Computational analysis of the transient movement of helices in sensory rhodopsin II.

MD simulation of sensory rhodopsin II was executed for three intermediates (ground-state, K-state, M-state) appearing in its photocycle. We observed a large displacement of the cytoplasmic side of helixF only in M-state among the three intermediates. This displacement was transmitted to TM2, and the cytoplasmic side of TM2 rotated clockwise. These transient movements are in agreement with the results of an EPR experiment. That is, the early stage of signal transduction in a sRII-HtrII complex was successfully reproduced by the in silico MD simulation. By analyzing the structure of the sRII-HtrII complex, the following findings about the photocycle of sRII were obtained: (1) The hydrogen bonds between helixF and other helices determine the direction of the movement of helixF; (2) three amino acids (Arg162, Thr189, Tyr199) are essential for sRII-HtrII binding and contribute to the motion transfer from sRII to HtrII; (3) after the isomerization of retinal, a major conformational change of retinal was caused by proton transfer from Schiff base to Asp75, which, in turn, triggers the steric collision of retinal with Trp171. This is the main reason for the movement of the cytoplasmic side of helixF.

Chloro(3,6,13,16-tetraethyl-2,7,12,17-tetramethylporphycenato-kappa4N)iron(III) chloroform solvate.

The X-ray crystallographic analysis of the title complex, chloro[3,10,13,20-tetraethyl-4,9,14,19-tetramethylpentacyclo[16.2.1.1(2,5).1(8,11).1(12,15)]tetracosa-2,4,6,8(23),9,12,14,16,18(21),19-decaene]iron(III) chloroform solvate, [Fe(C(33)H(37)N(4))Cl].CHCl(3), reveals a twisted macrocyclic framework with a slightly distorted rectangular pyramidal core, where the deviation of the central Fe(III) atom from the least-squares plane of the C(20)N(4) core is 0.594 (1) A. Some important bond distances are as follows: Fe-N 2.019 (3), 2.026 (3), 2.028 (3) and 2.034 (3) A; Fe-Cl 2.232 (1) A.

Mechanisms and surface chemical prediction of imipramine-induced hemolysis suppressed by modified cyclodextrins.

The suppression of imipramine hydrochloride (IMP)- induced hemolysis by native cyclodextrins (alpha-, beta-, and gamma-CDs) and beta-CD derivatives is measured as a function of CD concentration and is quantitatively correlated with the surface tension of the solution determined at 37.0 degrees C. The modified beta-CDs are more or less adsorbed onto the air-water interface and occupy larger areas than the wider rim of beta-CD. The surface tension data at low CD concentrations in the presence of 3 mM IMP allow us to estimate the 1:1 binding constants of IMP with CDs. Both the capabilities of hemolysis suppression and surface tension elevation for 3 mM IMP are strong in the order carboxymethyl-beta-CD (CM) > beta-CD approximately equal to 6-O-glucosyl-beta-CD (G(1)) > gamma-CD > 2-hydroxypropyl-beta-CD (HP) > alpha-CD > or = 2,6-di-O-methyl-beta-CD (DM). The suppression of IMP-induced hemolysis is ascribed to the decrease in the concentration of free IMP molecules. This concentration can be quantitatively estimated from the surface tension data determined at 37 degrees C. Therefore, the suppression of IMP-induced hemolysis by most of the CDs can be quantitatively predicted from these surface tension data, regardless of the kind and concentration of CD. However, alpha-CD, HP, and DM are outliers of this prediction. This failure for alpha-CD and HP is ascribed to their weaker competitive binding to IMP than to membrane phospholipid. Because DM has a strong hemolytic activity, it does not almost suppress the IMP-induced hemolysis.

(12,17-diethoxycarbonyl-2,3,6,7,11,18-hexamethylcorrphycenato-kappa4N)iodoiron(III) chloroform solvate.

The title complex, (diethyl 3,4,8,15,19,20-hexamethyl-21,22,23,24-tetraazopentacyclo[16.2.1.1(2,5).1(7,11).1(14,17)]tetracosa-1(20),2(22),3,5,7,9,11,13(24),14,16,18-undecaene-9,14-dicarboxylate-kappa4N)iodoiron(III) chloroform solvate, [Fe(C32H32N4O4)I].CHCl3, shows an almost planar arrangement of the corrphycene moiety with a slightly distorted trapezoid pyramidal core; the Fe(III) atom is 0.416 (1) A from the plane of the C20N4 system. The Fe-N distances are 2.049 (3), 2.044 (3), 2.079 (3) and 2.075 (3) A. The solvated chloroform forms a C-H...O hydrogen bond [C...O 3.107 (10) A] to an adjacent carbonyl O atom. This is the first X-ray structure analysis of a corrphycenatoiron(III) derivative.

Stoichiometric and microenvironmental effects on hydrolysis of propantheline and oxyphenonium bromides in cyclodextrin solutions.

The effects of alpha-, beta-, and gamma-cyclodextrins (CDs) on the basic hydrolysis of propantheline bromide (PB) and oxyphenonium bromide (OB) are analyzed in terms of the stoichiometry and microenvironments of their complexes. The rate constant of each species is evaluated with binding constant data for the 1:1, 1:2, and 2:1 complexes. The dielectric constant of the binding site of PB is estimated from the ultraviolet maximum wavelength in reference with the ethanol-water and dioxane-water systems. The energy-optimized structures of some complexes of PB with beta- and gamma-CD are obtained by molecular mechanics. Because the ester linkage of PB in the 1:1 complex with alpha-CD and in the 2:1 complex with gamma-CD is located near hydroxyls of the CD rim, these complexes catalyze the hydrolysis of PB. In contrast, the hydrolysis is inhibited by the formation of the 1:1 and 1:2 complexes of beta-CD and the 1:1 complex of gamma-CD because the ester linkage of PB is rather deeply incorporated into the CD cavities for these complexes. All the CDs inhibit the hydrolysis of OB. The rate constant of the 1:1 complex of OB and CD is in the decreasing order alpha-CD > gamma-CD > beta-CD. This order is consistent with that of the local dielectric constants of the binding sites.

Unusual spin state equilibrium of azide metmyoglobin induced by ferric corrphycene.

Myoglobin was reconstituted with the ferric complex of corrphycene, a novel porphyrin isomer with a rearranged tetrapyrrole array, to investigate the influence of porphyrin deformation on the equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states in the azide derivative. The azide affinity, 2.5 x 10(4) M(-1), was 1 order of magnitude lower than the corresponding values of a reference myoglobin containing an electron-deficient diformylheme similar to the corrphycene. Analysis of the visible absorption spectrum over a range of 0-40 degrees C reveals that the population of high-spin iron is 76-82% at room temperature for azide metmyoglobin complexed with ferric corrphycene. The unusual predominance of the high-spin state was verified from the infrared spectrum of coordinating azide, where the high-spin peak at 2046 cm(-1) is 4-fold larger in intensity than the 2023 cm(-1) low-spin band. Electron paramagnetic resonance at 15 K further indicated that the iron-histidine bond is cleaved to form a five-coordinate derivative in some fraction of the myoglobin. The remarkable high-spin bias of the spin equilibrium at room temperature and cleavage of the iron-histidine bond at 15 K could be explained in terms of the contracted and trapezoidal metallo core that weakens the iron-histidine bond of azide metmyoglobin bearing corrphycene.

Functional analysis of the iron(II) etiocorrphycene incorporated in the myoglobin heme pocket.

The iron(III) complex of 2,7,12,17-tetraethyl-3,6,11,18-tetra-methylcorrphycene, an isomeric heme, was complexed with apomyoglobin to examine the ligand binding ability of the novel macrocycle under physiological conditions. The reconstituted holoprotein was found to be functionally active at pH 7.4 and 20 degrees C and to bind oxygen and carbon monoxide reversibly with a half-saturation pressure at 6.7 and 3.5mmHg, respectively. Equilibrium affinities for these ligands are one to two orders of magnitude lower than those reported for native myoglobin. The functional anomaly was ascribed to the geometric and electronic strain on the iron(II) atom in the trapezoidal coordination core of corrphycene.

Ultraviolet spectroscopic estimation of microenvironments and bitter tastes of oxyphenonium bromide in cyclodextrin solutions.

The UV absorbance and bitter taste of oxyphenonium bromide (OB), an antiacetylcholine drug, in cyclodextrin (CD) solutions are measured, and the local environment of the binding site and the reduction of the bitter taste intensity are quantitatively estimated from the UV data. The UV spectrum of OB is changed with the addition of alpha-, beta-, and gamma-CD, because the phenyl group of OB is included into the CD cavity. The maximum wavelength, lambda(max), senses environmental changes of OB best among several spectral characteristics. From comparison of lambda(max) between a CD solution and the reference ethanol-water and dioxane-water systems, the dielectric constant of the binding site is evaluated. This value leads us to estimate the microenvironment and structure of the binding site. The suppression of the bitter taste of 4 mM OB by CDs is in the increasing order alpha-CD < gamma-CD < beta-CD. The extent of this suppression can be quantitatively predicted from the UV absorbance by assuming that the free OB molecule alone exhibits the bitter taste, regardless of the kind and concentration of CD. Some implications and limitations of the present approach are discussed.

Quantitative estimation of the bitter taste intensity of oxyphenonium bromide reduced by cyclodextrins from electromotive force measurements.

The bitter taste of oxyphenonium bromide, an antiacetylcholine drug, is suppressed by cyclodextrins. The extent of the suppression can be predicted from the electromotive force measurements with an oxyphenonium bromide-selective electrode. The relationship between the bitter taste intensity and the electromotive force holds true, regardless of the kind and concentration of natural and modified cyclodextrins. This result is explicable on the basis of the observation that both the bitter taste and the electric potential are determined by the concentration of free oxyphenonium bromide. Some implications and limitations of the present approach are discussed.

Structure and function of 6,7-dicarboxyheme-substituted myoglobin.

Myoglobin was reconstituted with 6,7-dicarboxy-1,2,3,4,5, 8-hexamethylheme, a compact synthetic heme with the shortest acid side chains, to pursue the structural and functional consequences after intensive disruption of the heme propionate-apoglobin linkages in the native protein. The electron-withdrawing carboxylate groups directly attached to the porphyrin ring lowered the oxygen affinity by 3-fold as compared with native myoglobin. Autoxidation of the oxy derivative to the ferric protein proceeded with 1.6 x 10(-)2 min-1 at pH 7.0 and 30 degrees C. The crystallographic structure of the cyanomet myoglobin with 1.9 A resolution shows that the heme adopts a unique orientation in the protein pocket to extend the two carboxylates toward solvent sphere. The native globin fold is conserved, and the conformations of globin side chains are almost intact except for those located nearby the heme 6,7-carboxylates. The 7-carboxylate only weakly interacts with Ser92 and His97 through two mediating water molecules. The 6-carboxylate, on the other hand, forms a novel salt bridge with Arg45 owing to conformational flexibility of the guanidinium side chain. The proton NMR shows that the small heme does not fluctuate about the iron-histidine bond even at 55 degreesC, suggesting that the salt bridge between Arg45 and heme 6-carboxylate is of critical importance to recognize and fix the heme in myoglobin.

Remarkable functional aspects of myoglobin induced by diazaheme prosthetic group.

The iron complex of beta,delta-diazamesoporphyrin III, a molecular hybrid of porphyrin and phthalocyanine, was incorporated into apomyoglobin to investigate novel biological aspects of myoglobin. The reconstituted ferric protein forms an internal hemichrome with the iron-bound distal histidine. The reduced ferrous protein has extraordinarily high affinities for O2 and CO. The ferrous myoglobin is capable of strong binding with pyridine, imidazole, cyanide, and azide, and reacts moderately with ammonia. The NO complex exhibited 5-coordinate to 6-coordinate transition over 150 min. The instability of 5-coordinate NO heme is consistent with a high affinity of imidazole to the ferrous heme. The kinetic analyses of the ferrous derivatives suggest the importance of the pi orbitals in neutral ligands as well as the negative charges in anionic ligands. A high affinity of imidazole to ferrous diazaheme accounts for the internal hemochrome formation in ferrous myoglobin containing phthalocyanines.

Functional comparison of the myoglobins reconstituted with symmetric deuterohemes.

Deuterohemins III and XIII were coupled with apomyoglobin to examine the influence of the modified heme-globin contacts on the functions of the reconstituted holoproteins. Owing to the molecular symmetry of the prosthetic groups, the resultant proteins are free from the heme orientational disorder. The coordination structures of the two reconstituted myoglobins were found to be normal and closely similar to each other. The equilibrium ligand bindings also resembled with each other in both ferric and ferrous states. The results demonstrate that the different local heme-globin contacts affect the structure and function of the reconstituted myoglobins only slightly. The results therefore suggest that the two asymmetric deuteroheme IX isomers, which are inverted about the alpha, gamma-meso carbon axis of the heme, also exhibit very similar functions in myoglobin.

Specific orientation of porphyrin at the binding site of catalytic antibody.

The catalytic antibody which catalyzes insertion of a cupric ion into mesoporphyrin bound ferric N-methyl mesoporhyrin and ferric mesoporhyrin to yield the respective complexes. The binding affinity of cyanide to the ferric iron in the complexes was compared with that of free ferric porphyrins. The results indicate that the one side of the porphyrin plane in the complex is thoroughly exposed to surrounding solvents and the other side interacts with the protein molecule. The cyanide binding suggests the specific orientation of distorted porphyrin at the binding site of the antibody.

Kinetic characterization of antibody-catalyzed insertion of a metal ion into porphyrin.

The insertion of a copper (II) ion into mesoporphyrin by a monoclonal catalytic antibody has been investigated kinetically by measuring the increase in Soret absorbance due to the production of copper (II)-mesoporphyrin. The initial rate of the reaction showed saturation kinetics as a function of the mesoporphyrin concentration, while it increased linearly with an increase in the copper (II) concentration. Based on observations, a scheme for the reaction was proposed: mesoporphyrin binds to the antibody to form a complex, and copper (II) binds to the complex to yield copper (II)-mesoporphyrin. Kinetic parameters for the respective steps were estimated, and the thermodynamic parameters were calculated. The binding of mesoporphyrin to the antibody was endothermic and entropically driven. This implies that hydrophobic interactions are an important factor in the binding. Free energy profiles for the antibody-catalyzed and uncatalyzed reactions were drawn by use of the obtained thermodynamic parameter values. The results demonstrate that the rate acceleration by, the antibody is ascribable to transition-state stabilization, and suggest that the structure of mesoporphyrin in the complex is more distorted than that of free mesoporphyrin.

Novel ligand binding properties of the myoglobin substituted with monoazahemin.

The iron complex of alpha-azamesoporphyrin XIII was combined with apomyoglobin to investigate influence of the meso nitrogen on ligand binding properties in the reconstituted protein. Stoichiometric complex formation between the two components was confirmed, and conservation of the native coordination structures in the resultant myoglobin was established with spectroscopic criteria and apparently normal ligand binding. The visible absorption spectra of various ferric and ferrous derivatives are characteristic with less intense Soret peaks and enhanced visible bands. The electron paramagnetic resonance spectrum with g = 5.2 suggests an anomalous intermediate spin (S = 3/2) character for the aquomet protein. The oxygen affinity of reduced azaheme myoglobin, 0.010 mm Hg, is 50 times larger than that of the native myoglobin. In addition, azaheme myoglobin forms stable complexes with imidazole, pyridine, or cyanide in ferrous state. All of these new properties were consistently explained in terms of stronger equatorial ligand field of the heme iron in a narrower coordination cavity. Similarities of azaheme to verdoheme were also pointed out.

Consequence of rapid heme rotation to the oxygen binding of myoglobin.

The myoglobin complexed with octamethylhemin was prepared. The visible absorption profiles are typical of general alkylhemin-substituted myoglobins, suggesting normal insertion of the hemin into the hydrophobic cavity. The NMR spectra of various ferric proteins were anomalous, without clearly resolved signals from the prosthetic group, most probably reflecting rapid heme rotation about the iron-histidine bond. The equilibrium and kinetic oxygen bindings were measured to examine the functional significance of the heme motion. Functional comparison with the myoglobin having immobile hemin indicates that rotation of the octamethylheme negligibly affects the myoglobin function. The results suggest that neither the heme peripheral contacts nor the proximal imidazole orientation about the heme normal is the dominant factor to control myoglobin function.

Structural analysis of the myoglobin reconstituted with iron porphine.

Sperm whale apomyoglobin was complexed with iron porphine to examine the influence of completely removed heme side chains on the entire molecular structure. Paramagnetic NMR peak from the proximal histidine of the deoxy protein ensured formation of the iron-histidine bond. Porphine pyrrole-proton NMR signals of the cyanmet and deoxy derivatives are unusually sharp single lines manifesting rapid heme rotation about the iron-histidine bond. X-ray crystallographic structure of the cyanmet derivative, determined with a final R factor of 0.21 for 11,808 independent reflections ranging from 7 to 1.8 A, was resolved at 1.8 A resolution. The result confirmed 1:1 coupling between apomyoglobin and iron porphine. The cyano ligand adopts a bent configuration with an Fe-C-N angle of 127 degrees and a Fe-CN distance of 1.89 A. The overall globin structure and side chain conformations are remarkably similar to those of native myoglobin despite intensive disruption of the original heme-globin interactions. The native apoprotein structure unexpectedly conserved even after iron porphine insertion demonstrates that the complex polypeptide fold of holomyoglobin is more inherent in the amino acid sequence than is generally believed.

Dynamic analysis of efficient heme rotation in myoglobin by NMR spectroscopy.

Sperm whale myoglobin was reconstituted with 1,4,5,8-tetramethylhemin. The hyperfine-shifted proton NMR signals from the prosthetic group exhibit remarkable pattern changes around 15 degrees C, while the globin resonances are normal to obey the Curie law. The NMR anomaly specifically observed for the heme signals suggests a slow to rapid rotational transition of the hemin about the iron-histidine bond. The temperature-dependent pattern changes were quantitatively analyzed by a dynamic NMR method. Two sets of analyses with the heme-methyl and pyrrole-proton lines consistently afforded delta H not equal to = 16.3 kcal/mol, delta S not equal to = 14.0 e.u., delta G not equal to = 12.1 kcal/mol at 298 K, and a frequency of 90 degrees heme rotation 5600 s-1 at 20 degrees C. The relatively large activation entropy suggests that structural rearrangements at the direct heme vicinity are involved and that efficient heme rotation is accomplished by a number of fluctuative local heme-globin contacts within a conserved crevice structure.