Nature of a H2O Molecule Confined in the Hydrophobic Interface between the Heme and G-Quartet Planes in a Heme-DNA Complex.

Heme binds selectively to the 3'-terminal G-quartet of all parallel G-quadruplex DNAs to form stable heme-DNA complexes. Interestingly, the heme-DNA complexes exhibit various spectroscopic and functional properties similar to those of hemoproteins. Since the nature of the axial ligands is crucial in determining the physicochemical properties of heme, identification and characterization of the axial ligands in a heme-DNA complex are essential to elucidate the structure-function relationship in the complex. NMR studies of a complex possessing a low-spin ferric heme with a water molecule (H2O) and cyanide ion (CN-) as the axial ligands allowed detailed characterization of the physicochemical nature of the axial H2O ligand. We found that the in-plane asymmetry of the heme electronic structure of the complex is not largely affected by the axial H2O coordination, indicating that the H2O confined in the hydrophobic interface between the heme and G-quartet planes of the complex rotates about the coordination bond with respect to the heme. The effect of the hydrogen(H)/deuterium(D) isotope replacement of the axial H2O on the heme electronic structure was manifested in the isotope shifts of paramagnetically shifted heme methyl proton signals of the complex in such a manner that three resolved peaks associated with axial H2O, HDO, and D2O were observed for each of the heme methyl proton signals. These findings provide not only the basis for an understanding of the nature of the unique axial H2O but also an insight into the molecular mechanism responsible for the control of the heme reactivity in the heme-DNA complex.

Effects of Heme Electronic Structure and Local Heme Environment on Catalytic Activity of a Peroxidase-Mimicking Heme-DNAzyme.

The catalytic cycle of a peroxidase-mimicking heme-DNAzyme involves an iron(IV)oxo porphyrin π-cation radical intermediate known as compound I formed through heterolytic O-O bond cleavage of an Fe3+-bound hydroperoxo ligand (Fe-OOH) in compound 0, like that of a heme enzyme such as horseradish peroxidase (HRP). Peroxidase assaying of complexes composed of chemically modified hemes possessing various electron densities of the heme iron atom (ρFe) and parallel-stranded tetrameric G-quadruplex DNAs of oligonucleotides d(TTAGGG), d(TTAGGGT), and d(TTAGGGA) was performed to elucidate the effects of the heme electronic structure and local heme environment on the catalytic activity of the heme-DNAzyme. The study revealed that the DNAzyme activity is enhanced through an increase in the ρFe and general base catalysis of the adenine base adjacent to the heme, which are reminiscent of the "push" and "pull" mechanisms in the catalytic cycle of HRP, respectively, and that the activity of the heme-DNAzyme can be independently controlled through the heme electronic structure and local heme environment. These findings allow a deeper understanding of the structure-function relationship of the peroxidase-mimicking heme-DNAzyme.

Reversible Redox System of 2-Oxypyritriphyrin(1.2.1) Accompanying Interconversion between 3-Pyridone and 3-Hydroxypyridine Units.

To develop a system in which a π-conjugation circuit switched by a redox reaction between 3-pyridone and 3-hydroxypyridine, a ring-contracted analog of oxypyriporphyrin, 2-oxypyritriphyrin(1.2.1) was synthesized for the first time. 2-Oxypyritriphyrin(1.2.1) contains a 14π aromatic conjugation circuit with the keto-form of the 3-oxypyridine ring. When 2-oxypyritriphyrin was treated with NaBH4 , not only the 3-oxypyridine group, but also the meso-carbons were also reduced to give a colorless porphyrinogen analog. The reduced compound could be oxidized again to provide 2-oxypyritripyhrin in a reversible manner.

Effect of the Electron Density of the Heme Fe Atom on the Nature of Fe-O2 Bonding in Oxy Myoglobin.

Mössbauer spectroscopy has been used to characterize oxygenated myoglobins (oxy Mbs) reconstituted with native and chemically modified 57Fe-enriched heme cofactors with different electron densities of the heme Fe atom (ρFe) and to elucidate the effect of a change in the ρFe on the nature of the bond between heme Fe and oxygen (O2), i.e., the Fe-O2 bond, in the protein. Quadrupole splitting (ΔEQ) was found to decrease with decreasing ρFe, and the observed ρFe-dependent ΔEQ confirmed an increase in the contribution of the ferric-superoxide (Fe3+-O2-) form to the resonance hybrid of the Fe-O2 fragment with decreasing ρFe. These observations explicitly accounted for the lowering of O2 affinity of the protein due to an increase in the O2 dissociation rate and a decrease in the autoxidation reaction rate of oxy Mb through decreasing H+ affinity of the bound ligand with decreasing ρFe. Therefore, the present study demonstrated the mechanism underlying the electronic control of O2 affinity and the autoxidation of the protein through the heme electronic structure. Carbon monoxide (CO) adducts of reconstituted Mbs (CO-Mbs) were similarly characterized, and we found that the resonance between the two canonical forms of the Fe-CO fragment was also affected by a change in ρFe. Thus, the nature of the Fe-ligand bond in the protein was found to be affected by the ρFe.

Deprotection of a benzyl unit induces a 22π aromatic macrocycle of 3-oxypyripentaphyrin(0.1.1.1.0) with strong NIR absorption.

We report aromaticity switching from a 6π pyridine ring to a 22π macrocyclic ring of 3-oxypyripentaphyrin(0.1.1.1.0). This system has potential applications in photodynamic therapy owing to macrocyclic aromaticity being selectively induced by protecting group removal and strong absorption bands produced in the NIR region especially in methanol.

meso-Diketopyripentaphyrin and Diketopyrihexaphyrin as Macrocyclic Tripyrrinone Ligands for NiII Ions.

We report expanded porphyrins with pyridine rings and two neighboring carbonyl groups, which allow NiII ions to coordinate to the tripyrrinone-type NNNO coordination structure with Ni-O bonds. The selectivity of tripyrrinone is superior to other pyrrolic or pyridinic cavities of expanded porphyrins. Introduction of α-carbonyl pyridine next to the tripyrrolic conjugated structure is a powerful strategy for regioselective metalation of flexible expanded porphyrinoids.

A Nuclear Resonance Vibrational Spectroscopic Study of Oxy Myoglobins Reconstituted with Chemically Modified Heme Cofactors: Insights into the Fe-O2 Bonding and Internal Dynamics of the Protein.

The molecular mechanism of O2 binding to hemoglobin (Hb) and myoglobin (Mb) is a long-standing issue in the field of bioinorganic and biophysical chemistry. The nature of Fe-O2 bond in oxy Hb and Mb had been extensively investigated by resonance Raman spectroscopy, which assigned the Fe-O2 stretching bands at ∼570 cm-1. However, resonance Raman assignment of the vibrational mode had been elusive due to the spectroscopic selection rule and to the limited information available about the ground-state molecular structure. Thus, nuclear resonance vibrational spectroscopy was applied to oxy Mbs reconstituted with 57Fe-labeled native heme cofactor and two chemically modified ones. This advanced spectroscopy in conjunction with DFT analyses gave new insights into the nature of the Fe-O2 bond of oxy heme by revealing the effect of heme peripheral substitutions on the vibrational dynamics of heme Fe atom, where the main Fe-O2 stretching band of the native protein was characterized at ∼420 cm-1.

Synergistic Effect of Distal Polar Interactions in Myoglobin and Their Structural Consequences.

In the L29F variant of myoglobin (Mb), the coordination of oxygen (O2) to the heme Fe atom is stabilized by favorable electrostatic interactions between the polar Fe-O2 moiety and the multipole of the phenyl ring of the Phe29 side chain (Phe29 interaction), in addition to the well-known hydrogen bond (H-bond) between the Fe-bound O2 and the 64th residue (distal H-bond; Carver, T. E.; Brantley, R. E., Jr.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem. 1992, 267, 14443-14450). The O2 and carbon monoxide (CO) binding properties and autoxidation of the L29F/H64L and L29F/H64Q variants reconstituted with a series of chemically modified heme cofactors were analyzed and then compared with those of native Mb, and the L29F, H64Q, and H64L variants similarly reconstituted with the chemically modified heme cofactors in order to elucidate the relationship between the Phe29 interaction and the distal H-bond that critically contributes to stabilization of Fe-bound O2. We found that the Phe29 interaction and distal H-bond act cooperatively to stabilize the Fe-bound O2 in such a manner that the Phe29 interaction strengthens with increasing strength of the distal H-bond. Comparison of the functional properties between the L29F and H64L variants indicated that the synergistic effect of the two interactions decreases the O2 dissociation and autoxidation rate constants of the protein by factors of ∼1/2000 and ∼1/400, respectively. Although the CO binding properties of the proteins were not greatly affected by the distal polar interactions, their synergistic effects were clearly and sharply manifested in the vibrational frequencies of the Fe-bound C-O stretching of the proteins.

Characterization of Catalytic Activities and Heme Coordination Structures of Heme-DNA Complexes Composed of Some Chemically Modified Hemes and an All Parallel-Stranded Tetrameric G-Quadruplex DNA Formed from d(TTAGGG).

Heme binds selectively to the 3'-terminal G-quartet (G6 G-quartet) of an all parallel-stranded tetrameric G-quadruplex DNA, [d(TTAGGG)]4, to form a heme-DNA complex. Complexes between [d(TTAGGG)]4 and a series of chemically modified hemes possessing a heme Fe atom with a variety of electron densities were characterized in terms of their peroxidase activities to evaluate the effect of a change in the electron density of the heme Fe atom (ρFe) on their activities. The peroxidase activity of a complex decreased with a decreasing ρFe, supporting the idea that the activity of the complex is elicited through a reaction mechanism similar to that of a peroxidase. In the ferrous heme-DNA complex, carbon monoxide (CO) can bind to the heme Fe atom on the side of the heme opposite the G6 G-quartet, and a water molecule (H2O) is coordinated to the Fe atom as another axial ligand, trans to the CO. The stretching frequencies of Fe-bound CO (νCO) and the Fe-C bond (νFe-C) of CO adducts of the heme-DNA complexes were determined to investigate the structural and electronic natures of the axial ligands coordinated to the heme Fe atom. Comparison of the νCO and νFe-C values of the heme-DNA complexes with those of myoglobin (Mb) revealed that the donor strength of the axial ligation trans to the CO in a complex is considerably weaker than that of the proximal histidine in Mb, as expected from the coordination of H2O trans to the CO in the complex.

Simulation Time Required for Diminishing the Initial Conformational Deviations among Protein Crystal Structures.

Multiple crystal structures of a single kind of protein can be generally separated into several groups from their conformational deviations. A major factor causing the structural separation is the space group of crystals, in which precipitating agents have a strong influence on the packing of proteins in a crystal. In this study, we examined whether the separated groups of protein crystal structures can be merged into one group by computer simulation without a precipitating agent. The crystal structures of hen egg-white lysozyme (HEWL), myoglobin (Mb), hemoglobin (Hb), and human serum albumin (HSA) were selected as samples for molecular dynamics (MD) simulation. For example, 25 MD simulations were performed for HEWL, with 25 computational models being built from different crystal structures. Cluster analysis was applied to 25 snapshot structures obtained at the same time point from the respective simulation trajectories and the cluster analysis was repeated every 5 ns during the simulations. As a result, the separated cluster groups were basically merged into one group with only a few exceptions. In HEWL, noticeable conformational changes from the crystal structures were observed after heating. The dependence of the simulated structures on the initial crystals was diminished, and all of the clusters were merged into one group at 20 ns of MD simulation. In Mb, all of the clusters were merged into one group at 10 ns. For Hb and HSA, the time necessary for merging the structures became longer. In Hb, the initial group separation gradually became ambiguous after pre-equilibration, and the time required for diminishing the dependence on the crystal structure was 130 ns except for one cluster group. In HSA, 160 ns was necessary for all of the clusters to be merged into one group. These times provide important index for judging the equilibration of protein simulations.

Analysis of Physicochemical Interaction of Aß40 with a GM1 Ganglioside-Containing Lipid Membrane.

The interaction of amyloid beta (Aß) peptides with the cell membrane is one of the factors enhancing Aß aggregation, which is closely related to neurodegenerative disease. In this work, we performed molecular dynamics (MD) simulation to investigate the initial stage of adhesion of Aß40 to a GM1 ganglioside-containing membrane. Conformational change of Aß40 due to interaction with the membrane was monitored and compared with that of Aß42 observed in the previous study. Multiple computational trials were executed to analyze the probability of Aß binding using a calculation model consisting of a GM1-containing mixed lipid membrane, a water layer, ions, and Aß40. A single long-time MD simulation was also carried out. It was suggested from the simulation that a cluster of sialic acids of GM1 head groups often caught the side chain of His13 or His14 of Aß40 in the early stage of the MD simulations. Afterward, the main chain of Leu34 formed many hydrogen bonds with gangliosides. These residues cooperatively work for Aß40 to be held on the lipid membrane. It is notable that Aß40 was observed to be deeply inserted into the head group region of the lipid membrane in some computational trials. In the insertion, Aß40 occasionally formed a hydrogen bond with sphingomyelin. The difference in the secondary structure between Aß40 and Aß42 was an important factor for Aß40 to be deeply inserted into the membrane.

Stable meso-Aryl ß-Alkyl Hybrid Sapphyrin with a Warped π-Conjugation Circuit and Neo-Confused Sapphyrin-Silver(I) Complex.

A meso-aryl and ß-alkyl substituted sapphyrin and its rhodium(I) and silver complexes were synthesized. This sapphyrin was so stable that the non-inverted and warped structure could be analyzed by X-ray crystallography even in its neutral state. Its bis-rhodium(I) complex has a more planar structure than the sapphyrin with enhanced aromaticity over the conjugation circuit. On the other hand, silver metalation of the sapphyrin caused a marked core rearrangement into a neo-confused sapphyrin derivative with a C(α)-N bond and a twisted macrocycle.

Simulation Study on Complex Conformations of Aß42 Peptides on a GM1 Ganglioside-Containing Lipid Membrane.

Aggregation and complex formation of amyloid beta (Aß) peptides on a neuronal cell membrane is a hallmark of neuro-disturbance diseases. In this work, we performed molecular dynamics (MD) simulations to investigate the initial stage of interactions of multiple Aß42 peptides on a GM1 ganglioside-containing membrane that mimics a micro-domain on the neuronal cell surface. Conformational changes of Aßs due to adhesion on the membrane and subsequent molecular interactions among the Aßs were monitored. It was suggested from results of the two 1.0 µs simulation trials that stable complexes of Aß peptides were not rapidly generated but that a steady binding of two Aßs was gradually formed. Observation of two Aßs that will be a complex with steady binding revealed that one Aß was bound to the membrane surface, while the other was attached to the first one without strong contact with the membrane. The motion of the first one was restricted and its conformational change was limited, with the basic side-chains of Arg5 and Lys28 working as anchors to hold the Aß helix region on the membrane. In contrast, the second one had high flexibility and showed diversity in its conformation. The second Aß can search for an energetically favorable binding position on the first one. A parallel ß-sheet structure was formed between the C-terminal sides of the two Aßs. Ala30 was critically important to lead the stable ß-sheet conformation at the C-terminal hydrophobic domains of Aßs. In the N-terminal sides, helix structures were kept in both Aßs.

[24]Pentaphyrin(2.1.1.1.1): A Strongly Antiaromatic Pentaphyrin.

[24]Pentaphyrin(2.1.1.1.1) 1 was synthesized by dehydrogenation of dihydropentaphyrin(2.1.1.1.1) 2 as the first example of vinylogous pentaphyrin. Pentaphyrin 1 takes a roughly planar structure and shows strong antiaromatic character, reflecting a 24π-conjugated circuit. In spite of the antiaromatic character and the relatively small circuit, 1 is stable under ambient conditions.

Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor.

The orientation of a CF3-substituted heme in sperm whale myoglobin and L29F, H64L, L29F/H64Q, and H64Q variant proteins has been investigated using 19F NMR spectroscopy to elucidate structural factors responsible for the thermodynamic stability of the heme orientational disorder, i.e., the presence of two heme orientations differing by a 180° rotation about the 5-15 meso axis, with respect to the protein moiety. Crystal structure of the met-aquo form of the wild-type myoglobin reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), determined at resolution of 1.25 Å, revealed the presence of the heme orientational disorder. Alterations of the salt bridge between the heme 13-propionate and Arg45(CD3) side chains due to the mutations resulted in equilibrium constants of the heme orientational disorder ranging between 0.42 and 1.4. Thus, the heme orientational disorder is affected by the salt bridge associated with the heme 13-propionate side chain, confirming the importance of the salt bridge in the heme binding to the protein.

Stable Non-fused [22]Pentaphyrins and A Fused [24]Pentaphyrin Displaying Crystal Polymorphism between Hückel and Möbius Structures.

Partially ß-substituted and meso-tetrakis(pentafluorophenyl)-substituted [22]pentaphyrins 11 and 12 were synthesized by acid-catalyzed condensation of a meso-C6 F5 -substituted tripyrrane dicarbinol with ß-alkylated dipyrromethanes. These [22]pentaphyrins are stable, allowing their characterization by NMR and UV/Vis spectroscopies, and X-ray crystallography, and display strong aromaticity due to 22π-electronic circuits. In methanol, ß,ß-diethoxycarbonylated pentaphyrin 12 underwent an N-fusion reaction to give N-fused pentaphyrin 13, which exhibits crystal polymorphism between Hückel and Möbius structures, depending upon the recrystallization solvent.

Characterization of Ground State Electron Configurations of High-Spin Quintet Ferrous Heme Iron in Deoxy Myoglobin Reconstituted with Trifluoromethyl Group-Substituted Heme Cofactors.

We introduced trifluoromethyl (CF3) group(s) as heme side chain(s) of sperm whale myoglobin (Mb) in order to characterize the electronic nature of heme Fe(II) in deoxy Mb using 19F NMR spectroscopy. On the basis of the anti-Curie behavior of CF3 signals, we found that the deoxy Mb is in thermal equilibrium between the 5B2, (dxy)2(dxz)(dyz)(dz2)(dx2-y2), and 5E, (dxy)(dxz)2(dyz)(dz2)(dx2-y2), states of the heme Fe(II), i.e., 5B2 ⇆ 5E. Analysis of the curvature in Curie plots has yielded for the first time ΔH and ΔS values of ∼-20 kJ mol-1 and ∼-60 J K-1 mol-1, respectively, for the thermal equilibrium. Thus, the 5E state is slightly dominant over the 5B2 one at 25 °C. These findings provide not only valuable information about the ground state electronic structure of the high-spin heme Fe(II) in deoxy native Mb but also an important clue for elucidating the mechanism responsible for acceleration of the spin-forbidden oxygenation of the protein.

[62]Tetradecaphyrin and Its Mono- and Bis-Zn(II) Complexes.

A coiled structure of meso-pentafluorophenyl-substituted [62]tetradecaphyrin 1 was revealed by X-ray structural analysis. Synthetic protocols were devised to form mono- and bis-Zn(II) complexes, 1 Zn and 1 Zn2 , selectively. The former displayed a trigonal-bipyramidal pentacoordinated Zn(II) ion as a rare case and a cyclic voltammogram exhibiting eleven reversible redox waves. The latter showed a Ci-symmetric structure with modest Hückel aromaticity owing to a 62 π-electronic circuit as the largest aromatic molecule to date.

Synthesis of 5,10-bis(Trifluoromethyl) Substituted ß-Octamethylporphyrins and Central-Metal-Dependent Solvolysis of Their meso-Trifluoromethyl Groups.

5,10-Bistrifluoromethyl substituted ß-octamethylporphyrins were synthesized via a scrambling side reaction of a dipyrromethane precursor in the presence of a large excess of trifluoroacetic acid. Compared with the trans-analogs, the cis-analogs of meso-trifluoromethyl ß-octaalkylporphyrin showed more red-shifted absorption bands. These meso-trifluoromethyl derivatives of ß-octaalkylporphyrins underwent smooth metalation, similar to other common porphyrins, however, the corresponding zinc complexes underwent a type of solvolysis, whereby the trifluoromethyl groups were converted into methoxycarbonyl groups by the methanol used as solvent. UV-visible absorption spectra and X-ray crystal structure analyses revealed that the presence of a methoxycarbonyl substituent did not influence the deformation of the molecular framework and its absorption properties; this is because the methoxycarbonyl has a planar and perpendicular geometry, as opposed to the relatively bulky trifluoromethyl substituent.

Conformational Fixation of a Rectangular Antiaromatic [28]Hexaphyrin Using Rationally Installed Peripheral Straps.

A rectangular [28]hexaphyrin bearing outer straps at the long side has been synthesized by SN Ar reaction of [26]hexaphyrin with allyl alcohol, intramolecular olefin metathesis by using Hoveyda-Grubbs second-generation catalyst, and reduction with NaBH4 . The peripheral straps enforce a rectangular conformation for the [28]hexaphyrin, which shows Hückel antiaromatic character, as confirmed by its planar X-ray structure, a strong paratropic ring current, characteristic UV/Vis/NIR absorption features, small electrochemical HOMO-LUMO gap, and very fast S1 decay.