Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase.

For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.     

Infrared absorption study of the heme pocket dynamics of carbonmonoxyheme proteins

The temperature dependencies of the infrared absorption CO bands of carboxy complexes of horseradish peroxidase (HRP(CO)) in glycerol/water mixture at pH 6.0 and 9.3 are interpreted using the theory of optical absorption bandshape. The bands' anharmonic behavior is explained assuming that there is a higher-energy set of conformational substates (CSS(h)), which are populated upon heating and correspond to the protein substates with disordered water molecules in the heme pocket. Analysis of the second moments of the CO bands of the carboxy complexes of myoglobin (Mb(CO)) and hemoglobin (Hb(CO)), and of HRP(CO) with benzohydroxamic acid (HRP(CO)+BHA), shows that the low energy CSS(h) exists also in the open conformation of Mb(CO), where the heme pocket is spacious enough to accommodate a water molecule. In the HRP(CO)+BHA and closed conformations of Mb(CO) and Hb(CO), the heme pocket is packed with BHA and different amino acids, the CSS(h) has much higher energy and is hardly populated even at the highest temperatures. Therefore only motions of these amino acids contribute to the band broadening. These motions are linked to the protein surface and frozen in the glassy matrix, whereas in the liquid solvent they are harmonic. Thus the second moment of the CO band is temperature-independent in glass and is proportional to the temperature in liquid. The temperature dependence of the second moment of the CO peak of HRP(CO) in the trehalose glass exhibits linear coupling to an oscillator. This oscillator can be a moving water molecule locked in the heme pocket in the whole interval of temperatures or a trehalose molecule located in the heme pocket.     

Observation of the keto tautomer of D-fructose in D(2)O using (1)H NMR spectroscopy

D-Fructose was analysed by NMR spectroscopy and previously unidentified (1)H NMR resonances were assigned to the keto and α-pyranose tautomers. The full assignment of shifts for the various fructose tautomers enabled the use of (1)H NMR spectroscopy in studies of the mutarotation (5-25°C) and tautomeric composition at equilibrium (5-50°C). The mutarotation of β-pyranose to furanose tautomers in D(2)O at a concentration of 0.18 M was found to have an activation energy of 62.6 kJmol(-1). At tautomeric equilibrium (20°C in D(2)O) the distribution of the β-pyranose, β-furanose, α-furanose, α-pyranose and the keto tautomers was found to be 68.23%, 22.35%, 6.24%, 2.67% and 0.50%, respectively. This tautomeric composition was not significantly affected by varying concentrations between 0.089 and 0.36 M or acidification to pH 3. Upon equilibrating at 6 temperatures between 5 and 50°C there was a linear relationship between the change in concentration and temperature for all forms.     

Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.     

Structural consequences of heme isomerism in monomeric hemoglobins from Glycera dibranchiata

Two-dimensional 1H-NMR methods have been used to assign heme and amino acid proton resonances in both isomeric states of the carbon monoxide complexes of two Glycera dibranchiata monomeric hemoglobins, HbA and HbB. For each hemoglobin, there are small differences in heme pocket structure in the two isomeric forms. The largest structural perturbations associated with heme isomerism involve residues close to pyrrole rings I and II. The positions relative to the heme of phenylalanine CD1 and the proximal histidine ligand are almost unaffected by heme isomerism. These residues probably play a key role in determining the location of the heme within the heme pocket.     

Variations of the tautomeric preferences and π-electron delocalization for the neutral and redox forms of purine when proceeding from the gas phase (DFT) to water (PCM)

Quantum-chemical calculations were performed for all possible nine neutral tautomers of purine and their oxidized and reduced forms in water {PCM//DFT(B3LYP)/6?311+G(d,p)} and compared to those in the gas phase {DFT(B3LYP)/6?311+G(d,p)}. PCM hydration influences geometries, π-electron delocalization, and relative energies of purine tautomers in different ways. Generally, the harmonic oscillator model of electron delocalization (HOMED) indices increase when proceeding from the gas phase to aequeous solution for the neutral and redox forms of purine. Their changes for the neutral and oxidized tautomers are almost parallel to the relative energies showing that aromaticity plays an important role in the tautomeric preferences. Tautomeric stabilities and tautomeric preferences vary when proceeding from the gas phase to water indicating additionally that intra- and intermolecular interactions affect tautomeric equilibria. The tautomeric mixture of neutral purine in the gas phase consists mainly of the N9H tautomer, whereas two tautomers (N9H and N7H) dominate in water. For oxidized purine, N9H is favored in the gas phase, whereas N1H in water. A gain of one electron dramatically changes the relative stabilities of the CH and NH tautomers that C6H and C8H dominate in the tautomeric mixture in the gas phase, whereas N3H in water. These variations show exceptional sensitivity of the tautomeric purine system on environment in the electron-transfer reactions.     

Investigation of tautomeric composition of cytosine by multiphoton excitation spectra. II. Two-photon absorption spectra

For the first time the quantum-mechanical calculations of intensity distribution in the two-photon absorption spectra of the six tautomeric forms of cytosine were performed. It has been confirmed that in the aqueous solution of cytosine (pH 3.0) there exist several tauromeric forms: canonical amino-oxo tautomeric form, cation, as well as cys-imino-oxo and cys-amino-hydroxy tautomers.     

Tautomeric energetics of xanthine oxidase substrates: xanthine, 2-oxo-6-methylpurine, and lumazine

The stability of the tautomers of each of the three important substrates of xanthine oxidase, xanthine, 2-oxo-6-methylpurine, and lumazine, was examined by quantum mechanical calculations. The geometries of these tautomers were optimized at the AM1, Hartree-Fock (HF/6-31G), and hybrid Hartree-Fock/density functional theory (B3LYP/6-31G(d)) levels of theory. The single point energies of some of the more stable tautomers for each of the substrates were calculated at the B3LYP/6-311 +G(2d,p) level of theory. The Conductor Polarized Continuum Model (CPCM) was used to evaluate the solvent effects on the relative stabilities of these tautomers. The calculations clearly identify the lowest energy tautomeric form for xanthine and lumazine. On the other hand, there appear to be three tautomers for 2-oxo-6-methylpurine, with only minor energetic differences in vacuo. In water, however, only one of them predominates. The lowest energy tautomers presumably represent the predominant tautomeric forms at the molybdenum center of xanthine oxidase during catalysis. Implications of these computational results are discussed in the context of enzyme catalysis.     

Sampling field heterogeneity at the heme of c-type cytochromes by spectral hole burning spectroscopy and electrostatic calculations

We report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes-namely yeast and horse heart cytochromes c-using a combination of hole burning Stark spectroscopy and electrostatic calculations. The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes. In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed. The yeast species, on the other hand, displays a single electronic origin. Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space. The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of "electrostatic substates" that they can sample, thus accounting for the different spectral populations observed in the two cytochromes.     

Role of tautomerism in RNA biochemistry

Heterocyclic nucleic acid bases and their analogs can adopt multiple tautomeric forms due to the presence of multiple solvent-exchangeable protons. In DNA, spontaneous formation of minor tautomers has been speculated to contribute to mutagenic mispairings during DNA replication, whereas in RNA, minor tautomeric forms have been proposed to enhance the structural and functional diversity of RNA enzymes and aptamers. This review summarizes the role of tautomerism in RNA biochemistry, specifically focusing on the role of tautomerism in catalysis of small self-cleaving ribozymes and recognition of ligand analogs by riboswitches. Considering that the presence of multiple tautomers of nucleic acid bases is a rare occurrence, and that tautomers typically interconvert on a fast time scale, methods for studying rapid tautomerism in the context of nucleic acids under biologically relevant aqueous conditions are also discussed.     

Application of pattern recognition techniques to the analysis of protein crystal structure data. I. Characteristics of per-residue side chain contact frequency distributions

A statistical study of amino acid side chain contact interactions was carried out using a data set based on 36 protein structures. For each type of amino acid, a distribution of per-residue inter-side-chain contacts was obtained, over the observed span of zero to 11 contacts per residue. Significant observations included the following: 1) The mean number of inter-side-chain contacts is proportional to side chain surface area with the exception of Lys and Arg. 2) The mean number of contacts was greater for amino acids in beta-sheet relative to alpha-helical regions. 3) The more polar or surface-loving amino acids exhibited non-normal distributions, whereas distributions for the non-polar or interior-loving amino acids fell within accepted limits of normality.     

The role of T56 in controlling the flexibility of the distal histidine in dehaloperoxidase-hemoglobin from Amphitrite ornata

The activation of dehaloperoxidase-hemoglobin (DHP) to form a ferryl intermediate requires the distal histidine, H55, to act as an acid base catalyst. The lack of ancillary amino acids in the distal pocket to assist in this process makes H55 even more important to the formation of active intermediates than in conventional peroxidases. Therefore, one can infer that the precise conformation H55 may greatly affect the enzymatic activity. Using site-direct mutagenesis at position T56, immediately adjacent to H55, we have confirmed that subtle changes in the conformation of H55 affect the catalytic efficiency of DHP. Mutating T56 to a smaller amino acid appears to permit H55 to rotate with relatively low barriers between conformations in the distal pocket, which may lead to an increase in catalytic activity. On the other hand, larger amino acids in the neighboring site appear to restrict the rotation of H55 due to the steric hindrance. In the case of T56V, which is an isosteric mutation, H55 appears less mobile, but forced to be closer to the heme iron than in wild type. Both proximity to the heme iron and flexibility of motion in some of the mutants can result in an increased catalytic rate, but can also lead to protein inactivation due to ligation of H55 to the heme iron, which is known as hemichrome formation. A balance of enzymatic rate and protein stability with respect to hemichrome formation appears to be optimum in wild type DHP (WT-DHP).     

Investigation of tautomeric composition of cytosine by multiphoton excitation spectra. I. Resonance hyper-Raman spectra

For the first time the quantum-mechanical calculations of intensity distribution in the resonance hyper-Raman spectra of the six tautomeric forms of cytosine were performed. It has been confirmed that in the aqueous solution of cytosine (pH 3.0) there exist several molecular structures: cytosine in the canonical amino-oxo tautomeric form, cation, as well as cys-imino-oxo and cys-amino-hydroxy tautomers; in the gaseous phase at 235°C cys-imino-oxotautomer dominates with the presence of small amount of cys-, trans-, amino-hydroxy tautomers and the caconical form of cytosine.     

Muscimol and some aza analogues: molecular orbital calculations relating to their potency as GABA mimetics

Ab initio molecular orbital calculations have been carried out on muscimol (1) and on two aza analogues, 5-aminomethylpyrazol-3-one (2) and 5-aminomethyl-1,2,4-triazol-3-one (3). Fully ionized species were studied in each case including the tautomeric forms of (2) and (3). The calculations reveal that the more stable tautomeric forms of (2) and (3) resemble muscimol in having a low energy barrier to rotation of the aminomethyl group (2–4 kcal mol^?1) but differ substantially from muscimol in the electronic character of the heterocyclic ring. The change in π-electron distribution in the more stable tautomers of (2) and (3) compared with muscimol is summarized by the dipole moment decrease (1) 7·75 au, (2) 6·62 au, (3) 5·81 au. In addition, the frontier molecular orbitals show analogous differences. On the other hand, the less stable tautomers of (2) and (3) are similar to muscimol in π-electron distribution and frontier orbitals, but have a high energy barrier to rotation of the aminomethyl group (11–14 kcal mol^?1). The calculated properties of the molecules are related to the ability of the three compounds to displace [³H]-GABA from the postsynaptic receptors of human cerebellum and it is suggested that the dominant influence on biochemical activity is the π-electron distribution in the heterocyclic ring.     

Variable temperature infrared spectroscopy of cytosine-guanine base pairs: tautomerism versus polarization.

This report describes an infrared (IR) spectroscopic study of a model cytosine-guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O6-methylguanosine (O6MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35,724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw,J.Am.Chem.Soc. 112,829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent, but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C = O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O6-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

On tautomerism of the cytosine molecule

Tautomerism of the cytosine molecule is discussed in connection with recent experimental matrix-isolation infrared spectroscopic measurements and recent ab initio calculations of relative stabilities of tautomers and of IR spectra for different tautomeric forms of the compound. Experimental IR spectra in the N-H and O-H stretching regions and in the C = O stretching region are presented for cytosine and for its several derivatives considered as model compounds. This experimental evidence, as well as the quantum-mechanical calculations (including both electron correlation and zero-point vibrational contributions), clearly indicate that two tautomers of cytosine, i.e. the amino-hydroxy and amino-oxo forms with the hydrogen atom at the N(1) position, exist in equilibrium when the cytosine molecule is isolated in an inert environment. The effect of the environment on the relative stabilities of several tautomers is also discussed briefly.     

The effect of tautomeric constant on the specificity of nucleotide incorporation during DNA replication: support for the rare tautomer hypothesis of substitution mutagenesis

The nucleoside analogue dP (6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) displays ambivalent hydrogen bonding characteristics whereby the imino tautomer of P can base-pair with adenine and its amino tautomer can base-pair with guanine. Fixed imino and amino tautomers of 6-methyl-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one (N-methyl P) have been synthesised and their structures obtained by X-ray crystallography. The tautomeric constant of N-methyl P has been calculated from pK(a) values of the fixed tautomers and the kinetic parameters for the incorporation of its 5'-triphosphate (dPTP) by exonuclease-free Klenow fragment of DNA polymerase I have been determined. A strong correlation between the tautomeric constant and the incorporation specificity of dPTP is found. These results lend support to the proposal that the minor tautomeric forms of the natural bases may play an important role in substitution mutagenesis during DNA replication. Furthermore, they imply that DNA polymerases impose specific steric requirements on the base-pair during nucleotide incorporation.     

DFT studies on one-electron oxidation and one-electron reduction for 2- and 4-aminopyridines

Quantum-chemical calculations {DFT(B3LYP)/6-311+G(d,p)} were performed for all possible tautomers (aromatic and nonaromatic) of neutral 2- and 4-aminopyridines and their oxidized and reduced forms. One-electron oxidation has no important effect on the tautomeric preference for 2-aminopyridine. The amine tautomer is favored. However, oxidation increases the stability of the imine NH tautomer, and its contribution in the tautomeric mixture cannot be neglected. In the case of 4-aminopyridine, one-electron oxidation increases the stability of both the amine and imine NH tautomers. Consequently, they possess very close energies. As major tautomers, they dictate the composition of the tautomeric mixture. The CH tautomers may be considered as very rare forms for both neutral and oxidized aminopyridines. A reverse situation takes place for the reduced forms of aminopyridines. One-electron reduction favors the C3 atom for the labile proton for both aminopyridines. This may partially explain the origin of the CH tautomers for the anionic states of nucleobases containing the exo NH(2) group.     

Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind