electron from the lowest oxidation state (S0) in the dark. Using quantum mechanical/molecular mechanical calculations, we investigated the redox potential (E m) of TyrD and its H-bond partner, D2-His189. The potential energy profile indicates that the release of a proton from the TyrD...D2-His189 pair leads to the formation of a low-barrier H-bond. The E m depends on the H+ position along the low-barrier H-bond, e.g., 680 mV when the H+ is at the D2-His189 moiety and 800 mV when the H+ is at the TyrD moiety, which can explain why TyrD mediates both the S0 to S1 oxidation and the S2 to S1 reduction.

Requirement of Chloride for the Downhill Electron Transfer Pathway from the Water-Splitting Center in Natural Photosynthesis.

In photosystem II (PSII), Cl- is a prerequisite for the second flash-induced oxidation of the Mn4CaO5 cluster (the S2 to S3 transition). We report proton transfer from the substrate water molecule via D1-Asp61 and electron transfer via redox-active D1-Tyr161 (TyrZ) to the chlorophyll pair in Cl--depleted PSII using a quantum mechanical/molecular mechanical approach. The low-barrier H-bond formation between the substrate water molecule and D1-Asp61 remained unaffected upon the depletion of Cl-. However, the binding site, D2-Lys317, formed a salt bridge with D1-Asp61, leading to the inhibition of the subsequent proton transfer. Remarkably, the redox potential (Em) of S2/S3 increased significantly, making electron transfer from S2 to TyrZ energetically uphill, as observed in Ca2+-depleted PSII. The uphill electron transfer pathway was induced by the significant increase in Em(S2/S3) caused by the loss of charge compensation for D2-Lys317 upon the depletion of Cl-, whereas it was induced by the significant decrease in Em(TyrZ) caused by the rearrangement of the water molecules at the Ca2+ binding moiety upon the depletion of Ca2+.

Mechanism of the formation of proton transfer pathways in photosynthetic reaction centers

In photosynthetic reaction centers from purple bacteria (PbRCs) from Rhodobacter sphaeroides, the secondary quinone QB accepts two electrons and two protons via electron-coupled proton transfer (PT). Here, we identify PT pathways that proceed toward the QB binding site, using a quantum mechanical/molecular mechanical approach. As the first electron is transferred to QB, the formation of the Grotthuss-like pre-PT H-bond network is observed along Asp-L213, Ser-L223, and the distal QB carbonyl O site. As the second electron is transferred, the formation of a low-barrier H-bond is observed between His-L190 at Fe and the proximal QB carbonyl O site, which facilitates the second PT. As QBH2 leaves PbRC, a chain of water molecules connects protonated Glu-L212 and deprotonated His-L190 forms, which serves as a pathway for the His-L190 reprotonation. The findings of the second pathway, which does not involve Glu-L212, and the third pathway, which proceeds from Glu-L212 to His-L190, provide a mechanism for PT commonly used among PbRCs.

Energetics of Ionized Water Molecules in the H-Bond Network near the Ca2+ and Cl- Binding Sites in Photosystem II.

In photosystem II, water oxidation occurs at the oxygen-evolving complex (OEC). The presence of a hydronium ion (H3O+) was proposed at the Cl- binding site and Ca2+-depleted OEC. Using a quantum mechanical/molecular mechanical approach, we report the stability of H3O+ in the PSII protein environment. Neither release of the proton from ligand water molecule W2 at the OEC nor formation of H3O+ at Cl- is energetically favorable. In contrast, H3O+ can exist at the Ca2+-depleted OEC. Even when H3O+ exists in Ca2+-depleted PSII, the H-bond network of the redox-active tyrosine (TyrZ) remains unaltered, retaining the unusually short low-barrier H-bond with D1-His190, and the redox potential of TyrZ, Em(TyrZ), remains unaltered. These observations explain why the oxidation of the Ca2+-depleted Mn4O5 cluster by TyrZ (i.e., the S2 to S3 transition) is not inhibited at low pH. It seems likely that Ca2+ plays a role in not only (i) maintaining the H-bond network and facilitating TyrZ oxidation [tuning Em(TyrZ)] but also (ii) providing the valence of +2, decreasing the pKa of the ligand molecule (W1), and facilitating the release of the proton from W1 in the S2 to S3 transition together with Cl-.

Pivotal role of the redox-active tyrosine in driving the water splitting catalyzed by photosystem II.

Photosynthetic water oxidation is catalyzed by the Mn4Ca cluster in photosystem II (PSII). The nearby redox-active tyrosine (YZ) serves as a direct electron acceptor of the Mn4Ca cluster and it forms a low-barrier H-bond (LBHB) with a neighboring histidine residue (D1-His190). Experimental evidence indicates that YZ oxidation triggers changes in the hydrogen bonding network that precede proton abstraction from the Mn4Ca cluster. In order to characterize such changes, we compare ab initio molecular dynamics simulations of different states of the catalytic cycle of PSII with dynamics of isolated tyrosine models (namely, p-cresol) in different oxidation states. The systematic comparison of the H-bond networks in different simulated systems suggests that the YZ oxidation leads to a water hydration pattern which is more similar to that of the neutral p-cresol rather than that of the p-cresol anion. Our simulations also reveal the twofold nature of the interactions between YZ and the Mn4Ca cluster. Firstly, the YZ oxidation triggers rapid structural changes of the H-bond pattern in the proximity of the cluster which have been observed to propagate on the ps time scale on the Ca2+ hydration shell up to other water molecules in the proximity of the cluster. Secondly, it is clear that YZ interacts with the Mn4Ca cluster also through Coulombic interactions mediated by CP43-Arg357 through the remaining positive charge of the pair. Our results are able to identify, for the first time, the structural rearrangements guided by the oxidation of YZ necessary for the evolution of the water splitting reaction in PSII. Based on these findings, we propose a mechanism of structural changes which is functional towards the progression of the catalytic cycle in PSII.

Redox Potential of the Oxygen-Evolving Complex in the Electron Transfer Cascade of Photosystem II.

In photosystem II (PSII), water oxidation occurs in the Mn4CaO5 cluster with the release of electrons via the redox-active tyrosine (TyrZ) to the reaction-center chlorophylls (PD1/PD2). Using a quantum mechanical/molecular mechanical approach, we report the redox potentials (Em) of these cofactors in the PSII protein environment. The Em values suggest that the Mn4CaO5 cluster, TyrZ, and PD1/PD2 form a downhill electron transfer pathway. Em for the first oxidation step, Em(S0/S1), is uniquely low (730 mV) and is ∼100 mV lower than that for the second oxidation step, Em(S1/S2) (830 mV) only when the O4 site of the Mn4CaO5 cluster is protonated in S0. The O4-water chain, which directly forms a low-barrier H-bond with the Mn4CaO5 cluster and mediates proton-coupled electron transfer in the S0 to S1 transition, explains why the second lowest oxidation state, S1, is the most stable and S0 is converted to S1 even in the dark.

Probing phenol dimer in molecular complex: Role of nitro group and stabilizing agent

The phenol dimer is a prototype molecular cluster system and is considered as a low-barrier H-bond (LBHB) interaction. The phenol dimer influences and catalyses various transformation in biological system but not identified in solid state yet. In present study, p-nitrophenol dimer (NPD) has been identified in the solid state. N-methylpiperidine (MP) is used to stabilise the NPD dimer and formed a stable MPH(PDA) molecular complex. Instead of p-nitrophenol dimer (NPD), the effect of the nitro group, the role of N-methylpiperidine (MP) and H-bonding interaction in MPH(PDA) have been investigated directly by experimental and theoretical studied. It is observed that extent of π electron delocalisation on p-nitrophenol play an important role on stability of p-nitrophenol dimer (NPD).

Mechanism of Radical Formation in the H-Bond Network of D1-Asn298 in Photosystem II.

In photosystem II (PSII), redox-active tyrosine Z (TyrZ) forms a low-barrier H-bond with Nε of D1-His190. The PSII crystal structures show that Nδ of D1-His190 donates an H-bond to the carbonyl O of D1-Asn298. However, at a level of ∼2 Å resolution, a clear discrimination between the -NH2 and -C═O groups of the asparagine side chain may not be possible based on the electron density map. Using quantum mechanical/molecular mechanical calculations, we investigated the energetics of the D1-Asn298 conformations. In the D1-Asn298-rotated conformation, where the amide N group donates an H-bond to deprotonated Nδ of D1-His190, oxidation of S2 resulted in formation of a neutral radical, either TyrZ• or D1-His190•. This suggests that in the D1-Asn298-rotated conformation, the redox potential ( Em) values of TyrZ/D1-His190 are lower than the Em of the Mn4CaO5 cluster due to deprotonated D1-His190. The large disorder of a water molecule (water 1117A) at D1-Asn298 in the crystal structure as well as the absence of water 1117A in the Sr2+-substituted crystal structure may be associated with coexistence of the two D1-Asn298 conformations in the crystals.

O2 evolution and recovery of the water-oxidizing enzyme

In photosystem II, light-induced water oxidation occurs at the Mn4CaO5 cluster. Here we demonstrate proton releases, dioxygen formation, and substrate water incorporation in response to Mn4CaO5 oxidation in the protein environment, using a quantum mechanical/molecular mechanical approach and molecular dynamics simulations. In S2, H2O at the W1 site forms a low-barrier H-bond with D1-Asp61. In the S2-to-S3 transition, oxidation of OW1H– to OW1•–, concerted proton transfer from OW1H– to D1-Asp61, and binding of a water molecule Wn-W1 at OW1•– are observed. In S4, Wn-W1 facilitates oxo-oxyl radical coupling between OW1•– and corner μ-oxo O4. Deprotonation via D1-Asp61 leads to formation of OW1=O4. As OW1=O4 moves away from Mn, H2O at W539 is incorporated into the vacant O4 site of the O2-evolved Mn4CaO4 cluster, forming a μ-oxo bridge (Mn3–OW539–Mn4) in an exergonic process. Simultaneously, Wn-W1 is incorporated as W1, recovering the Mn4CaO5 cluster.

Exceedingly Large Stokes Shift Induced by Low‐Barrier H‐Bond‐Assisted Internal Charge Transfer

AbstractBy only replacing phenyl group with N‐methylpyridinium in a 2‐(2‐hydroxyphenyl)benzothiazole (HBT) derivative HBT‐s‐Ph, which is an excited state intramolecular proton transfer (ESIPT)‐active dye, the conjugated HBT–N‐methylpyridinium hybrid HBT‐Py, emits high‐efficiency fluorescence (Φf: 29 %) with an exceedingly large Stokes shift (213 nm) even in aqueous solution. Emission spectroscopy and computational calculations indicate that this typical photophysical property owes to low‐barrier H‐bond (LBHB)‐assisted internal charge transfer (ICT), not ESIPT.

Formation of an unusually short hydrogen bond in photoactive yellow protein

The photoactive chromophore of photoactive yellow protein (PYP) is p-coumaric acid (pCA). In the ground state, the pCA chromophore exists as a phenolate anion, which is H-bonded by protonated Glu46 (OGlu46–OpCA=~2.6Å) and protonated Tyr42. On the other hand, the OGlu46–OpCA H-bond was unusually short (OGlu46–OpCA=2.47Å) in the intermediate pRCW state observed in time-resolved Laue diffraction studies. To understand how the existence of the unusually short H-bond is energetically possible, we analyzed the H-bond energetics adopting a quantum mechanical/molecular mechanical (QM/MM) approach based on the atomic coordinates of the PYP crystal structures. In QM/MM calculations, the OGlu46–OpCA bond is 2.60Å in the ground state, where Tyr42 donates an H-bond to pCA. In contrast, when the hydroxyl group of Tyr42 is flipped away from pCA, the H-bond was significantly shortened to 2.49Å in the ground state. The same H-bond pattern reproduced the unusually short H-bond in the pRCW structure (OGlu46–OpCA=2.49Å). Intriguingly, the potential-energy profile resembles that of a single-well H-bond, suggesting that the pKa values of the donor (Glu46) and acceptor (pCA) moieties are nearly equal. The present results indicate that the “equal pKa” requirement for formation of single-well or low-barrier H-bond (LBHB) is satisfied only when Tyr42 does not donate an H-bond to pCA, and argue against the possibility that the OGlu46–OpCA bond is an LBHB in the ground state, where Tyr42 donates an H-bond to pCA.

H Atom Positions and Nuclear Magnetic Resonance Chemical Shifts of Short H Bonds in Photoactive Yellow Protein

Recent neutron diffraction studies on photoactive yellow protein (PYP) proposed that the H bond between protonated Glu46 and the chromophore-ionized p-coumaric acid (pCA) is a low-barrier H bond (LBHB) mainly because the H atom position was assigned at the midpoint of the O(Glu46)-O(pCA) bond. However, the (1)H nuclear magnetic resonance (NMR) chemical shift (δ(H)) was 15.2 ppm, which is lower than the values of 17-19 ppm for typical LBHBs. We evaluated the dependence of δ(H) on an H atom position in the O(Glu46)-O(pCA) bond in the PYP ground state by using a quantum mechanical/molecular mechanical (QM/MM) approach. The calculated chemical shift unambiguously suggested that a δ(H) of 15.2 ppm for the O(Glu46)-O(pCA) bond in NMR studies should correspond to the QM/MM geometry (δ(H) = 14.5 ppm), where the H atom belongs to the Glu moiety, rather than the neutron diffraction geometry (δ(H) = 19.7 ppm), where the H atom is near the midpoint of the donor and acceptor atoms.

Energetics of short hydrogen bonds in photoactive yellow protein

Recent neutron diffraction studies of photoactive yellow protein (PYP) proposed that the H bond between protonated Glu46 and the chromophore [ionized p-coumaric acid (pCA)] was a low-barrier H bond (LBHB). Using the atomic coordinates of the high-resolution crystal structure, we analyzed the energetics of the short H bond by two independent methods: electrostatic pK(a) calculations and a quantum mechanical/molecular mechanical (QM/MM) approach. (i) In the QM/MM optimized geometry, we reproduced the two short H-bond distances of the crystal structure: Tyr42-pCA (2.50Å) and Glu46-pCA (2.57Å). However, the H atoms obviously belonged to the Tyr or Glu moieties, and were not near the midpoint of the donor and acceptor atoms. (ii) The potential-energy curves of the two H bonds resembled those of standard asymmetric double-well potentials, which differ from those of LBHB. (iii) The calculated pK(a) values for Glu46 and pCA were 8.6 and 5.4, respectively. The pK(a) difference was unlikely to satisfy the prerequisite for LBHB. (iv) The LBHB in PYP was originally proposed to stabilize the ionized pCA because deprotonated Arg52 cannot stabilize it. However, the calculated pK(a) of Arg52 and QM/MM optimized geometry suggested that Arg52 was protonated on the protein surface. The short H bond between Glu46 and ionized pCA in the PYP ground state could be simply explained by electrostatic stabilization without invoking LBHB.

IR Spectrum of the H5O2+ Cation in the Context of Proton Disolvates L−H+−L

The H(5)O(2)(+) ion has been studied in chlorocarbon, benzene, and weakly coordinating anion environments to bridge the gap between the gas-phase and traditional condensed-phase investigations. Symmetrical cations of the type [H(5)O(2)(+) x 4Solv] are formed via H-bonding with the terminal O-H groups. In the infrared spectrum, the nu(s)OH and nu(as)OH vibrations behave in a manner similar to those of common water molecules: the stronger is the H-bonding interaction with the surroundings, the lower is the frequency shift. A consistent pattern of IR bands from the central O-H(+)-O group is identified, regardless of the strength of the interaction of H(5)O(2)(+) with its environment. Three intense bands develop: a (860-995 cm-1), b (1045-1101 cm(-1)), and c (1672-1700 cm(-1)), as well as two weak bands, d ( approximately 1300 cm(-1)) and e ( approximately 1400-1500 cm(-1)). These fingerprint bands are highly characteristic for vibrations of O-H-O group irrespective of formal charge. They are seen in symmetrical proton disolvates of the type L-H(+)-L, where L is an O-atom donor (alcohol, ether, ketone, phosphate, etc.), and in [A-H-A](-) acid salts (A(-) = oxyanion). The commonality is equivalency of the two O-atoms, a short O...O distance (ca. 2.40 Angstrom), and a flat-bottomed potential well for the bridging proton, that is, a short, strong, low-barrier H-bond. Assignments for bands a-e are suggested in an attempt to resolve inconsistencies between experimental and calculated data.

Review: Contributions of NMR spectroscopy to the study of hydrogen bonds in serine protease active sites

This article is a chronology of NMR studies over the past three decades which have led to our present understanding of active site H-bonding in serine proteases. The story of H-bonding in this highly scrutinized family of enzymes, as revealed by NMR and x-ray crystallography, is to a large extent the foundation upon which our understanding of the catalytic mechanism of this archetypical enzyme system rests. Various theories have come into and out of favor as each bit of hard-earned evidence has been established, particularly with regard to the active-site imidazole, its state of protonation, its acid–base and tautomeric equilibrium constants and its H-bonding. Specific bioincorporation of 15N and 13C isotopic labels at the active site has proven especially useful in revealing the locations of protons and H-bond interactions through chemical shift and spin-coupling information, the latter useful also for spectral editing that reveals only the resonances of interest. However, the final chapter in the serine protease mechanism is yet to unfold. Currently competing proposals for the catalytic mechanism, one involving a low-barrier H-bond and the other a reaction driven histidine ring flip, are presented and discussed. Copyright © 2001 John Wiley & Sons, Ltd.