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Biochemistry Nov 2000N5-Methyltetrahydrofolate (CH(3)-H(4)folate) donates a methyl group to the cob(I)alamin cofactor in the reaction catalyzed by cobalamin-dependent methionine synthase...
N5-Methyltetrahydrofolate (CH(3)-H(4)folate) donates a methyl group to the cob(I)alamin cofactor in the reaction catalyzed by cobalamin-dependent methionine synthase (MetH, EC 2.1.1.3). Nucleophilic displacement of a methyl group attached to a tertiary amine is a reaction without an obvious precedent in bioorganic chemistry. Activation of CH(3)-H(4)folate by protonation prior to transfer of the methyl group has been the favored mechanism. Protonation at N5 would lead to formation of an aminium cation, and quaternary amines such as 5,5-dimethyltetrahydropterin have been shown to transfer methyl groups to cob(I)alamin. Because CH(3)-H(4)folate is an enamine, protonation could occur either at N5 to form an aminium cation or on a conjugated carbon with formation of an iminium cation. We used (13)C distortionless enhancement by polarization transfer (DEPT) NMR spectroscopy to infer that CH(3)-H(4)folate in aqueous solution protonates at N5, not on carbon. CH(3)-H(4)folate must eventually protonate at N5 to form the product H(4)folate; however, this protonation could occur either upon formation of the binary enzyme-CH(3)-H(4)folate complex or later in the reaction mechanism. Protonation at N5 is accompanied by substantial changes in the visible absorbance spectrum of CH(3)-H(4)folate. We have measured the spectral changes associated with binding of CH(3)-H(4)folate to a catalytically competent fragment of MetH over the pH range from 5.5 to 8.5. These studies indicate that CH(3)-H(4)folate is bound in the unprotonated form throughout this pH range and that protonated CH(3)-H(4)folate does not bind to the enzyme. Our observations are rationalized by sequence homologies between the folate-binding region of MetH and dihydropteroate synthase, which suggest that the pterin ring is bound in the hydrophobic core of an alpha(8)beta(8) barrel in both enzymes. The results from these studies are difficult to reconcile with an S(N)2 mechanism for methyl transfer and suggest that the presence of the cobalamin cofactor is important for CH(3)-H(4)folate activation. We propose that protonation of N5 occurs after carbon-nitrogen bond cleavage, and we invoke a mechanism involving oxidative addition of Co(1+) to the N5-methyl bond to rationalize our results.
Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Conserved Sequence; Dihydropteroate Synthase; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Protein Structure, Secondary; Protons; Sequence Homology, Amino Acid; Solutions; Spectrophotometry, Ultraviolet; Substrate Specificity; Tetrahydrofolates; Vitamin B 12
PubMed: 11076529
DOI: 10.1021/bi001431x -
The Journal of Physical Chemistry. B Nov 2021We generalize the Kirkwood-Shumaker theory of protonation fluctuation for an anisotropic distribution of dissociable charges on a globular protein. The fluctuations of...
We generalize the Kirkwood-Shumaker theory of protonation fluctuation for an anisotropic distribution of dissociable charges on a globular protein. The fluctuations of the total charge and the total dipole moment, in contrast to their average values, depend on the same proton occupancy correlator, thus exhibiting a similar dependence also on the solution pH. This has important consequences for the Kirkwood-Shumaker interaction and its dependence on the bathing solution conditions.
Topics: Electric Capacitance; Proteins; Protons
PubMed: 34784480
DOI: 10.1021/acs.jpcb.1c08200 -
Physical Chemistry Chemical Physics :... Oct 2011The Trp-cage miniprotein is a 20 amino acid peptide that exhibits many of the properties of globular proteins. In this protein, the hydrophobic core is formed by a...
The Trp-cage miniprotein is a 20 amino acid peptide that exhibits many of the properties of globular proteins. In this protein, the hydrophobic core is formed by a buried Trp side chain. The folded state is stabilized by an ion pair between aspartic acid and an arginine side chain. The effect of protonating the aspartic acid on the Trp-cage miniprotein folding/unfolding equilibrium is studied by explicit solvent molecular dynamics simulations of the protein in the charged and protonated Asp9 states. Unbiased Replica Exchange Molecular Dynamics (REMD) simulations, spanning a wide temperature range, are carried out to the microsecond time scale, using the AMBER99SB forcefield in explicit TIP3P water. The protein structural ensembles are studied in terms of various order parameters that differentiate the folded and unfolded states. We observe that in the folded state the root mean square distance (rmsd) from the backbone of the NMR structure shows two highly populated basins close to the native state with peaks at 0.06 nm and 0.16 nm, which are consistent with previous simulations using the same forcefield. The fraction of folded replicas shows a drastic decrease because of the absence of the salt bridge. However, significant populations of conformations with the arginine side chain exposed to the solvent, but within the folded basin, are found. This shows the possibility to reach the folded state without formation of the ion pair. We also characterize changes in the unfolded state. The equilibrium populations of the folded and unfolded states are used to characterize the thermodynamics of the system. We find that the change in free energy difference due to the protonation of the Asp amino acid is 3 kJ mol(-1) at 297 K, favoring the charged state, and resulting in ΔpK(1) = 0.5 units for Asp9. We also study the differences in the unfolded state ensembles for the two charge states and find significant changes at low temperature, where the protonated Asp side chain makes multiple hydrogen bonds to the protein backbone.
Topics: Amino Acid Sequence; Molecular Dynamics Simulation; Molecular Sequence Data; Peptides; Protein Folding; Protein Stability; Protons
PubMed: 21773639
DOI: 10.1039/c1cp21193e -
The Journal of Physical Chemistry. A Dec 2005The gas-phase basicities of a representative set of hydroxy- and methoxycarbonyl compounds (hydroxyacetone, 1, 3-hydroxybutanone, 2, 3-hydroxy-3-methylbutanone, 3,...
The gas-phase basicities of a representative set of hydroxy- and methoxycarbonyl compounds (hydroxyacetone, 1, 3-hydroxybutanone, 2, 3-hydroxy-3-methylbutanone, 3, 1-hydroxy-2-butanone, 4, 4-hydroxy-2-butanone, 5, 5-hydroxy-2-pentanone, 6, methoxyacetone, 7, 3-methoxy-2-butanone, 8, 4-methoxy-2-butanone, 9, and 5-methoxy-2-pentanone, 10) were experimentally determined by the equilibrium method using Fourier transform ion cyclotron resonance and high-pressure mass spectrometry techniques. The latter method allows the measurement of proton transfer equilibrium constants at various temperatures and thus the estimate of both the proton affinities and the protonation entropies of the relevant species. Quantum chemical calculations at the G3 and the B3LYP/6-311+G(3df,2p)//6-31G(d) levels of theory were undertaken in order to find the most stable structures of the neutrals 1-10 and their protonated forms. Conformational and vibrational analyses have been done with the aim of obtaining a theoretical estimate of the protonation entropies.
Topics: Acetone; Butanones; Gases; Molecular Structure; Phase Transition; Protons; Spectroscopy, Fourier Transform Infrared; Thermodynamics
PubMed: 16366636
DOI: 10.1021/jp054955l -
Inorganic Chemistry Apr 2016The complexes Fe2(pdt)(CNR)6 (pdt(2-) = CH2(CH2S(-))2) were prepared by thermal substitution of the hexacarbonyl complex with the isocyanides RNC for R = C6H4-4-OMe (1),...
The complexes Fe2(pdt)(CNR)6 (pdt(2-) = CH2(CH2S(-))2) were prepared by thermal substitution of the hexacarbonyl complex with the isocyanides RNC for R = C6H4-4-OMe (1), C6H4-4-Cl (2), Me (3). These complexes represent electron-rich analogues of the parent Fe2(pdt)(CO)6. Unlike most substituted derivatives of Fe2(pdt)(CO)6, these isocyanide complexes are sterically unencumbered and have the same idealized symmetry as the parent hexacarbonyl derivatives. Like the hexacarbonyls, the stereodynamics of 1-3 involve both turnstile rotation of the Fe(CNR)3 as well as the inversion of the chair conformation of the pdt ligand. Structural studies indicate that the basal isocyanide has nonlinear CNC bonds and short Fe-C distances, indicating that they engage in stronger Fe-C π-backbonding than the apical ligands. Cyclic voltammetry reveals that these new complexes are far more reducing than the hexacarbonyls, although the redox behavior is complex. Estimated reduction potentials are E1/2 ≈ -0.6 ([2](+/0)), -0.7 ([1](+/0)), and -1.25 ([3](+/0)). According to DFT calculations, the rotated isomer of 3 is only 2.2 kcal/mol higher in energy than the crystallographically observed unrotated structure. The effects of rotated versus unrotated structure and of solvent coordination (THF, MeCN) on redox potentials were assessed computationally. These factors shift the redox couple by as much as 0.25 V, usually less. Compounds 1 and 2 protonate with strong acids to give the expected μ-hydrides [H1](+) and [H2](+). In contrast, 3 protonates with [HNEt3]BAr(F)4 (pKa(MeCN) = 18.7) to give the aminocarbyne [Fe2(pdt)(CNMe)5(μ-CN(H)Me)](+) ([3H](+)). According to NMR measurements and DFT calculations, this species adopts an unsymmetrical, rotated structure. DFT calculations further indicate that the previously described carbyne complex [Fe2(SMe)2(CO)3(PMe3)2(CCF3)](+) also adopts a rotated structure with a bridging carbyne ligand. Complex [3H](+) reversibly adds MeNC to give [Fe2(pdt)(CNR)6(μ-CN(H)Me)](+) ([3H(CNMe)](+)). Near room temperature, [3H](+) isomerizes to the hydride [(μ-H)Fe2(pdt)(CNMe)6](+) ([H3](+)) via a first-order pathway.
Topics: Carbolines; Coordination Complexes; Crystallography, X-Ray; Cyanides; Electrons; Ferric Compounds; Isomerism; Magnetic Resonance Spectroscopy; Models, Molecular; Protons
PubMed: 26999632
DOI: 10.1021/acs.inorgchem.5b02789 -
Annual Review of Biophysics 2015Electrostatics play an important role in many aspects of protein chemistry. However, the accurate determination of side chain proton affinity in proteins by experiment... (Review)
Review
Electrostatics play an important role in many aspects of protein chemistry. However, the accurate determination of side chain proton affinity in proteins by experiment and theory remains challenging. In recent years the field of nuclear magnetic resonance spectroscopy has advanced the way that protonation states are measured, allowing researchers to examine electrostatic interactions at an unprecedented level of detail and accuracy. Experiments are now in place that follow pH-dependent (13)C and (15)N chemical shifts as spatially close as possible to the sites of protonation, allowing all titratable amino acid side chains to be probed sequence specifically. The strong and telling response of carefully selected reporter nuclei allows individual titration events to be monitored. At the same time, improved frameworks allow researchers to model multiple coupled protonation equilibria and to identify the underlying pH-dependent contributions to the chemical shifts.
Topics: Hydrogen-Ion Concentration; Nuclear Magnetic Resonance, Biomolecular; Proteins; Protons; Static Electricity
PubMed: 25747592
DOI: 10.1146/annurev-biophys-083012-130351 -
Biophysical Journal Aug 2016Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For...
Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both redox enzyme and proton pump, proton transfer out of either of the channels toward the BNC or into the protein toward a proton loading site, and ultimately through the membrane, must be highly regulated. The O→E intermediate of cytochrome c oxidase is the first redox state in its catalytic cycle, where proton transfer through the K-channel, from K362 to Y288 at the BNC, is important. Molecular dynamics simulations of this intermediate with 16 different combinations of protonation states of key residues in the D- and K-channel show the mutual impact of the two proton-conducting channels to be protonation state-dependent. Strength as well as means of communication, correlations in positions, or connections along the hydrogen-bonded network, change with the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286, which would in principle allow proton transfer to the BNC, but no proton pumping until a proton has reached E286.
Topics: Electron Transport Complex IV; Hydrogen Bonding; Molecular Dynamics Simulation; Protein Conformation; Protons
PubMed: 27508434
DOI: 10.1016/j.bpj.2016.06.038 -
Physical Chemistry Chemical Physics :... Mar 2022The protonation site of molecules can be varied by their surrounding environment. Gas-phase studies, including the popular techniques of infrared spectroscopy and ion...
Collision-assisted stripping for determination of microsolvation-dependent protonation sites in hydrated clusters by cryogenic ion trap infrared spectroscopy: the case of benzocaineH(HO).
The protonation site of molecules can be varied by their surrounding environment. Gas-phase studies, including the popular techniques of infrared spectroscopy and ion mobility spectrometry, are a powerful tool for the determination of protonation sites in solvated clusters but often suffer from inherent limits for larger hydrated clusters. Here, we present collision-assisted stripping infrared (CAS-IR) spectroscopy as a new technique to overcome these problems and apply it in a proof-of-principle experiment to hydrated clusters of protonated benzocaine (HBC), which shows protonation-site switching depending on the degree of hydration. The most stable protomer of HBC in the gas phase (O-protonated) is interconverted into its most stable protomer in aqueous solution (N-protonated) upon hydration with three water molecules. CAS-IR spectroscopy enables us to unambiguously assign protonation sites and quantitatively determine the relative abundance of various protomers.
Topics: Benzocaine; Ion Mobility Spectrometry; Protons; Spectrophotometry, Infrared; Water
PubMed: 35199812
DOI: 10.1039/d1cp05762f -
Physical Chemistry Chemical Physics :... Nov 2020Photosynthetic water oxidation takes place through the light-driven cycle of five intermediates (S-S) of the water oxidizing complex (WOC), which consists of the MnCaO...
Protonation structure of the photosynthetic water oxidizing complex in the S state as revealed by normal mode analysis using quantum mechanics/molecular mechanics calculations.
Photosynthetic water oxidation takes place through the light-driven cycle of five intermediates (S-S) of the water oxidizing complex (WOC), which consists of the MnCaO cluster and surrounding amino acid residues in photosystem II. Clarifying the protonation structures of the MnCaO cluster and its water ligands (W1-W4) is essential for understanding the molecular mechanism of water oxidation. Here, we performed normal mode analysis of WOC in the S and S states using quantum mechanics/molecular mechanics calculations and simulated an S-minus-S infrared difference spectrum focusing on the symmetric COO stretching (νCOO) region. The calculated spectrum by an S model, in which O4 of the MnCaO cluster is protonated and W2 is HO, and a corresponding S state with deprotonated O4 best reproduced the νCOO features of the experimental spectrum, whereas models with protonated O5 showed poor agreement. In addition, comparison of the calculated coordination distances of the water ligands with the experimental data by X-ray diffraction analysis indicates that W2 is most probably not OH but HO both in the S and S states. The present calculations thus strongly suggest that the S state has a protonation structure of O4-H and W2 = HO. The O4-H structure in the S state supports the view that this proton is released through the O4-water chain immediately after electron transfer during the S→ S transition.
Topics: Models, Molecular; Oxidation-Reduction; Photosystem II Protein Complex; Protons; Quantum Theory; Water
PubMed: 33084674
DOI: 10.1039/d0cp04079g -
International Journal of Molecular... Jul 2016A series of a new type of tetracyclic carbazolequinones incorporating a carbonyl group at the ortho position relative to the quinone moiety was synthesized and analyzed...
A series of a new type of tetracyclic carbazolequinones incorporating a carbonyl group at the ortho position relative to the quinone moiety was synthesized and analyzed by tandem electrospray ionization mass spectrometry (ESI/MS-MS), using Collision-Induced Dissociation (CID) to dissociate the protonated species. Theoretical parameters such as molecular electrostatic potential (MEP), local Fukui functions and local Parr function for electrophilic attack as well as proton affinity (PA) and gas phase basicity (GB), were used to explain the preferred protonation sites. Transition states of some main fragmentation routes were obtained and the energies calculated at density functional theory (DFT) B3LYP level were compared with the obtained by ab initio quadratic configuration interaction with single and double excitation (QCISD). The results are in accordance with the observed distribution of ions. The nature of the substituents in the aromatic ring has a notable impact on the fragmentation routes of the molecules.
Topics: Benzoquinones; Gases; Ions; Ketones; Protons; Spectrometry, Mass, Electrospray Ionization; Thermodynamics
PubMed: 27399676
DOI: 10.3390/ijms17071071