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Acta Crystallographica. Section D,... Jun 2022Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and...
Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and Asp52 interacts with the positively charged substrate, suggesting different protonation states of these residues. However, neutron crystallographic studies thus far have not provided a consistent picture of the protonation states of these residues. Only one study succeeded in observing the active protonation states of Glu35 and Asp52 in the triclinic crystal system. However, their active states in the most widely studied tetragonal crystal system are still unknown. The application of the D/H contrast technique in neutron crystallography improves the ability to locate exchangeable D/H atoms in proteins. In the present study, DO and HO solvent crystals were prepared. Each neutron data set was collected for only five days by combining a time-of-flight diffractometer (iBIX) and the spallation neutron source at the Japan Proton Accelerator Research Complex. The D/H contrast map provided better visualization of the D/H atoms in HEWL than the conventional neutron scattering length density map. The neutron D/H contrast map demonstrated the alternative protonation of the OE1 and OE2 atoms in the carboxyl group of Glu35. This alternative protonation occurs in the absence of a substrate, where high selectivity of the protonation site does not occur. In this case, only the OE1-HE1 bond attacks the substrate in an equilibrium between OE1-HE1 and OE2-HE2, or the H ion of the OE2-HE2 bond moves to the OE1 atom just before or after substrate binding to initiate the catalytic reaction. In contrast, the carboxyl group of Asp52 is not protonated. Protonation of the carboxyl group was not observed for other Asp and Glu residues. These results are consistent with results from NMR spectroscopy and explain the protonation states at the active site in the apo form of HEWL.
Topics: Crystallography; Models, Molecular; Muramidase; Neutrons; Protons
PubMed: 35647923
DOI: 10.1107/S2059798322004521 -
Physical Chemistry Chemical Physics :... Feb 2022Spatial, temporal, and remote control of proton chemistry can be achieved by using photoacids, which are molecules that transform from weak to strong acids under light.... (Review)
Review
Spatial, temporal, and remote control of proton chemistry can be achieved by using photoacids, which are molecules that transform from weak to strong acids under light. Most of proton chemistry is driven by a high concentration of protons ([H]), which is difficult to obtain using excited-state photoacids. Metastable-stable state photoacids (mPAHs) can reversibly generate a high [H] under visible light with a moderate intensity. It has been widely applied in different fields, fueling dissipative assemblies, driving molecular machines, controlling organic reactions, powering nanoreactors, curing diseases, manipulating DNA and proteins, developing smart materials, capturing carbon dioxide in air This article compares mPAH with excited-state photoacid as well as common acids HCl to explain its advantages. Recent studies on the thermal dynamics, kinetics, and photoreaction of mPAHs are reported. The advantages and disadvantages of the three types of mPAHs, merocyanine, indazole, and TCF mPAHs, are compared with regard to photo-induced [H], switching rate, and other properties. The mechanisms of controlling or driving functional systems, which involve acid-base reactions, acid catalyzed reactions, ionic bonding, coordination bonding, hydrogen bonding, ion exchange, cation-π interaction, solubility, swellability, permeability, and pH change in biosystems, are described. Applications of mPAHs in the chemical, material, energy, biotechnology and biomedical fields published in the past 5 years are reviewed. Prospects in the development and application of mPAHs are discussed.
Topics: Acids; Kinetics; Light; Protons
PubMed: 35129187
DOI: 10.1039/d1cp05627a -
Accounts of Chemical Research Jan 2021Hydrogenases are metalloenzymes that catalyze proton reduction and H oxidation with outstanding efficiency. They are model systems for bioinorganic chemistry, including...
Hydrogenases are metalloenzymes that catalyze proton reduction and H oxidation with outstanding efficiency. They are model systems for bioinorganic chemistry, including low-valent transition metals, hydride chemistry, and proton-coupled electron transfer. In this Account, we describe how photochemistry and infrared difference spectroscopy can be used to identify the dynamic hydrogen-bonding changes that facilitate proton transfer in [NiFe]- and [FeFe]-hydrogenase.[NiFe]-hydrogenase binds a heterobimetallic nickel/iron site embedded in the protein by four cysteine ligands. [FeFe]-hydrogenase carries a homobimetallic iron/iron site attached to the protein by only a single cysteine. Carbon monoxide and cyanide ligands in the active site facilitate detailed investigations of hydrogenase catalysis by infrared spectroscopy because of their strong signals and redox-dependent frequency shifts. We found that specific redox-state transitions in [NiFe]- and [FeFe]-hydrogenase can be triggered by visible light to record extremely sensitive "light-minus-dark" infrared difference spectra monitoring key amino acid residues. As these transitions are coupled to protonation changes, our data allowed investigation of dynamic hydrogen-bonding changes that go well beyond the resolution of protein crystallography.In [NiFe]-hydrogenase, photolysis of the bridging hydride ligand in the Ni-C state was followed by infrared difference spectroscopy. Our data clearly indicate the formation of a protonated cysteine residue as well as hydrogen-bonding changes involving a glutamic acid residue and a "dangling water" molecule. These findings are in excellent agreement with crystallographic analyses of [NiFe]-hydrogenase. In [FeFe]-hydrogenase, an external redox dye was used to accumulate the Hred state. Infrared difference spectra indicate hydrogen-bonding changes involving two glutamic acid residues and a conserved arginine residue. While crystallographic analyses of [FeFe]-hydrogenase in the oxidized state failed to explain the rapid proton transfer because of a breach in the succession of residues, our findings facilitated a precise molecular model of discontinued proton transfer.Comparing both systems, our data emphasize the role of the outer coordination sphere in bimetallic hydrogenases: we suggest that protonation of a nickel-ligating cysteine in [NiFe]-hydrogenase causes the notable preference toward H oxidation. On the contrary, proton transfer in [FeFe]-hydrogenase involves an adjacent cysteine as a relay group, promoting both H oxidation and proton reduction. These observations may guide the design of organometallic compounds that mimic the catalytic properties of hydrogenases.
Topics: Carbon Monoxide; Catalytic Domain; Hydrogen; Hydrogen Bonding; Hydrogenase; Iron-Sulfur Proteins; Light; Oxidation-Reduction; Protons
PubMed: 33326230
DOI: 10.1021/acs.accounts.0c00651 -
Biomolecules Nov 2022The transmembrane transport of weak acid and base metabolites depends on the local pH conditions that affect the protonation status of the substrates and the... (Review)
Review
The transmembrane transport of weak acid and base metabolites depends on the local pH conditions that affect the protonation status of the substrates and the availability of co-substrates, typically protons. Different protein designs ensure the attraction of substrates and co-substrates to the transporter entry sites. These include electrostatic surface charges on the transport proteins and complexation with seemingly transport-unrelated proteins that provide substrate and/or proton antenna, or enzymatically generate substrates in place. Such protein assemblies affect transport rates and directionality. The lipid membrane surface also collects and transfers protons. The complexity in the various systems enables adjustability and regulation in a given physiological or pathophysiological situation. This review describes experimentally shown principles in the attraction and facilitation of weak acid and base transport substrates, including monocarboxylates, ammonium, bicarbonate, and arsenite, plus protons as a co-substrate.
Topics: Protons; Biological Transport; Membrane Transport Proteins; Hydrogen-Ion Concentration
PubMed: 36551222
DOI: 10.3390/biom12121794 -
The Journal of Chemical Physics Aug 2022Acid ionization constants (pK's) of titratable amino acid side chains have received a large amount of experimental and theoretical attention. In many situations,...
Acid ionization constants (pK's) of titratable amino acid side chains have received a large amount of experimental and theoretical attention. In many situations, however, the rates of protonation and deprotonation, k and k, may also be important, for example, in understanding the mechanism of action of proton channels or membrane proteins that couple proton transport to other processes. Protonation and deprotonation involve the making and breaking of covalent bonds, which cannot be studied by classical force fields. However, environment effects on the rates should be captured by such methods. Here, we present an approach for estimating deprotonation rates based on Warshel's extension of Marcus's theory of electron transfer, with input from molecular simulations. The missing bond dissociation energy is represented by a constant term determined by fitting the pK value in solution. The statistics of the energy gap between protonated and deprotonated states is used to compute free energy curves of the two states and, thus, free energy barriers, from which the rate can be deduced. The method is applied to Glu, Asp, and His in bulk solution and select membrane proteins: the M2 proton channel, bacteriorhodopsin, and cytochrome c oxidase.
Topics: Amino Acids; Aspartic Acid; Bacteriorhodopsins; Hydrogen-Ion Concentration; Kinetics; Protons
PubMed: 36050014
DOI: 10.1063/5.0101960 -
ACS Chemical Neuroscience Jan 2022Methylations in living cells are methyl groups attached to amino acids, DNA, RNA, and so on. However, their biochemical roles have not been fully defined. A theory has...
Methylations in living cells are methyl groups attached to amino acids, DNA, RNA, and so on. However, their biochemical roles have not been fully defined. A theory has been postulated that methylation leads to hyperconjugation, and the electron-donating feature weakens a nearby chemical bond, which increases the bond length of C-N of 5-methylcytosine, therefore weakening the C-N bond and resulting in stronger protonation or hydrogen bonding of the N nitrogen atom. Protonation can give rise to the generation of mutagenic and carcinogenic strong acids such as HCl, which are also capable of solubilizing stressful, insoluble, and stiff salts. Insoluble and rigid salts such as calcium oxalate and/or calcium phosphate were recently proposed as a primary cause of some neurodegenerative disorders. Protonation of nitrogen atoms in 5-methylcytosine enhances the interaction with negatively charged phosphate groups and contributes to the formation of compact heterochromatin. The electronegativity of the oxygen atoms in the modifications of 5-hydroxymethylcytosine or 5-formylcytosine can shorten the lengths of adjacent bonds with no increase of cation affinity in N. The carboxyl group in 5-carboxylcytosine is a weak acid capable of antagonizing mutagenic HCl and modestly helping solubilize insoluble salts. Electron delocalization of the methyl group in N4-methylcytosine results in a lower affinity of N to cations. The positive charge at N in the resonance structure of 3-methylcytosine is lessened by the electron-donating attribute of the methyl group attached to the N atom, consequently reducing acid formation. The electron delocalization of three methyl groups decreases the positive charge in the amino nitrogen in the side group of lysine 4 in histone H3, weakening interactions with phosphate groups and consequently activating gene expression. The carbonyl oxygen in 8-oxo-7,8-dihydroguanine draws protons and accumulates HCl, accounting for its moderate mutation propensity and potential capacity to solubilize stiff salts. The biochemical insight will further our understanding on the crosstalk of genetics and epigenetics in the etiology of neurodegenerative diseases.
Topics: Amino Acids; Epigenesis, Genetic; Humans; Hydrogen Bonding; Neurodegenerative Diseases; Protons
PubMed: 35000390
DOI: 10.1021/acschemneuro.1c00701 -
Nature Communications Sep 2022Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P...
Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60:Arg289) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange.
Topics: Anion Transport Proteins; Endothelial Cells; Humans; Ligands; Lysophospholipids; Protons; Signal Transduction; Sphingosine
PubMed: 36055994
DOI: 10.1038/s41467-022-32759-2 -
Protein Science : a Publication of the... Apr 2021Acid-base reactions that are exceedingly unfavorable under standard conditions can be catalytically important at enzyme active sites. For example, in triose phosphate... (Review)
Review
Acid-base reactions that are exceedingly unfavorable under standard conditions can be catalytically important at enzyme active sites. For example, in triose phosphate isomerase, a glutamate side chain (nominal pK ≈ 4 in solution) can in fact deprotonate a CH group that is vicinal to a carbonyl (pK ≈ 18 in solution). This is true because of three distinct interactions: (a) ground state pK shifts due to environment polarity and electrostatics; (b) dramatic increases in effective molarity due to optimization of proximity and orientation; and (c) transition state pK shifts due to binding interactions and the formation of strong low barrier hydrogen bonds. In this report, we review the literature showing that the sum of these three effects supplies more than enough free energy to push forward proton transfer reactions that under standard conditions are exceedingly nonspontaneous and slow.
Topics: Hydrogen Bonding; Models, Molecular; Protons; Static Electricity
PubMed: 33554401
DOI: 10.1002/pro.4037 -
Organic Letters Jan 2023A novel class of stable monoareno-pentalenes is introduced that have an olefinic proton on each five-membered ring of the pentalene subunit. Their synthesis was...
A novel class of stable monoareno-pentalenes is introduced that have an olefinic proton on each five-membered ring of the pentalene subunit. Their synthesis was accomplished via a regioselective carbopalladation cascade reaction between -arylacetyleno -dibromoolefins and TIPS-acetylene. These molecules could be experimental probes of magnetic (anti)aromaticity effects.
Topics: Protons; Molecular Structure; Alkenes; Acetylene
PubMed: 36576234
DOI: 10.1021/acs.orglett.2c03752 -
The Ulster Medical Journal Jan 2021
Topics: History, 20th Century; Humans; Nobel Prize; Protons
PubMed: 33642630
DOI: No ID Found