Prosthetic weight acceptance mechanics in transtibial amputees wearing the Single Axis, Seattle Lite, and Flex Foot

We discuss the use of ab initio quantum mechanical methods in drug metabolism studies. These methods require only the positions and atomic numbers of the atoms to be specified and offer greater transferability than conventional molecular modeling techniques. This fact, coupled with the accuracy of our approach, permits 'computational experiments' to be performed, allowing details of reaction mechanisms to be understood. We review the application of these methods to the cytochrome P450 superfamily of enzymes. There is much interest in understanding the mechanisms of these enzymes due to their participation in a wide range of metabolic processes including drug activation/deactivation. We find that our methods accurately reproduce the low- to high-spin transition of the haem Fe on binding of a substrate. Furthermore, we identify a new mechanism for the suppression of this spin transition, namely the shortening of the bond between the Fe atom and the coordinated S atom of the cysteine axial ligand. These results indicate that ab initio molecular modeling may be usefully applied in the study of drug metabolism and that further study of intermediate states in the P450 reaction cycle would be beneficial, particularly those which are not accessible using conventional experimental approaches.     

Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells

In this article the complexation of anhydrotetracycline (AHTC), the major toxic decomposition product of the antibiotic tetracycline, with Al(III) has been investigated using the AM1 semiempirical and ab initio Hartree-Fock levels of theory. Different modes of complexation have been considered with the structure of tetra- and pentacoordinated complexes being fully optimized. In the gas phase, processes ii and iii, which lead to the complexes with stoichiometry MHL2+, are favored. Structure II ([AlLH2(OH)(H2O)]2+) has the metal coordinated to the O11 and O12 groups and the O3 group protonated and is the global minimum on the potential energy surface for the interaction. In water solution, the Al(III) is predicted to form predominantly a tetracoordinated complex at the Oam and O3 site (V) of the AHTC with the stoichiometry MH2L3+ (process i). The experimental proposal is the complexed form with the metal ion coordinated to the O11-O12 moiety (site II). The intramolecular proton transfer, which leads to the most stable Al(III)-AHTC MHL2+ complex, has not been considered by the experimentalists. The experimental structure was found to be unfavorable in our calculations in both gas phase and water solution. All the semiempirical results are in perfect agreement with the ab initio calculations. So, we suggest that the experimental assignments should be revised, taking into account the results obtained in the present study.     

Three-dimensional structural analysis of avian-teratogenic pyrimidine dialkyl phosphate esters and their analogs by molecular orbital calculations

The most stable structures of avian-teratogenic pyrimidinyl dialkyl phosphates and phosphorothionates at semi-empirical molecular orbital (MO) AM1 method level were obtained to get net charges of nitrogen (N) and phosphorus (P), distances between 1-N and P, and twist angles between plane of the heterocycle and that made by C-O-P. These geometries of typical compounds were optimized by ab initio MO calculations at level of HF/6-31 G*. In the most potent teratogens, distances between 1-N and P were 4.3-5.4 A. Compounds of about 3 A has no teratogenicity.     

Hydrogen atom abstraction of 3,5-disubstituted analogues of paracetamol by horseradish peroxidase and cytochrome P450

1. The formation of free radicals during enzyme catalysed oxidation of eight 3,5-disubstituted analogues of paracetamol (PAR) has been studied. A simple peroxidase system as well as cytochrome P450-containing systems were used. Radicals were detected by electron spin resonance (ESR) on incubation of PAR and 3,5-diCH3-, 3,5-diC2H5-, 3,5-ditC4H9-, 3,5-diOCH3-, 3,5-diSCH3-, 3,5-diF-, 3,5-diCl- and 3,5-diBr-substituted analogues of PAR with horseradish peroxidase in the presence of hydrogen peroxide (H2O2). Initial analysis of the observed ESR spectra revealed all radical species to be phenoxy radicals, based on the absence of dominant nitrogen hyperfine splittings. No radicals were detected in rat liver cytochrome P450-containing microsomal or reconstituted systems. 2. To rationalize the observed ESR spectra, hydrogen atom abstraction of PAR and four of the 3,5-disubstituted analogues (3,5-diCH3-, 3,5-diOCH3-, 3,5-diF- and 3,5-diCl-PAR) was calculated using ab initio calculations, and a singlet oxygen atom was used as the oxidizing species. The calculations indicated that for all compounds studied an initial hydrogen atom abstraction from the phenolic hydroxyl group is favoured by approximately 125 kJ/mol over an initial hydrogen atom abstraction from the acetylamino nitrogen atom, and that after hydrogen abstraction from the phenolic hydroxyl group, the unpaired electron remains predominantly localised at the phenoxy oxygen atom (+/-85%). 3. The experimental finding of phenoxy radicals in horseradish peroxidase/H2O2 incubations paralleled these theoretical findings. The failure to detect experimentally phenoxy radicals in cytochrome P450-catalysed oxidation of any of the eight 3,5-disubstituted PAR analogues is more likely due to the reducing effects that agents like NADPH and protein thiol groups have on phenoxy radicals rather than on the physical instability of the respective substrate radicals.     

The association rate constant for heme binding to globin is independent of protein structure

Rate constants for CO-heme binding to 35 different recombinant apomyoglobins and several other apoproteins were measured in an effort to understand the factors governing heme affinity and the velocity of the association reaction. Surprisingly, the rate constant for the binding of monomeric heme is approximately 1 x 10(8) M-1 s-1 regardless of the structure or overall affinity of the apoprotein for iron-porphyrin. Major differences between the proteins are reflected primarily in the rates of dissociation of the prosthetic group. Slow phases observed in the reaction of CO heme with excess apomyoglobin result from formation of nonspecific heme-protein complexes which must dissociate before heme can bind specifically in the heme pocket. Once the specific heme-globin complex is formed, the heme pocket rapidly collapses around the porphyrin, simultaneously forming the bond between the proximal His93 and the heme iron atom. The overall affinity of sperm whale apomyoglobin for hemin is approximately 1 x 10(14) M-1. Nonspecific hydrophobic interactions between the porphyrin and the apolar heme cavity account for a factor of 10(5)-10(7). Covalent bond formation between Fe3+ and His93(F8) provides an additional factor of 10(3)-10(4). Specific interactions with conserved amino acids in the heme pocket contribute the final factor of 10(3)-10(4).     

Factors affecting contrasting results between self-reported and performance-based levels of physical limitation

Molecular electrostatic potential (MEP) maps of certain 3-methoxy flavone derivatives having different anti-picornavirus activities have been studied. Geometries of the molecules were optimised and charge distributions computed using the AM1 molecular orbital method. Hybridization displacement charges (HDC) were combined with the L?wdin charge distributions to compute the MEP maps. Reliability of the method of computing MEP maps was tested by studying certain other molecules for which ab initio MEP results are available. The anti-picornavirus activities of the flavones have been shown to be related with negative MEP values in two regions, one near the 3-methoxy group and another in a diagonally opposite region near the substituent attached to the C7 atom of the molecules.     

Rotational Spectra of the Isotopic Species of Chloroform: Experimental and Ab Initio Structures

The millimeter-wave spectra of three different samples of chloroform (CHCl3, CDCl3, and 13CHCl3) have been measured between 145 and 470 GHz which corresponds to J values between 22 and 70. We report accurate rotational and centrifugal distortion constants for the ground vibrational states of 11 isotopic species. The experimental ro, rs, rIepsilon, rrhom, and rz structures have been determined using the determined rotational constants. The structure has also been calculated ab initio at the SCF, MP2, RQCISD, and B3LYP levels using triple zeta polarized basis sets. The experimental results are found in excellent agreement with the ab initio predictions. An approximate equilibrium structure has been obtained by combining the experimental results and the ab initio calculations: re(C-H) = 1.080 (2) ?, re(C-Cl) = 1.760 (2) ?, and anglee(HCCl) = 108.23 (2) degrees. Copyright 1998 Academic Press.     

Absorption and magnetic circular dichroism spectra of nonequilibrium states of hemoproteins. III. Complexes of peroxidase

Absorption and magnetic curcular dichroism spectra of nonequilibrium states of peroxidase and its complexes with F-, N3-, CN- produced by reduction of oxidased forms of proteins by thermalysed electrons at 77 degrees K were studied. Mixtures of high spin and low spin ferroforms were found in nonequilibrium states of peroxidase and complexes with F- and N3-, the content of the high spin ferroform increasing as follows: N3- complex less than peroxidase less than fluorine complex. Only low spin ferroforms was found after low temperature reduction of the cyanide complex. The existence of the low spin ferroform in equilibrium states of peroxidase and its complex with F- was explained by location of iron near the porphyrine plane. In the case of azide and cyanide complexes the existence of the low spin form is due to the presence of these ligands in heme iron's coordination sphere. The temperature relaxation of all nonequilibrium forms was investigated and a possible mechanism of the process is proposed.     

Nonenzymatic and enzymatic hydrolysis of alkyl halides: a theoretical study of the SN2 reactions of acetate and hydroxide ions with alkyl chlorides

The SN2 displacements of chloride ion from CH3Cl, C2H5Cl, and C2H4Cl2 by acetate and hydroxide ions have been investigated, using ab initio molecular orbital theory at the HF/6-31+G(d), MP2/6-31+G(d), and MP4/6-31+G(d) levels of theory. The central barriers (calculated from the initial ion-molecule complex) of the reactions, the differences of the overall reaction energies, and the geometries of the transition states are compared. Essential stereochemical changes before and after the displacement reactions are described for selected cases. The gas phase reactions of hydroxide with CH3Cl, C2H5Cl, and C2H4Cl2 have no overall barrier, but there is a small overall barrier for the reactions of acetate with CH3Cl, C2H5Cl, and C2H4Cl2. A self-consistent reaction field solvation model was used to examine the SN2 reactions between methyl chloride and hydroxide ion and between 1,2-dichloroethane and acetate in solution. As expected, the reactions in polar solvent have a large barrier. However, the transition state structures determined by ab initio calculations change only slightly in the presence of a highly polar solvent as compared with the gas phase. We also calibrated the PM3 method for future study of an enzymatic SN2 displacement of halogen.     

Mutation of the distal arginine in Coprinus cinereus peroxidase--structural implications

Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron. Spectroscopic techniques, in contrast to X-ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment. Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0. This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51-->Leu, Asn, Gln, and Lys, respectively). These mutations cause changes in the coordination and spin states of the heme iron, and in the v(Fe-Im) stretching frequency. The variations are explained in terms of pH-dependent changes, charge location, size and hydrogen-bonding acceptor/donor properties of the residue at position 51. The present work indicates that the hydrogen-bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.     

New Rydberg-Rydberg Transitions of the ArH and ArD Molecules

We report observations and analyses of bands in the spectra of the ArH and ArD molecules due to emission from the 4f Rydberg complexes. For ArD, the observed bands are 4f --> 5s and 4f --> 3dlambda for all three components lambda = 0, 1, and 2 of the 3d state. These give direct information on all 14 spin-orbit components of the 4f complex. For ArH, the data is more limited because the levels of the 5s state are broadened by predissociation, and only the lambda = 2 component of the 4f --> 3dlambda transitions is seen clearly, although a number of lines could be assigned to the 4f --> 3dpi band, which is strongly overlapped by the 6s --> 4p band. The 4f complexes of both isotopes are good example of Hund's case (d) coupling. The 4f orbitals are essentially non-core-penetrating, so that spin-orbit coupling is negligible for them, and the zero-rotation splitting agrees well with calculations from the ab initio quadrupole moment and anisotropic polarizability of the core. The orbit-rotation coupling parameter xi4f gives a value of the core electric dipole moment that is in reasonable agreement with ab initio and previous experimental values. A summary of the presently known states of ArH and ArD is presented. Copyright 1998 Academic Press.     

Oxidation of tetrahydro-beta-carboline by cytochrome P-450cam--determination and rationalisation of product distribution

Cytochrome P-450cam oxidises 1,2,3,4-tetrahydro-beta-carboline, an indolic alkaloid. We report here measurements of the product distribution of this oxidation. To rationalise the experimental results, ab initio quantum-chemistry calculations of the product stabilities and molecular-dynamics calculations of the substrate-binding mode in the active site were performed. The calculations suggest that the product distribution is influenced by both the relative intrinsic gas-phase stabilities of the monohydroxy products and by conformational rearrangement of the active site on substrate binding.     

Structural fluctuations of myoglobin from normal-modes, M?ssbauer, Raman, and absorption spectroscopy

A normal-mode analysis of carbon monoxymyoglobin (MbCO) and deoxymyoglobin (Mb) with 170 water molecules is performed for (54)Fe and (57)Fe. A projection is defined that extracts iron out-of-plane vibrational modes and is used to calculate spectra that can be compared with those from resonance Raman scattering. The calculated spectra and the isotopic shift (57)Fe versus (54)Fe agree with the experimental data. At low temperatures the average mean square fluctuations (MSFs) of the protein backbone atoms agree with molecular dynamics simulation. Below 180 K the MSFs of the heme iron agree with the data from Mossbauer spectroscopy. The MSFs of the iron atom relative to the heme are an order of magnitude smaller than the total MSFs of the iron atom. They agree with the data from optical absorption spectroscopy. Thus the MSFs of the iron atom as measured by Mossbauer spectroscopy can be used to probe the overall motion of the heme within the protein matrix, whereas the Gaussian thermal line broadening of the Soret band and the resonance Raman bands can be used to detect local intramolecular iron-porphyrin motions.     

Theoretical study of the distal-side steric and electrostatic effects on the vibrational characteristics of the FeCO unit of the carbonylheme proteins and their models

The vibronic theory of activation and quantum chemical intermediate neglect of differential overlap (INDO) calculations are used to study the activation of carbon monoxide (change of the C-O bond index and force field constant) by the imidazole complex with heme in dependence on the distortion of the porphyrin ring, geometry of the CO coordination, iron-carbon and iron-imidazole distances, iron displacement out of the porphyrin plane, and presence of the charged groups in the heme environment. It is shown that the main contribution to the CO activation stems from the change in the sigma donation from the 5 sigma CO orbital to iron, and back-bonding from the iron to the 2 pi orbital of CO. It follows from the results that none of the studied distortions can explain, by itself, the wide variation of the C-O vibrational frequency in the experimentally studied model compounds and heme proteins. To study the dependence of the properties of the FeCO unit on the presence of charged groups in the heme environment, the latter are simulated by the homogeneous electric field and point charges of different magnitude and location. The results show that charged groups can strongly affect the strength of the C-O bond and its vibrational frequency. It is found that the charges located on the distal side of the heme plane can affect the Fe-C and C-O bond indexes (and, consequently, the Fe-C and C-O vibrational frequencies), both in the same and in opposite directions, depending on their position. The theoretical results allow us to understand the peculiarities of the effect of charged groups on the properties of the FeCO unit both in heme proteins and in their model compounds.     

High Strength Stainless Austenitic CrMnN steels ‐ Part III: Electronic Properties

Some results of ab initio calculations and experimental studies of the C, N and C+N effect on the electron structure of austenitic CrMn steels are presented. It is shown that the total electron energy per atom decreases due to alloying in the sequence of carbon→nitrogen→carbon+nitrogen, which suggests a corresponding increase in the thermodynamic stability of the austenite. Alloying with nitrogen and nitrogen+carbon increases the density of electron states at the Fermi level in comparison with interstitial‐free and carbon‐alloyed steel. Measurements of conduction electron spin resonance (CESR) allow separating the contributions from free electrons which are responsible for the metallic character of interatomic bonds and from localized electrons involved in the covalent bonds. It is shown that, in contrast to carbon, nitrogen enhances the metallic character of atomic interactions with a maximum of the concentration of free electrons at some critical content of nitrogen (about 2 at.%). The combined alloying with carbon+nitrogen leads to two effects: (i) a larger concentration of free electrons and (ii) a shift of the critical content of interstitials towards higher values. The obtained results of the theoretical and experimental studies of the electron structure were used as a physical background for the development of super‐high–strength stainless austenitic steels.     

An artificial cytochrome P450 that hydroxylates unactivated carbons with regio- and stereoselectivity and useful catalytic turnovers

A catalyst has been synthesized comprising a manganese porphyrin carrying four beta-cyclodextrin groups. It catalyzes the hydroxylation of substrates of appropriate size carrying tert-butylphenyl groups that can hydrophobically bind into the cyclodextrin cavities. In one example as many as 650 catalytic turnovers are seen before the catalyst is oxidatively destroyed, and with a rate comparable to that of typical cytochrome P450 enzymes. In another example, a steroid derivative is regio- and stereoselectively hydroxylated at a single unactivated carbon atom, but more slowly and with fewer turnovers. The carbon attacked is not the most chemically reactive, and the selectivity is determined by the geometry of the catalyst-substrate complex. Nonbinding substrates are not reactive under the conditions used, and substrates with more flexible binding geometries give more than a single product.     

Cloning and expression of cDNA for soluble form of rat heme oxygenase-1

Heme oxygenase catalyzes the oxidation of heme to biliverdin and carbon monoxide. The gene encoding the truncated soluble rat heme oxygenase-1 (Metl-Pro267) was cloned. The enzyme protein was expressed in E. coli JM109 and purified to homogeneity. The molecular weight of the recombinant enzyme was 30 kDa as assessed by SDS-polyacrylamide gel electrophoresis. From a 3-L culture, about 90 mg of the purified enzyme was routinely obtained. The dependency of the heme oxygenase reaction catalyzed by the soluble enzyme on the NADPH-cytochrome P-450 reductase concentrations and the effect of catalase on the reaction were examined to compare with the purified membrane-bound form of heme oxygenase-1 (Yoshida and Kikuchi, 1978b). The activity of the soluble enzyme was inhibited at high concentrations of NADPH-cytochrome P-450 reductase and the inhibition was not alleviated by addition of catalase unlike the membrane-bound form. The ferric iron of the heme-heme oxygenase complex was in a typical high spin state at acidic to neutral pH (pH 6.5-7.0) but conversion to low spin state was observed at basic pH (pH 9-10). The heme bound to heme oxygenase was converted to biliverdin at a stoichiometric ratio of unity in the presence of NADPH-cytochrome P-450 reductase system. During the heme degradation of the heme-heme oxygenase complex under atmospheric oxygen, several intermediates, that is, oxygenated heme and verdoheme, were spectrally discriminated.     

Characteristics of streptolysin S hemolysis

The MCD spectra of ferri- and ferro-heme in high and low spin states and zinc porphyrin for reference have been measured in the ultraviolet region. Zinc porphyrin offered an A term at 325 nm attributable to the N transition. A ferrous low-spin complex with CO or CN exhibited MCD spectra composed of B terms around 325 nm. This striking difference from that of zinc porphyrin in the ultraviolet region is noted from the fact that their Soret and visible MCD's gave A terms similar to those of zinc porphyrin. A ferric complex with high spin had MCD spectrum different from that of low spin, the former showing a negative C term at 287 nm and a B term at 339 nm, but the latter positive C terms at 275 nm and 320 nm. Some of these Faraday parameters were ascribed to charge-transfer transitions.     

PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases

Theoretical quantum mechanical ab initio Hartree-Fock calculations on molecular systems, modeling processes related to the specificity of thymidylate synthase inactivation are reported. We considered several steps of the methylation of the substrate dUMP and 4- or 5-mono- and 4,5-bisubstituted dUMP analogs, as well. The following reactions were modeled: the cysteine residue (Cys198 in the L.casei enzyme) nucleophilic attack on the substrate and the substrate C(5)-H proton abstraction. The substrate was modeled by the 1-methyluracil molecule and its structural analogs. The cysteine Cys198 residue was modeled by the methylmercaptane molecule. The substrate-enzyme binary complex was modeled by the 1-methyl-5,6-dihydro-6-thiomethyl-uracil (P1) molecule. The present theoretical calculations suggest that the cysteine nucleophilic attack on the substrate may result in the SH-group addition to the pyrimidine C(5)=C(6) bond in the course of a weakly exothermic reaction. The formerly presumed enolate carbanion appeared to be weakly stable or unstable and it can readily split into the thiol and pyrimidine residues. The s2-thio- (P2) and s2,4-dithio- (P3) substrate analogs should form stable thiolate anions after cysteine residue attachment to the C(6) position of the pyrimidine ring. Studies of the deformed P1 molecule interacting with a water molecule bound to the pyrimidine C(4)=O carbonyl residue allow a suggestion that this water molecule may be directly involved in the C(5)-H proton abstraction and may serve as a proton transmitter between the substrate and the proton acceptor residue, possibly located on the cofactor N10-nitrogen. Interaction of the pyrimidine C(4)=O group, or its modification, with the N5,10-methylenetetrahydrofolate N(10) nitrogen atom is suggested as an additional factor influencing the inhibition process.     

Electron affinities of p-benzoquinone, p-benzoquinone imine and p-benzoquinone diimine, and spin densities of their p-benzosemiquinones computed by several quantum chemical models

Restricted and unrestricted (U) Hartree-Fock (HF), second-order M?ller-Plesset perturbation (MP2), density functional (DF), hybrid HF/DF and semiempirical (half-electron (HE) method) models have been used to calculate adiabatic electron affinities (EAad values) of p-benzoquinone (I), p-benzoquinone imine (VI) and p-benzoquinone diimine (XI), as well as expectation values () and spin density distributions in the radical anions of I, VI and XI. The AM1/AM1-HE and ab initio calculated structures are found to be in accord with each other. The ROHF/6-31G(d) method gave the poorest EAad result. The UHF and UMP2 wave functions were found to be substantially spin contaminated (for the radicals) and the accuracies of the EAad values calculated were also poor. The use of molecular energies obtained after spin annihilation did not lead to significant improvement of the UHF and UMP2 results. In contrast to the ROHF, UHF and UMP2 results, the DF(USVWN, UBVWN, UBLYP) and hybrid HF/DF(UB3LYP) methods, as well as the AM1-HE, gave much better results. The calculated EAad values decreased, as predicted by most of the models, in the order EAad(I) > EAad(VI) > EAad(XI). The differences in the EAs, EAad(I)-EAad(VI) and EAad(I)-EAad(XI), were consistently predicted to be about 8-9 and 17-18 kcal/mol, respectively, by the DF, B3LYP and AM1-HE models. The performance of the PM3 and SAM1 models was not as good as the AM1 model. Of all the methods tested, the B3LYP/6-311G(d,p) model is concluded to give the most accurate quantitative trend (I(42.6) > VI(33.1) > XI(23.7)) in EAad. The predicted trend in EA can satisfactorily be rationalized by the calculated LUMO orbital energies, atomic charges and spin density distributions. Analysis of the spin density data predicts that phenoxyl- and anilino-type radical anions predominate in the p-benzosemiquinones of I and XI, respectively, while both phenoxyl- and anilino-type radicals contribute to the structure of the p-benzosemiquinone of VI, with the anilino-type predominating.