Local Electrostatic Environment Research Articles

Ester carbonyl vibration as a sensitive probe of protein local electric field.

The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to gaining a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard, since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric-field map will find use in various applications. Furthermore, we show that, when situated in a non-hydrogen-bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, its application to amyloid fibrils formed by Aβ(16-22) revealed that the interior of such β-sheet assemblies has an ε value of approximately 5.6.

Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

AbstractThe ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to gaining a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non‐natural amino acids, L‐aspartic acid 4‐methyl ester and L‐glutamic acid 5‐methyl ester, is a convenient and sensitive probe in this regard, since its frequency correlates linearly with the local electrostatic field for both hydrogen‐bonding and non‐hydrogen‐bonding environments. We expect that the resultant frequency–electric‐field map will find use in various applications. Furthermore, we show that, when situated in a non‐hydrogen‐bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, its application to amyloid fibrils formed by Aβ16–22 revealed that the interior of such β‐sheet assemblies has an ε value of approximately 5.6.

IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

A variety of IR-active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon-deuterium bonds (CD), cyano groups (CN), and azides (N3 ); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N-terminal Src homology 3 domain of the murine adapter protein Crk-II (nSH3) with CD bonds site-selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two-state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N3 probes at five of the same positions. In agreement with previous studies, the position-dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface-exposed positions. Thus, unlike CD labels, which are both sensitive to their environment and non-perturbative, CN and N3 probes should be used with caution.

De Novo-Designed Metallopeptides with Type 2 Copper Centers: Modulation of Reduction Potentials and Nitrite Reductase Activities

Enzymatic reactions involving redox processes are highly sensitive to the local electrostatic environment. Despite considerable effort, the complex interactions among different influential factors in native proteins impede progress toward complete understanding of the structure-function relationship. Of particular interest is the type 2 copper center Cu(His)3, which may act as an electron transfer center in peptidylglycine α-hydroxylating monooxygenase (PHM) or a catalytic center in copper nitrite reductase (CuNiR). A de novo design strategy is used to probe the effect of modifying charged amino acid residues around, but not directly bound to, a Cu(His)3 center embedded in three-stranded coiled coils (TRI-H)3 [TRI-H = Ac-G WKALEEK LKALEEK LKALEEK HKALEEK G-NH2]. Specifically, the peptide TRI-EH (=TRI-HK22E) alters an important lysine to glutamate just above the copper binding center. With a series of TRI-EH peptides mutated below the metal center, we use a variety of spectroscopies (EPR, UV-vis, XAS) to show a direct impact on the protonation equilibria, copper binding affinities, reduction potentials, and nitrite reductase activities of these copper-peptide complexes. The potentials at a specific pH vary by 100 mV, and the nitrite reductase activities range over a factor of 4 in rates. We also observe that the affinities, potentials, and catalytic activities are strongly influenced by the pH conditions (pH 5.8-7.4). In general, Cu(II) affinities for the peptides are diminished at low pH values. The interplay among these factors can lead to a 200 mV shift in reduction potential across these peptides, which is determined by the pH-dependent affinities of copper in both oxidation states. This study illustrates the strength of de novo protein design in elucidating the influence of ionizable residues on a particular redox system, an important step toward understanding the factors that govern the properties of this metalloenzyme with a goal of eventually improving the catalytic activity.

Computational Infrared and Two-Dimensional Infrared Photon Echo Spectroscopy of Both Wild-Type and Double Mutant Myoglobin-CO Proteins

The CO stretching mode of both wild-type and double mutant ( T67R / S92D ) MbCO (carbonmonoxymyoglobin) proteins is an ideal infrared (IR) probe for studying the local electrostatic environment inside the myoglobin heme pocket. Recently, to elucidate the conformational switching dynamics between two distinguishable states, extensive IR absorption, IR pump-probe, and two-dimensional (2D) IR spectroscopic studies for various mutant MbCO's have been performed by the Fayer group. They showed that the 2D IR spectroscopy of the double mutant, which has a peroxidase enzyme activity, reveals a rapid chemical exchange between two distinct states, whereas that of the wild-type does not. Despite the fact that a few simulation studies on these systems were already performed and reported, such complicated experimental results have not been fully reproduced nor described in terms of conformational state-to-state transition processes. Here, we first develop a distributed vibrational solvatochromic charge model for describing the CO stretch frequency shift reflecting local electric potential changes. Then, by carrying out molecular dynamic simulations of the two MbCO's and examining their CO frequency trajectories, it becomes possible to identify a proper reaction coordinate consisting of His64 imidazole ring rotation and its distance to the CO ligand. From the 2D surfaces of the resulting potential of mean forces, the spectroscopically distinguished A1 and A3 states of the wild-type as well as two more substates of the double mutant are identified and their vibrational frequencies and distributions are separately examined. Our simulated IR absorption and 2D IR spectra of the two MbCO's are directly compared with the previous experimental results reported by the Fayer group. The chemical exchange rate constants extracted from the two-state kinetic analyses of the simulated 2D IR spectra are in excellent agreement with the experimental values. On the basis of the quantitative agreement between the simulated spectra and experimental ones, we further examine the conformational differences in the heme pockets of the two proteins and show that the double mutation, T67R / S92D , suppresses the A1 population, restricts the imidazole ring rotation, and increases hydrogen-bond strength between the imidazole Nε-H and the oxygen atom of the CO ligand. It is believed that such delicate change of distal His64 imidazole ring dynamics induced by the double mutation may be responsible for its enhanced peroxidase catalytic activity as compared to the wild-type myoglobin.

Synergistic effects of mutations in cytochrome P450cam designed to mimic CYP101D1.

A close orthologue to cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo-hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam, whereas this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme, about 15-20 Å away, Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing are substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV-vis spectroscopy. Individually, the mutants have little effect on activity or structure, but in combination there is a major drop in enzyme activity. This loss in activity is due to the mutants being locked in the low-spin state, which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well-separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states.

Infrared Probes Based on Nitrile-Derivatized Prolines: Thermal Insulation Effect and Enhanced Dynamic Range

Vibrational chromophores that are sensitive to local electrostatic environment are useful probes of structural variations of proteins on subnanosecond time scales, but their short vibrational lifetimes often limit their applicability. Here we explore a possibility to increase the lifetime of nitrile probes by introducing heavy atoms between the probe and protein side chains. Stereoisomers of thiocyanato- and selenocyanato-derivatized prolines, Pro-SCN and Pro-SeCN, are synthesized, and their CN stretch lifetimes in D2O and chloroform are measured with polarization-controlled IR pump–probe spectroscopy. The measured lifetimes of 170–330 ps for Pro-SeCN are three to four times longer than those for Pro-SCN, indicating that selenium atom is more effective than sulfur atom in blocking the intramolecular vibrational relaxation pathways of the CN stretch mode. This is further confirmed by carrying out nonequilibrium molecular dynamics simulations of the vibrational relaxation processes. Given the crucial role of the proline residue in determining protein structures, we anticipate that the Pro-SeCN probe can be an excellent site-specific probe of changes in protein local environment.

Electrostatic frequency shifts in amide I vibrational spectra: Direct parameterization against experiment

The interpretation of protein amide I infrared spectra has been greatly assisted by the observation that the vibrational frequency of a peptide unit reports on its local electrostatic environment. However, the interpretation of spectra remains largely qualitative due to a lack of direct quantitative connections between computational models and experimental data. Here, we present an empirical parameterization of an electrostatic amide I frequency map derived from the infrared absorption spectra of 28 dipeptides. The observed frequency shifts are analyzed in terms of the local electrostatic potential, field, and field gradient, evaluated at sites near the amide bond in molecular dynamics simulations. We find that the frequency shifts observed in experiment correlate very well with the electric field in the direction of the C=O bond evaluated at the position of the amide oxygen atom. A linear best-fit mapping between observed frequencies and electric field yield sample standard deviations of 2.8 and 3.7 cm(-1) for the CHARMM27 and OPLS-AA force fields, respectively, and maximum deviations (within our data set) of 9 cm(-1). These results are discussed in the broader context of amide I vibrational models and the effort to produce quantitative agreement between simulated and experimental absorption spectra.

Study of Self Catalyzed Protein Glycation on Monoclonal Antibody A (MAb A)

Protein glycation is known as a nonenzymatic reaction between reducing sugars, and amine groups. It may lead to functional changes of a protein. Recently, a site specific glycation on lysine Kn of MAb A was reported, with a hypothesis that MAb A self catalysis in reaction with glucoses at specific site. To elucidate the mechanism of the site specific glycation, an in vitro forced glycation study was employed to generate fully glycated MAb A. Subsequently, characterization of the glycated MAb A and its thermal degradation products were performed, through boronate affinity HPLC, ESI‐Mass spectrometry, Ion‐exchange HPLC, and peptide map. Finally, molecular dynamics (MD) simulations were used to map the local electrostatic environment around Kn under glycation conditions. The in vitro study showed that Kn is preferably glycated up to 90%, with four other lysine sites glycated at various low levels. Thermal stress study demonstrated that the main degradation pathway of highly glycated MAb A is hydrolysis of the Schiff base, the reverse reaction of glycation. Glycated Kn was preferably hydrolyzed as compared to the other glycated lysine sites. These results demonstrated the site specific glycation at Kn is self catalyzed. MD analysis suggested the observed self catalysis is caused by a strong electron‐donating environment surrounding Kn with three aspartates potentially involved in Kn reaction with a glucose.

Benzonitrile as a Probe of Local Environment in Ionic Liquids

Because of its sensitivity to chemical and electrostatic characteristics, nitrile group as an infrared (IR) probe to monitor the local structure, folding kinetics, and electrostatic environment of protein, or solvation of molecular solvents, has attracted increasing attention. Herein, by choosing benzonitrile and imidazolium ionic liquids (ILs) as the IR probe and model ILs, respectively, we report that the nitrile stretching vibration (νCN) could be utilized as a simple and substantial IR probe to monitor the local environment of ILs such as hydrogen bonding (H-bonding) as well as intrinsic electric field. In 1-alkyl-3-methylimidazolium-based non-hydroxyl ILs, the νCN is in a "free" state, and is less affected by the alkyl chain, while it significantly decreases with the effective anion charge. In 1-(2-hydroxyethyl)-3-methylimidazolium-based hydroxyl ILs, however, a distinct anion-dependent νCN forming H-bonding with the hydroxyl is also observed besides the "free" νCN band. The "free" component of νCN can be further employed to determine the intrinsic electric field in both non-hydroxyl (directly) and hydroxyl (indirectly by subtracting H-bonding contribution) ILs by using vibrational Stark effect. Moreover, the result suggests that benzonitrile is preferentially located in the charge domain in ILs and it could be a more suitable probe to report the ionic network rather than the nonpolar domain in ILs.

Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy.

Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pKa of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.

Vibrational solvatochromism and electrochromism. II. Multipole analysis

Small infrared probe molecules have been widely used to study local electrostatic environment in solutions and proteins. Using a variety of time- and frequency-resolved vibrational spectroscopic methods, one can accurately measure the solvation-induced vibrational frequency shifts and the timescales and amplitudes of frequency fluctuations of such IR probes. Since the corresponding frequency shifts are directly related to the local electric field and its spatial derivatives of the surrounding solvent molecules or amino acids in proteins, one can extract information on local electric field around an IR probe directly from the vibrational spectroscopic results. Here, we show that, carrying out a multipole analysis of the solvatochromic frequency shift, the solvatochromic dipole contribution to the frequency shift is not always the dominant factor. In the cases of the nitrile-, thiocyanato-, and azido-derivatized molecules, the solvatochromic quadrupole contributions to the corresponding stretch mode frequency shifts are particularly large and often comparable to the solvatochromic dipole contributions. Noting that the higher multipole moment-solvent electric field interactions are short range effects in comparison to the dipole interaction, the H-bonding interaction-induced vibrational frequency shift can be caused by such short-range multipole-field interaction effects. We anticipate that the present multipole analysis method specifically developed to describe the solvatochromic vibrational frequency shifts will be useful to understand the intermolecular interaction-induced vibrational property changes and to find out a relationship between vibrational solvatochromism and electrochromism of IR probes in condensed phases.

Studying the Effect of Site-Specific Hydrophobicity and Polarization on Hydrogen Bond Energy of Protein Using a Polarizable Method.

Quantification of backbone hydrogen bond energies in protein folding has remained elusive despite extensive theoretical and experimental investigations over the past 70 years. This is due to difficulties in experimental mutagenesis study as well as the lack of quantitatively reliable methods in theoretical calculation. Recent advance in experiment has enabled accurate measurement of site-specific backbone hydrogen bond energy in protein. In the present work, we developed an accurate and practical polarizable method to study site-specific hydrogen bond energies in the PIN WW domain. Excellent quantitative agreement between our calculated hydrogen bonding energy and recent experimental measurement is obtained. The direct comparison between theory and experiment helps uncover the microscopic mechanism of experimentally observed context dependent hydrogen bond contribution to protein stability in beta-sheet. In particular, our study reveals two effects that act in a cooperative manner to impact the strength of a hydrogen bond. One is the dynamic stability of the hydrogen bond determined by nearby solvent molecules, and the other is the polarization state of the hydrogen bond influenced by local electrostatic environment. The polar character of the hydrogen bond results in strong coupling between hydrophobic and polarization interactions in a cooperative manner. This nonadditive character in hydrogen bonding should help us better understand the microscopic mechanism in protein folding. Our study also investigated the possible structural effect of backbone amide to ester mutation which should be helpful to experimentalists using this technique in mutagenesis study.

Calculating Elastic Constants and the Effects of Curvature on the Binding of Lipid Chain Anchors to DPPC/DOPC/Cholesterol Model Lipid Bilayers

Using a theoretical model of a bilayer membrane containing cholesterol, dipalmitoyl-phophatidylcholine (DPPC), and dioleoylphosphatidylcholine (DOPC) that qualitatively reproduces phase diagrams of giant unilaminar vesicles (GUVs) of the same three components [R. Elliott, I. Szleifer, and M. Schick, Phys. Rev. Lett., 96, p.098101 (2006)], we calculate the bending and saddle-splay force constants in Gel, liquid-ordered (lo), and liquid-disordered (ld) phases. The molecular theory employed in our study allows us to determine the effects of the mode of membrane bending deformation on the value of the elastic constants for different phases. The effect of “blocked” vs “free” exchange of lipids across the two monolayers on the values of the bending constant is as high as 50 kbT in the ld phase to as high as 200 kbT in the lo phase. These results show that one must strongly consider the mode of deformation in regard to the mechanical properties of lipid bilayers. For example, if cholesterol is allowed to flip-flop and the other lipid species are “blocked”, then the bending elastic constant is 20-40 kbT larger then the case where all of the lipid species are allowed to be exchanged from leaflet to leaflet.We will also present results on how the curvature of lipid vesicles determines the amount of binding of molecules with lipid tail anchors. By explicitly determining the chemical potential difference of species across a curved bilayer under different modes of deformation, we are able to calculate the equilibrium binding concentrations of lipid tail anchors as a function of membrane curvature, concentration of lipids, and local electrostatic environment.

Unified Description of Optical Properties and Photostability of Fluorescent Proteins by Means of the Chromophore-Protein Electrostatic Interactions

Fluorescent proteins (FPs) are widely used in two-photon microscopy as genetically-targeted probes. Understanding the mechanisms of their two-photon absorption (2PA) and photobleaching is therefore of prime importance for creation of brighter and more photostable mutants.We study the effect of the local electrostatic environment of the FP chromophore on its optical properties and photostability. To this end, we select the series of red FPs, including DsRed and a number of its Fruits derivatives, all possessing the same red anionic chromophore but different local surrounding.2PA spectroscopy provided us a direct experimental access to the absolute values of the local electric field on the chromophore site [1]. Using crystallography data available for several of the proteins, we perform here a series of QM/MM calculations of local electric potentials and fields on the chromophore site and determine the “hot” spot on the chromophore where the calculated fields almost quantitatively match the experimental data. This procedure also allows us to validate the protonation state of certain amino acids near the chromophore.We further analyze our experimental 2PA spectra of the Fruits [2] and show that the local electric field at the “hot” spot of the chromophore drives their 2PA absorptivity. The variations of the field result in different vibronic band intensities and eventually are responsible for the 2PA (vibronic) peak amplitude.We finally present our experimental data on photobleaching kinetics and demonstrate that there is a correlation between the rate of chromophore degradation and local electrostatic potential on the chromophore.Our results suggest the directions of mutagenesis that can simultaneously improve the 2PA brightness and photostability.

Human Placental Alkaline Phosphatase‐Mediated Hydrolysis Correlates Tightly with the Electrostatic Contribution from Tail Group

Human placental alkaline phosphatase has been identified as a hydrolase that is significantly overexpressed on the surface of various solid tumor cells, and is therefore a suitable prodrug design target for non-invasive cancer imaging and therapy. Structure-based prediction of enzymatic activities is essential for rational prodrug design. We have been probing the catalytic proficiency--(k(cat) /K(M) )/k(w)--of placental alkaline phosphatase toward several widely diverse substrate structures experimentally and correlating these results to in silico predictions that are based on the free energy estimates obtained from docking of each substrate structure with placental alkaline phosphatase. We have found that electrostatic contribution from the tail group is the most crucial factor to determine the catalytic efficiencies of the substrates. The electrostatic contribution and the total binding energy of the tail group are well correlated with catalytic efficiencies (R² = 0.79 and 0.89, respectively). However, hydrophobic contribution from the tail group does not correlate with the catalytic efficiencies (negative correlation, R² = 0.27). This supports the prior hypothesis stating that alkaline phosphatase-mediated differential hydrolysis of its substrates is attributable to the differential interactions with the tail group, determined by the electrostatic contributions from the non-bridging oxygen atoms. Calculation of the electrostatic potentials within the active site of human placental alkaline phosphatase also suggests that the local positive electrostatic environment may account for its capability to distinguish various substrates. Our study is likely to have immediate implications in the design of prodrugs against human placental alkaline phosphatase and other esterases overexpressed by human tumor cells.

Quantifying the Stabilizing Energy of the Intraprotein Hydrogen Bond Due to Local Mutation

MD simulation of the WW domain of PIN based on a dynamically adjusted polarized protein-specific force field from quantum fragment calculations is carried out in both wild and VAL22ALA mutant states. The result shows that the geometry of the Arg14-TYR23 hydrogen bond is conserved upon mutation of VAL22 to ALA. However, the electrostatic energy of this hydrogen bond in the mutant is found to be 0.6 kcal/mol weaker than in the wild state, in close agreement with the experimentally measured upper limit of 1.2 kcal/mol. Analysis shows that the weakened energy of this hydrogen bond in the mutant is due to its dynamically changed polarization resulting from an altered local electrostatic environment near the hydrogen bond which becomes more exposed to the solvent than in the wild.

Direct Measurement of the Membrane Dipole Field in Bicelles Using Vibrational Stark Effect Spectroscopy

Electrostatic fields in lipid bilayer membranes influence the structure and function of membrane-associated proteins. We present here the first direct measurement of the membrane dipole electrostatic field in lipid bicelles using vibrational Stark effect spectroscopy, in which a nitrile oscillator’s vibrational frequency changes in response to its local electrostatic environment. We synthesized α-helical peptides containing the unnatural amino acid p-cyanophenylalanine (CN-Phe) at four locations along the helix. This peptide was intercalated into bicelles 5 and 15 nm in radius composed of mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). Changes in the vibrational absorption energy of the nitrile probe at each position along the helical axis were used to determine changes in the local electrostatic field of the probe. We measured the magnitude of the membrane dipole electrostatic field to be −6 MV/cm, changing rapidly near the membrane surface and more slowly in the low dielectric membrane interior.

Crystal Structure of Leishmania major Peroxidase and Characterization of the Compound I Tryptophan Radical

The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX). One common feature is a key Trp residue, Trp(208) in LmP and Trp(191) in CCP, that is situated adjacent to the proximal His heme ligand in CCP, APX, and LmP. In CCP, Trp(191) forms a stable cationic radical after reaction with H(2)O(2) to form Compound I; in APX, the radical is located on the porphyrin ring. In order to clarify the role of Trp(208) in LmP and to further probe peroxidase structure-function relationships, we have determined the crystal structure of LmP and have studied the role of Trp(208) using electron paramagnetic resonance spectroscopy (EPR), mutagenesis, and enzyme kinetics. Both CCP and LmP have an extended section of β structure near Trp(191) and Trp(208), respectively, which is absent in APX. This region provides stability to the Trp(191) radical in CCP. EPR of LmP Compound I exhibits an intense and stable signal similar to CCP Compound I. In the LmP W208F mutant, this signal disappears, indicating that Trp(208) forms a stable cationic radical. In LmP conversion of the Cys(197) to Thr significantly weakens the Compound I EPR signal and dramatically lowers enzyme activity. These results further support the view that modulation of the local electrostatic environment controls the stability of the Trp radical in peroxidases. Our results also suggest that the biological role of LmP is to function as a cytochrome c peroxidase.

Structures of Neutral and Protonated Water Clusters Confined in Predesigned Hosts: A Quantum Mechanical/Molecular Mechanical Study

Hybrid quantum mechanical/molecular mechanical (QM/MM) calculations have been carried out to investigate the structures of the neutral (H(2)O)(21) and protonated H(+)(H(2)O)(21) clusters confined in the crystal hosts. The influence of other cocrystallized species and the local electrostatic environments of the crystal hosts on the structures of water clusters has been analyzed. For the neutral (H(2)O)(21) cluster in the tetrahedral host, its low-lying structures are found to exist as a dodecahedral cage with one interior water molecule, which is in good accord with the corresponding X-ray data. The confined (H(2)O)(21) cluster possesses the main structural features of the lowest-energy structure of the free (H(2)O)(21) cluster in the gas phase. For the protonated H(+)(H(2)O)(21) cluster confined in the cubic cavity, its low-lying structures are found to have a common hexahedral (H(2)O)(20) shell, which is consistent with the experimental X-ray structure, but the position of the additional H(2)O (or the H(3)O(+) ion) in different low-lying structures is different, while the H(3)O(+) ion is situated at the center of the cage in the corresponding X-ray structure. The overall shape of the confined H(+)(H(2)O)(21) cluster is significantly different from that of the free cluster in the gas phase, and the confined cluster has much less intrinsic hydrogen bonds (H-bonds) than the free cluster, due to the need to form extrinsic H-bonds with acetonitrile molecules in the crystal host. The local electrostatic environment of the crystal host is found to exert a negligible influence on the structure of the (H(2)O)(21) cluster but play a significant role in modulating the structure of the H(+)(H(2)O)(21) cluster. This may be attributed to the fact that the protonated water cluster is much more easily polarized by the external electrostatic field of the crystal host than the neutral water cluster.