久保 稔

Among the four types of hemoglobin (Hb) M with a substitution of a tyrosine (Tyr) for either the proximal (F8) or distal (E7) histidine in the alpha or beta subunits, only Hb M Saskatoon (beta E7Tyr) assumes a hexacoordinate structure and its abnormal subunits can be reduced readily by methemoglobin (metHb) reductase. This is distinct from the other three M Hbs. To gain new insight into the cause of the difference, we examined the ionization states of E7 and F8 Tyrs by UV resonance Raman (RR) spectroscopy and Fe-O(Tyr) bonding by visible RR spectroscopy. Hb M Iwate (alpha F8Tyr), Hb M Boston (alpha E7Tyr), and Hb M Hyde Park (beta F8Tyr) exhibited two extra UV RR bands at 1,603 cm(-1) (Y8a') and 1,167 cm(-1) (Y9a') arising from deprotonated (ionized) Tyr, but Hb M Saskatoon displayed the UV RR bands of protonated (unionized) Tyr at 1,620 and 1,175 cm(-1) in addition to those of deprotonated Tyr. Evidence for the bonding of both ionization states of Tyr to the heme in Hb M Saskatoon was provided by visible RR spectroscopy. These results indicate that beta E7Tyr of Hb M Saskatoon is in equilibrium between protonated and deprotonated forms, which is responsible for facile reducibility. Comparison of the UV RR spectral features of metHb M with that of metHb A has revealed that metHb M Saskatoon and metHb M Hyde Park are in the R (relaxed) structure, similar to that of metHb A, whereas metHb M Iwate, metHb M Boston and metHb M Milwaukee are in the T (tense) quaternary structure.

The structure around the metal centre of a Ni(II) complex with an N2S2 square-planar geometry, 1, prepared as a model compound of the NiSOD active site was drastically changed upon addition of potassium superoxide (KO2).

Metal-dioxygen adducts, such as metal-superoxo and -peroxo species, are key intermediates often detected in the catalytic cycles of dioxygen activation by metalloenzymes and biomimetic compounds. The synthesis and spectroscopic characterization of an end-on nickel(II)-superoxo complex with a 14-membered macrocyclic ligand was reported previously. Here we report the isolation, spectroscopic characterization, and high-resolution crystal structure of a mononuclear side-on nickel(III)-peroxo complex with a 12-membered macrocyclic ligand, [Ni(12-TMC)(O(2))](+) (1) (12-TMC = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane). In contrast to the end-on nickel(II)-superoxo complex, the nickel(III)-peroxo complex is not reactive in electrophilic reactions, but is capable of conducting nucleophilic reactions. The nickel(III)-peroxo complex transfers the bound dioxygen to manganese(II) complexes, thus affording the corresponding nickel(II) and manganese(III)peroxo complexes. Our results demonstrate the significance of supporting ligands in tuning the geometric and electronic structures and reactivities of metal-O(2) intermediates that have been shown to have biological as well as synthetic usefulness in biomimetic reactions.

The nickel(II) complexes 1(X) supported by bis[(pyridin-2-yl)methyl]benzylamine tridentate ligands carrying m-substituted phenyl groups (X = OMe, Me, H, Cl, NO2) at the 6-position of pyridine donor groups (L-X, N,N-bis [(6-m-substituted-phenylpyridin-2-yl)methyl]benzylamine) have been synthesized and characterized. The X-ray crystallographic analyses have revealed that [Ni-II(L-H)(CH3CN)(H2O)](ClO4)(2) (1(H)), [Ni-II(L-OMe)(CH3CN)(MeOH)](ClO4)(2) (1(OMe)), [Ni-II(L-Me)(CH3CN)(H2O)](ClO4)(2) (1(Me)), and [Ni-II(L-Cl)(CH3CN)(H2O)](ClO4)(2) (1(Cl)) have a five-coordinate square pyramidal geometry, whereas [Ni-II(L-NO2)(CH3CN)(2)(H2O)](ClO4)(2) (1(NO2)) exhibits a six-coordinate octahedral geometry having an additional CH3CN co-ligand. H NMR spectra of the nickel(II) complexes 1(X) in CD3CN have indicated that all the complexes have a high spin ground state. The nickel(II) complexes 1(X) react with hydrogen peroxide (H2O2) in acetone to give bis(mu-oxo)dinickel(III) complexes 2(X) exhibiting a characteristic UV-vis absorption band at similar to 420 nm. In the case of 2(H), a resonance Raman band ascribable to a Ni2O2 core vibration was observed at 611 cm(-1) that shifted to 586 cm(-1) upon (H2O2)-O-18. The bis(mu-oxo)dinickel(III) intermediates 2(X) undergo an efficient aromatic ligand hydroxylation reaction, producing a mononuclear nickel(II)-phenolate complexes 4(X) via a putative intermediate (mu-phenoxo)(mu-hydroxo)dinickel (II) (3(X)). The kinetic studies on the aromatic ligand hydroxylation process including m-substituent effects (Hammett analysis) and kinetic deuterium isotope effects (KIE) have indicated that the reaction of 2(X) to 3(X) involves an electrophilic aromatic substitution mechanism, where C-O bond formation and C-H bond cleavage occur in a concerted manner. Intermediate 3(H) was detected by ESI-MS during the course of the reaction, and the final product 4(H) was characterized by elemental analysis, ESI-MS, and X-ray crystallographic analysis.

Mononuclear copper (II)-superoxo complexes 2(X)-OO(center dot) having triplet (S = 1) ground states were obtained via reaction Of O(2) with the copper(I) starting materials 1(X) supported by tridentate tigands L(X) [1-(2-p-X-phenethyl)-5-(2-pyridin-2-ylethyl)-1,5-diazacyclooctane; X = CH(3), H, NO(2)] in various solvents. The superoxo complexes 2(X)-OO(center dot) mimic the structure [tetrahedral geometry with an end-on (eta(1))-bound O(2)(center dot-)] and the aliphatic C-H bond activation chemistry of peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase.

A series of (mu-eta(2):eta(2)-disulfido) dinickel(II) complexes 2(n) have been synthesized by the reaction of Na2S2 and nickel(II) complexes 1(n) supported by tris[(pyridin-2-yl) methyl] amine (TPA; 1(0)) [(6-methylpyridin-2-yl) methyl] bis[(pyridin-2-yl) methyl] amine (Me(1)TPA; 1(1)), bis[(6-methylpyridin-2-yl) methyl][(pyridin-2-yl) methyl] amine (Me(2)TPA; 1(2)) and tris[(6-methylpyridin-2-yl) methyl] amine (Me(3)TPA; 1(3)), respectively, and characterised by UV-vis and resonance Raman spectroscopy. X-ray crystallographic analyses on 2(2) and 2(3) supported by Me(2)TPA and Me(3)TPA, respectively, have revealed that the Ni2S2 core is largely bent (similar to 30 degrees) along the S-S axis, being in sharp contrast to the planar Ni2S2 core structure of the (mu-eta(2):eta(2)-disulfido) dinickel(II) complexes reported so far. The UV-vis spectra of 2(0) and 2(1) supported by TPA and Me(1)TPA, respectively, exhibiting an intense absorption band at similar to 360 nm together with a shoulder around 400 nm and a weak and broad absorption band around 450-600 nm, are very close to those of 2(2) and 2(3), suggesting that 2(0) and 2(1) also exhibit a similar distorted Ni2S2 core structure. Resonance Raman spectra of 2(n) showed a characteristic S-S stretching vibration mode at similar to 450 cm(-1) with an isotope shift boolean AND nu(S-32-S-34) = 10-15 cm(-1). The reaction of 2(n) with (p-Me-C6H4)(3)P gave (p-Me-C6H4)(3)P=S quantitatively based on 2(n). Hammett analysis on the sulfur atom transfer process from 2(n) to the phosphine derivatives [(p-X-C6H4)P; X = OMe, Me, H and Cl] has indicated that the reaction involves an electrophilic ionic mechanism. Moreover, the order of reactivity of 2(n) toward PPh3 has been found as 2(0) > 2(3) > 2(1) > 2(2). On the basis of these results, the ligand effects of Me(n)TPA on the structure and reactivity of the nickel(II) complexes have been discussed.

The structure and H2O2-reactivity of a series of copper(II) complexes supported by tris[(pyridin-2-yl)methyl]amine (TPA) derivatives having a phenyl group at the 6-position of pyridine donor group(s) [(6-phenylpyridin-2-yl)methyl]bis[(pyridin-2-yl)methyl]amine (Ph(1)TPA), bis[(6-phenylpyridin-2-yl)methyl][(pyridin-2-yl)methyl]amine (Ph(2)TPA), and tris[(6-phenylpyridin-2-yl)methyl]amine (Ph(3)TPA) have systematically been examined to get insights into the aromatic substituent (6-Ph) effects on the coordination chemistry of TPA ligand system. The X-ray crystallographic analyses have revealed that [Cu-II(TPA)(CH3CN)](ClO4)(2) (CuTPA) and [Cu-II(Ph(3)TPA)(CH3CN)](ClO4)(2) (3) exhibit a trigonal bipyramidal structure, whereas [Cu-II(Ph(1)TPA)(CH3CN)](ClO4)(2) (1) shows a slightly distorted square pyramidal structure and [Cu-II(Ph(2)TPA)(CH3CN)](ClO4)(2) (2) has an intermediate structure between trigonal bipyramidal and square pyramidal. On the other hand, the UV-vis and ESR data have suggested that all the copper(II) complexes have a similar trigonal bipyramidal structure in solution. The redox potentials of CuTPA, 1, 2, and 3 have been determined as E-1/2 = -0.34, -0.28, -0.16, and -0.04 mV vs Ag/AgNO3, respectively, demonstrating that introduction of each 6-Ph group causes positive shift of E-1/2 about 0.1 V. Notable difference in H2O2-reactivity has been found among the copper(II) complexes. Namely, CuTPA and 1 afforded mononuclear copper(II)-hydroperoxo complexes CuTPA-OOH and 1-OOH, respectively, whereas complex 2 provided bis(mu-oxo)dicopper(III) complex 2-oxo. On the other hand, copper(II) complex 3 was reduced to the corresponding copper(I) complex 3(red). On the basis of the H2O2-reactivity together with the X-ray structures and the redox potentials of the copper(II) complexes, the substituent effects of 6-Ph are discussed in detail.

The low-frequency mode activity of metal loporphyrins has been studied for iron porphine-halides (Fe(P)(X), X = CI, Br) and nitrophorin 4 (NP4) using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). It is confirmed that the mode symmetry selection rules for FCS are the same as for Raman scattering and that both Franck-Condon and Jahn-Teller mode activities are observed for Fe(P)(X) under Soret resonance conditions. The DFT-calculated low-frequency (20-400 cm(-1)) modes, and their frequency shifts upon halide substitution, are in good agreement with experimental Raman and coherence data, so that mode assignments can be made. The doming mode is located at similar to 80 cm(-1) for Fe(P)(CI) and at similar to 60 cm(-1) for Fe(P)(Br). NP4 is also studied with coherence techniques, and the NO-bound species of ferric and ferrous NP4 display a mode at similar to 30-40 cm(-1) that is associated with transient heme doming motion following NO photolysis. The coherence spectra of three ferric derivatives of NP4 with different degrees of heme ruffling distortion are also investigated. We find a mode at similar to 60 cm(-1) whose relative intensity in the coherence spectra depends quadratically on the magnitude of the ruffling distortion. To quantitatively account for this correlation, a new "distortion-induced" Raman enhancement mechanism is presented. This mechanism is unique to low-frequency "soft modes" of the molecular framework that can be distorted by environmental forces. These results demonstrate the potential of FCS as a sensitive probe of dynamic and functionally important nonplanar heme vibrational excitations that are induced by the protein environmental forces or by the chemical reactions in the aqueous phase.

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths,with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm-1, which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.

Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm(-1)) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above similar to 50 cm(-1) and very weak activity below similar to 50 cm(-1). Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm(-1), while HRPCN displays a strong oscillation at higher frequency (96 cm(-1)) that can be correlated with the saddling distortion observed, in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm(-1), probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm(-1) that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T less than or similar to 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out.

Femtosecond coherence spectroscopy is applied to a series of ferric heme protein samples. The low-frequency vibrational spectra that are revealed show dominant oscillations near 40 cm(-1). MbCN is taken as a typical example of a histidine-ligated, six-coordinate, ferric heme and a comprehensive spectroscopic analysis is carried out. The results of this analysis reveal a new heme photoproduct species, absorbing near 418 nm, which is consistent with the photolysis of the His(93) axial ligand. The photoproduct undergoes subsequent rebinding/recovery with a time constant of similar to 4 ps. The photoproduct lineshapes are consistent with a photolysis quantum yield of 75-100%, although the observation of a relatively strong six-coordinate heme coherence near 252 cm(-1) (assigned to nu(9) in the MbCN Raman spectrum) suggests that the 75% lower limit is much more likely. The phase and amplitude excitation profiles of the low-frequency mode at 40 cm(-1) suggest that this mode is strongly coupled to the IMbCN photoproduct species and it is assigned to the doming mode of the transient penta-coordinated material. The absolute phase of the 40 cm(-1) mode is found to be pi/2 on the red side of 418 nm and it jumps to 3 pi/2 as excitation is tuned to the blue side of 418 nm. The absolute phase of the 40 cm(-1) signal is not explained by the standard theory for resonant impulsive stimulated Raman scattering. New mechanisms that give a dominant momentum impulse to the resonant wavepacket, rather than a coordinate displacement, are discussed. The possibilities of heme iron atom recoil after photolysis, as well as ultrafast nonradiative decay, are explored as potential ways to generate the strong momentum impulse needed to understand the phase properties of the 40 cm(-1) mode.

Reaction of beta-diketiminate copper(II) complexes and Na(2)S(2) resulted in formation of (mu-eta(2):eta(2)-disulfido)dicopper(II) complexes (adduct formation) or beta-diketiminate copper(I) complexes (reduction of copper(II)) depending on the substituents of the supporting ligands. In the case of sterically less demanding ligands, adduct formation occurred to provide the (mu-eta(2):eta(2)-disulfido)dicopper(II) complexes, whereas reduction of copper(II) took place to give the corresponding copper(I) complexes with sterically more demanding beta-diketiminate ligands. Spectroscopic examinations of the reactions at low temperature using UV-vis and ESR as well as kinetic analysis have suggested that a 1 : 1 adduct LCu(II)-S-SNa with an end-on binding mode is initially formed as a common intermediate, from which different reaction pathways exist depending on the steric environment of the metal-coordination sphere provided by the ligands. Thus, with the sterically less demanding ligands, rearrangement of the disulfide adduct from end-on to side-on followed by self-dimerisation occurs to give the (mu-eta(2):eta(2)-disulfido)dicopper(II) complexes, whereas such an intramolecular rearrangement of the disulfide co-ligand does not take place with the sterically more demanding ligands. In this case, homolytic cleavage of the Cu(II)-S bond occurs to give the reduced copper(I) product. The steric effects of the supporting ligands have been discussed on the basis of detailed analysis of the crystal structures of the copper(II) starting materials.

A subnanosecond time-resolved ultraviolet (UV) resonance Raman system has been developed to study protein structural dynamics. The system is based on a 1 kHz Nd:YLF-pumped Ti:Sapphire regenerative amplifier with harmonic generation that can deliver visible (412, 440, 458, and 488 nm) and UV (206, 220, 229, and 244 nm) pulses. A subnanosecond (0.2 ns) tunable near-infrared pulse from a custom-made Ti:Sapphire oscillator is used to seed the regenerative amplifier. A narrow linewidth of the subnanosecond pulse offers the advantage of high resolution of UV resonance Raman spectra, which is critical to obtain site-specific information on protein structures. By combination with a I m single spectrograph equipped with a 3600 grooves/mm holographic grating and a custom-made prism prefilter, the present system achieves excellent spectral (<10 cm(-1)) and frequency (similar to 1 cm(-1)) resolutions with a relatively high temporal resolution (<0.5 ns). We also report the application of this system to two heme proteins, hemoglobin A and CooA, with the 4,40 nm pump and 220 nm probe wavelengths. For hemoglobin A, a structural change during the transition to the earliest intermediate upon CO photodissociation is successfully observed, specifically, nanosecond cleavage of the A-E interhelical hydrogen bonds within each subunit at Trp alpha 14 and Trp beta 15 residues. For CooA, on the other hand, rapid structural distortion (<0.5 ns) by CO photodissociation and nanosecond structural relaxation following CO geminate recombination are observed through the Raman bands of Phe and Trp residues located near the heme. These results demonstrate the high potential of this instrument to detect local protein motions subsequent to photoreactions in their active sites.

HemAT is a signal transducer protein responsible for aerotaxis control of some bacteria and archaea, which contains a heme-containing globin domain as the sensor of its physiological effector, O(2). The interaction between the heme-bound ligand and the surrounding amino acid residue(s) plays a crucial role for selective sensing of O(2) and signal transduction by HemAT. In this work, we have elucidated by resonance Raman spectroscopy how O(2) and CO interact with HemAT-Hs and HemAT-Rr, HemAT from Halobacterium salinarum and Rhodospirillum rubrum, respectively. HemAT-Hs and HemAT-Rr showed three conformers in the O(2)-bound form, as is the case of HemAT-Bs, HemAT from Bacillus subtilis. Though the hydrogen bonding patterns observed in the three conformers were the same for HemAT-Bs, HemAT-Hs, and HemAT-Rr, the involved residues for the hydrogen bonding interaction were different from one another. Copyright (C) 2008 Society of Porphyrins & Phthalocyanines.

Femtosecond coherence spectroscopy is used to probe low frequency (20-400 cm(-1)) modes of the ferrous heme group in solution, with and without 2-methyl imidazole (2MeIm) as an axial ligand. The results are compared to heme proteins (CPO, P450(cam), HRP, Mb) where insertion of the heme into the protein results in redistribution of the low frequency spectral density and in (similar to 60%) longer damping times for the coherent signals. The major effect of imidazole ligation to the ferrous heme is the "softening'' of the low frequency force constants by a factor of similar to 0.6 +/- 0.1. The functional consequences of imidazole ligation are assessed and it is found that the enthalpic CO rebinding barrier is increased significantly when imidazole is bound. The force constant softening analysis, combined with the kinetics results, indicates that the iron is displaced by only similar to 0.2 angstrom from the heme plane in the absence of the imidazole ligand, whereas it is displaced by similar to 0.4 angstrom when imidazole ( histidine) is present. This suggests that binding of imidazole ( histidine) as an axial ligand, and the concomitant softening of the force constants, leads to an anharmonic distortion of the heme group that has significant functional consequences.

The ultrafast electron transfer occurring upon Soret excitation of three new porphyrin-ferrocene (XP-Fc) dyads has been studied by femtosecond up-conversion and pump-probe techniques. In the XP-Fc dyads (XP-Fcs) designed in this study, the ferrocene moiety is covalently bonded to the meso positions of 3,5-di-tert-butylphenyl zinc porphyrin (BPZnP-Fc), pentafluorophenyl zinc porphyrin (FPZnP-Fc), and 3,5-di-tert-butylphenyl free-base porphyrin (BPH2P-Fc). Charge separation and recombination in the XP-Fcs were confirmed by transient absorption spectra, and the lifetimes of the charge-separated states were estimated from the decay rate of the porphyrin radical anion band to be approximately 20 ps. The charge-separation rates of the XP-Fcs were found to be > 10(13) s(-1) from the S-2 state and 6.3 x 10(12) s(-1) from the S-1 state. Charge separation from the S-2 state was particularly efficient for BPZnP-Fc, whereas the main reaction pathway was from the S-1 state for BPH2P-Fc. Charge separation from the S-2 and S-1 states occurred at virtually the same rate in benzene and tetrahydrofuran and was much faster than their solvation times. Analysis of these results using semiquantum Marcus theory indicates that the magnitude of the electronic-tunneling matrix element is rather large and far outside the range of nonadiabatic approximation. The pump-probe data show the presence of vibrational coherence during the reactions, suggesting that wavepacket dynamics on the adiabatic potential energy surface might regulate the ultrafast reactions.

Ultrafast electron transfer in a porphyrin-ferrocene dyad in which the ferrocene moiety is directly linked at the meso-position of the porphyrin was studied using femtosecond up-conversion and pump-probe techniques. The time constant of electron transfer was similar to 110 fs both in benzene and tetrahydrofuran. Vibrational coherence of the 130-, 200-, and 250-cm(-1) modes, which were assigned to the translation and rotation of ferrocene, was observed, and was found to be phase shifted with respect to the vibrational coherence of the 330-cm(-1) mode. The relation between this behavior of vibrational modes and electron transfer is discussed. (c) 2006 Elsevier B.V. All rights reserved.

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O-2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector, O-2, from other gas molecules to generate the aerotactic signal, but the detailed mechanism of the selective O-2 sensing is not obvious. In this study, we measured electronic absorption, electron paramagnetic resonance (EPR), and resonance Raman spectra of HemAT-Bs to elucidate the mechanism of selective O-2 sensing by HemAT-Bs. Resonance Raman spectroscopy revealed the presence of a hydrogen bond between His86 and the heme propionate only in the O-2-bound form, in addition to that between Thr95 and the heme-bound O-2. The disruption of this hydrogen bond by the mutation of His86 caused the disappearance of a conformer with a direct hydrogen bond between Thr95 and the heme-bound O-2 that is present in WT HemAT-Bs. On the basis of these results, we propose a model for selective O-2 sensing by HemAT-Bs as follows. The formation of the hydrogen bond between His86 and the heme propionate induces a conformational change of the CE-loop and the E-helix by which Thr95 is located at the proper position to form the hydrogen bond with the heme-bound O-2. This stepwise conformational change would be essential to selective O-2 sensing and signal transduction by HemAT-Bs.

The UV and visible resonance Raman spectra are reported for CooA from Rhodospirillum rubrum, which is a transcriptional regulator activated by growth in a CO atmosphere. CO binding to heme in its sensor domain causes rearrangement of its DNA-binding domain, allowing binding of DNA with a specific sequence. The sensor and DNA-binding domains are linked by a hinge region that follows a long C-helix. UV resonance Raman bands arising from Trp-110 in the C-helix revealed local movement around Trp-110 upon CO binding. The indole side chain of Trp-110, which is exposed to solvent in the CO-free ferrous state, becomes buried in the CO-bound state with a slight change in its orientation but maintains a hydrogen bond with a water molecule at the indole nitrogen. This is the first experimental data supporting a previously proposed model involving displacement of the C-helix and heme sliding. The UV resonance Raman spectra for the CooA-DNA complex indicated that binding of DNA to CooA induces a further displacement of the C-helix in the same direction during transition to the complete active conformation. The Fe-CO and C-O stretching bands showed frequency shifts upon DNA binding, but the Fe-His stretching band did not. Moreover, CO-geminate recombination was more efficient in the DNA-bound state. These results suggest that the C-helix displacement in the DNA-bound form causes the CO binding pocket to narrow and become more negative.

Previous experiments showed that the expression and phosphorylation levels of cyclic AMP-response element binding protein (CREB) are important factors that regulate oligodendrocyte differentiation. The present study was designed to determine whether CREB phosphorylation advances oligodendrocyte differentiation or vice versa and to identify the protein kinase that primarily regulates CREB phosphorylation. We examined the expression and phosphorylation levels of CREB in developing oligodendrocytes at a specific differentiation stage by double-immunocytochemical staining with specific differentiation markers and antibody for phosphorylated CREB. We found that the CREB expression level increased along oligodendrocyte differentiation, and that its phosphorylated level was highest in immature oligodendrocytes. We also showed that CREB phosphorylation was regulated principally by protein kinase C (PKC) activity in immature oligodendrocytes. Our findings suggest that CREB phosphorylation is dependent on a PKC signaling cascade, and this phosphorylation activates CREB-mediated transcription and advances the differentiation of immature to mature oligodendrocytes. (c) 2005 Wiley-Liss, Inc.

Conformational changes of proteins are dominated by the excitation and relaxation processes of their vibrational states. To elucidate the mechanism of receptor activation, the conformation dynamics of receptors must be analyzed in response to agonist-induced vibrational excitation. In this study, we chose the bending vibrational mode,of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous studies, and we investigated picosecond dynamics of the glutamate receptor caused by the vibrational excitation of Arg485 via molecular dynamics simulations. The vibrational excitation energy in Arg485 in the ligand-binding site initially flowed into Lys730, and then into the J-helix at the subunit interface of the ligand-binding domain. Consequently, the atomic displacement in the subunit interface around an intersubunit hydrogen bond was evoked in about 3 ps. This atomic displacement may perturb the subunit packing of the receptor, triggering receptor activation.

Conformational changes of receptors are dominated by the excitation of their collective motions. The most likely energy source of this excitation is considered to be a collision of an agonist with the binding site of a receptor and a consequential excitation of their vibrational modes. In the present study, as an approach to elucidating the mechanism for receptor activation, we chose both the symmetric stretching vibration of the 1C-carboxyl group of glutamate and the bending vibration of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous study, and we quantum-mechanically calculated the vibrational excitation probability of these two vibrations by glutamate collision. The excitation probability of these two vibrations was found to exceed 0.5 about 260 fs after the onset of collision within the first-order perturbation approximation. Taking into account that the period of these vibrations is about 20 fs, we can expect that the vibrational excitation occurs in Arg485 after tens of vibrations during the collision. This vibrational energy may redistribute to the collective motions of the receptor, resulting in global conformational changes in the receptor. We also confirmed that the charge transfer was small between an agonist and the binding site of GluR2. The glutamate receptor may be oscillatory systems that require the energy injection into the specific vibrational modes of the specific amino acid residues to trigger their activation. Such an injection of energy is provided by agonist collision. (C) 2003 Elsevier B.V. All rights reserved.

To understand the mechanism of molecular recognition of glutamate receptors, we calculated the dipole moments, net charges, and electrostatic potentials of the agonists and antagonists of the glutamate receptors under aqueous conditions by the ab initio molecular orbital method at the HF/6-311++G(3df,2pd) level. All of the ligands had negative net charges at both termini and, consequently, formed negative electrostatic potentials at these termini. We further calculated the net charges and electrostatic potential of the S1S2 lobes of the glutamate receptor subunit GluR2 under aqueous conditions by the semempirical PM3 molecular orbital method, and found that the ligand-binding cleft of the S1S2 lobes formed a primarily positive electrostatic potential. A strongly positive electrostatic potential was formed particularly around Arg485 in the S1 lobe. A negative electrostatic potential was observed only in a small region around Glu657 in the S2 lobe in the ligand-binding cleft. When a ligand approaches the ligand-binding cleft, it may proceed to Arg485 in the S1 lobe, due to both repulsion by the negative electrostatic potential of Glu657 as well as the attraction of the strongly positive electrostatic potential of Arg485 itself. (C) 2003 Elsevier B.V. All rights reserved.

Normal mode coordinates of vibrational states associated with one electronic state are generally different from those of vibrational states associated with other electronic states in polyatomic molecules (normal coordinate mixing). This has prevented the multidimensional Franck-Condon integrals from being widely used in spite of their importance. We introduce a simple, noncumbersome numerical computer method for calculating those integrals despite including a mixing of the normal coordinates in the harmonic oscillator approximation on the basis of the expressions by Sharp and Rosenstock. We also introduce more simple expressions of Sharp-Rosenstock's formulas. (C) 2003 American Institute of Physics.

Even though glutamic acid contains only one more carboxyl group than gamma-aminobutyric acid (GABA), these neurotransmitters are recognized by their own specific receptors. To understand the ligand-recognition mechanism of the receptors, we must determine the geometric and electronic structures of GABA and glutamic acid in aqueous conditions using the ab initio calculation. The results of the present study showed that the stable structure of GABA was the extended form, and it attracted both cations and anions. Glutamic acid only attracted cations and was stabilized in four forms in aqueous conditions: Type 1 (an extended form), Type 2 (a rounded form), and Types 3 and 4 (twisted forms of Type 1). The former two types had low energy and the energy barrier between them was estimated to be small. These results showed that most free glutamic acid is present as Type a, Type 2, and transient forms. The present results therefore suggest that the flexibility of the geometric structures of ligands should be taken into account when we attempt to elucidate the mechanism of recognition between ligands and receptors, in addition to the physicochemical characteristics of ligands and receptors.

To understand the mechanism of activation of a receptor by its agonist, the excitation and relaxation processes of the vibrational states of the receptor should be examined. As a first approach to this problem, we calculated the normal vibrational modes of agonists (glutamate and kainate) and an antagonist (6-cyano-7-nitroquinoxaline-2,3-dione: CNQX) of the glutamate receptor, and then investigated the vibrational interactions between kainate and the binding site of glutamate receptor subunit GluR2 by use of a semiempirical molecular orbital method (MOPAC2000-PM3). We found that two local vibrational modes of kainate, which were also observed in glutamate but not in CNQX, interacted through hydrogen bonds with the vibrational modes of GluR2: (i) the bending vibration of the amine group of kainate, interacting with the stretching vibration of the carboxyl group of Glu705 of GluR2, and (ii) the symmetric stretching vibration of the carboxyl group of kainate, interacting with the bending vibration of the guanidinium group of Arg485, We also found collective modes with low frequency at the binding site of GluR2 in the kainate-bound state. The vibrational energy supplied by an agonist may flow from the high-frequency local modes to the low-frequency collective modes in a receptor, resulting in receptor activation.

As a first approach to understanding the mechanism for the recognition of a ligand by its receptor, we first calculated the electronic and structural states of ionized gamma -aminobutyric acid (GABA) and ionized glutamic acid using the ab initio method with the 6311++G (3df, 2pd) basis set. We paid special attention to the physicochemical characteristics of these molecules, such as the electric dipole moment, electrostatic potential, and electrostatic force, Even though GABA and glutamic acid are known to exert completely opposite influences in the mammalian brain by binding their specific receptors, the only difference in their chemical structures is that glutamic acid contains one more carboxyl group than GABA. As a result, we succeeded in showing that a difference of only one carboxyl group induces significant differences in the electronic and structural states between these molecules. These differences have a crucial influence on the electric dipole moments, the electrostatic potentials, and the electrostatic forces. The most remarkable finding of the present research is that the electrostatic potential formed by glutamic acid is composed of only negative parts, while that formed by GABA is separated into positive and negative parts. These results strongly suggest that GABA can approach either positively or negatively charged amino acids by adjusting its own orientation, while glutamic acid can approach only a positively charged binding site.