Reversible Switching of Fluorophore Property Based on Intrinsic Conformational Transition of Adenylate Kinase during Its Catalytic Cycle

Adenylate kinase shows a conformational transition (OPEN and CLOSED forms) during substrate binding and product release to mediate the phosphoryl transfer between ADP and ATP/AMP. The protein motional characteristics will be useful to construct switching systems of fluorophore properties caused by the catalytic cycle of the enzyme. This paper demonstrates in situ reversible switching of a fluorophore property driven by the conformational transition of the enzyme. The pyrene-conjugated mutant adenylate kinase is able to switch the monomer/excimer emission property of pyrene on addition of ADP or <i>P</i>¹<i>P</i>⁵-di­(adenosine-5′)­pentaphosphate (Ap₅A, a transition state analog). The observation under the dilute condition (∼0.1 μM) indicates that the emission spectral change was caused by the motion of a protein molecule and not led by protein–protein interactions through π–π stacking of pyrene rings. The switching can be reversibly conducted by using hexokinase-coupling reaction. The fashion of the changes in emission intensities at various ligand concentrations is different between ADP, Mg²⁺-bound ADP, and Mg²⁺-bound Ap₅A. The emission property switching is repeatable by a sequential addition of a substrate in a one-pot process. It is proposed that the property of a synthetic molecule on the enzyme surface is switchable in response to the catalytic cycle of adenylate kinase

DNA Cleavage by the Photocontrolled Cooperation of Zn^II Centers in an Azobenzene-Linked Dizinc Complex

We synthesized a new photoactive dinuclear zinc­(II) complex by linking two zinc centers with a ligand containing an azobenzene chromophore and investigated the DNA cleavage activities of its trans and cis forms. The trans structure of the dinuclear zinc complex was determined by X-ray crystallography, where each zinc center is situated in an octahedral coordination environment comprised of three nitrogen atoms from the ligand and three oxygen atoms from two nitrate ions. The dinuclear zinc complex containing the azobenzene chromophore was photoisomerizable between the trans and cis forms. The binding affinities of the trans and cis complexes with calf thymus (CT)-DNA were similar. Although the DNA cleavage activity of the trans complex was negligible, the cis complex was able to cleave DNA. We attribute the efficient activity of the cis complex to the cooperation of the two closely located zinc centers and the inactivity of the trans complex to the two metal centers positioned far away from each other. The DNA cleavage activity of the cis complex exhibited a pH-dependent bell-shaped profile, which has been observed in the hydrolytic cleavage of DNA by zinc complexes. The DNA cleavage activity was not inhibited by a major groove binder, methyl green, but decreased significantly by a minor groove binder, 4′,6-diamidino-2-phenylindole, indicating that the dinuclear zinc complex binds to the minor groove of DNA. The present work shows the importance of the cooperation of two zinc ions for hydrolytic DNA cleavage, which can be photoregulated by linking the two metal centers with a photoisomerizable spacer, such as an azobenzene chromophore

Floral traits and pollinator visitations in experiment 1

Floral traits and pollinator visitations in experiment

DNA Cleavage by the Photocontrolled Cooperation of Zn^II Centers in an Azobenzene-Linked Dizinc Complex

We synthesized a new photoactive dinuclear zinc­(II) complex by linking two zinc centers with a ligand containing an azobenzene chromophore and investigated the DNA cleavage activities of its trans and cis forms. The trans structure of the dinuclear zinc complex was determined by X-ray crystallography, where each zinc center is situated in an octahedral coordination environment comprised of three nitrogen atoms from the ligand and three oxygen atoms from two nitrate ions. The dinuclear zinc complex containing the azobenzene chromophore was photoisomerizable between the trans and cis forms. The binding affinities of the trans and cis complexes with calf thymus (CT)-DNA were similar. Although the DNA cleavage activity of the trans complex was negligible, the cis complex was able to cleave DNA. We attribute the efficient activity of the cis complex to the cooperation of the two closely located zinc centers and the inactivity of the trans complex to the two metal centers positioned far away from each other. The DNA cleavage activity of the cis complex exhibited a pH-dependent bell-shaped profile, which has been observed in the hydrolytic cleavage of DNA by zinc complexes. The DNA cleavage activity was not inhibited by a major groove binder, methyl green, but decreased significantly by a minor groove binder, 4′,6-diamidino-2-phenylindole, indicating that the dinuclear zinc complex binds to the minor groove of DNA. The present work shows the importance of the cooperation of two zinc ions for hydrolytic DNA cleavage, which can be photoregulated by linking the two metal centers with a photoisomerizable spacer, such as an azobenzene chromophore

Photocontrol of Spatial Orientation and DNA Cleavage Activity of Copper(II)-Bound Dipeptides Linked by an Azobenzene Derivative

Copper(II) ion-bound CysGly dipeptides linked by an azobenzene derivative were photoisomerized between the trans and cis forms. The two copper(II) ion centers were positioned close to each other in the cis form, whereas they were far away from each other in the trans form. The copper complex in the cis form exhibited DNA cleavage activity, whereas the activity in the trans form was negligible. The DNA cleavage activity of the cis form is attributed to the cooperation of the closely located copper(II) centers. The present results show the photocontrol of the cooperation of metal ions for DNA cleavage

A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating the Access of Water Molecules to the Substrate-Binding Site

A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating the Access of Water Molecules to the Substrate-Binding Sit

FT-IR Characterization of the Light-Induced Ni-L2 and Ni-L3 States of [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F

Different light-induced Ni-L states of [NiFe] hydrogenase from its Ni-C state have previously been observed by EPR spectroscopy. Herein, we succeeded in detecting simultaneously two Ni-L states of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F by FT-IR spectroscopy. A new light-induced ν_CO band at 1890 cm^–1 and ν_CN bands at 2034 and 2047 cm^–1 were detected in the FT-IR spectra of the H₂-activated enzyme under N₂ atmosphere at basic conditions, in addition to the 1910 cm^–1 ν_CO band and 2047 and 2061 cm^–1 ν_CN bands of the Ni-L2 state. The new bands were attributed to the Ni-L3 state by comparison of the FT-IR and EPR spectra. The ν_CO and ν_CN frequencies of the Ni-L3 state are the lowest frequencies observed among the corresponding frequencies of standard-type [NiFe] hydrogenases in various redox states. These results indicate that a residue, presumably Ni-coordinating Cys546, is protonated and deprotonated in the Ni-L2 and Ni-L3 states, respectively. Relatively small Δ<i>H</i> (6.4 ± 0.8 kJ mol^–1) and Δ<i>S</i> (25.5 ± 10.3 J mol^–1 K^–1) values were obtained for the conversion from the Ni-L2 to Ni-L3 state, which was in agreement with the previous proposals that deprotonation of Cys546 is important for the catalytic reaction of the enzyme

A Supramolecular Receptor of Diatomic Molecules (O₂, CO, NO) in Aqueous Solution

A per-O-methylated β-cyclodextrin dimer, Py2CD, was conveniently prepared via two steps: the Williamson reaction of 3,5-bis(bromomethyl)pyridine and β-cyclodextrin (β-CD) yielding 2A,2′A-O-[3,5-pyridinediylbis(methylene)bis-β-cyclodextrin (bisCD) followed by the O-methylation of all the hydroxy groups of the bisCD. Py2CD formed a very stable 1:1 complex (Fe(III)PCD) with [5,10,15,20-tetrakis(p-sulfonatophenyl)porphinato]iron(III) (FeIIITPPS) in aqueous solution. Fe(III)PCD was reduced with Na2S2O4 to afford the FeIITPPS/Py2CD complex (Fe(II)PCD). Dioxygen was bound to Fe(II)PCD, the P1/2O2 values being 42.4 ± 1.6 and 176 ± 3 Torr at 3 and 25 °C, respectively. The konO2 and koffO2 values for the dioxygen binding were determined to be 1.3 × 107 M−1s−1 and 3.8 × 103 s−1, respectively, at 25 °C. Although the dioxygen adduct was not very stable (KO2 = konO2/koffO2 = 3.4 × 103 M−1), no autoxidation of the dioxygen adduct of Fe(II)PCD to Fe(III)PCD was observed. These results suggest that the encapsulation of FeIITPPS by Py2CD strictly inhibits not only the extrusion of dioxygen from the cyclodextrin cage but also the penetration of a water molecule into the cage. The carbon monoxide affinity of Fe(II)PCD was much higher than the dioxygen affinity; the P1/2CO, konCO, koffCO, and KCO values being (1.6 ± 0.2) × 10−2 Torr, 2.4 × 106 M−1s−1, 4.8 × 10−2 s−1, and 5.0 × 107 M−1, respectively, at 25 °C. Fe(II)PCD also bound nitric oxide. The rate of the dissociation of NO from (NO)Fe(II)PCD ((5.58 ± 0.42) × 10−5 s−1) was in good agreement with the maximum rate ((5.12 ± 0.18) × 10−5 s−1) of the oxidation of (NO)Fe(II)PCD to Fe(III)PCD and NO3−, suggesting that the autoxidation of (NO)Fe(II)PCD proceeds through the ligand exchange between NO and O2 followed by the rapid reaction of (O2)Fe(II)PCD with released NO, affording Fe(II)PCD and the NO3− anion inside the cyclodextrin cage

Nature of Cysteine-Based Re(V)O(N₂S₂) Radiopharmaceuticals at Physiological pH Ascertained by Investigation of a New Complex with a <i>M</i><i>eso</i> N₂S₂ Ligand Having Carboxyl Groups Anti to the Oxo Group

X-ray structural characterization of a new isomer of ReO(TMECH3) revealed that it is anti-ReO(dl-TMECH3) (1). (dl-TMECH6 is meso-tetramethyl-ethylene-dicysteine prepared from racemic penicillamine (penH4), the subscript on H indicating the number of dissociable protons; anti denotes the geometric isomer having both carboxyl groups anti to the oxo ligand.) In 1, one carboxyl is deprotonated and coordinated trans to the oxo ligand, and the other is protonated and dangling. The 1H NMR spectrum (assigned by 2D methods) of 1 at pH 4 in aqueous solution revealed that the structure of 1 is the same as in the solid state except for deprotonation of the dangling carboxyl group, affording the monoanion. All chelate ring protons and methyl groups are inequivalent and give sharp signals. As the pH was raised above 7, the 1D 1H NMR signals of the monoanion broadened. Broadening was severe for the methyl and ethylene signals of the tridentate half of the monoanion, and these signals were replaced by new signals for the dianion. The changes suggested a rate process that was intermediate on the NMR time scale, such as CO2- ligation/deligation. With increasing pH the dianion signals sharpened up to pH ∼8 and then broadened up to pH ≈ 10. Finally, the spectrum at pH 10.8 showed only half the number of signals. Each signal was at the midpoint shift between two corresponding signals observed at lower pH, indicating a time averaging between the halves of the dl-TMEC ligand, but no change in protonation state. Two ReO stretching bands (923 and 933 cm-1) with a constant intensity ratio of ∼1 were observed for the dianion. These results can be explained if the dianion exists detectably only as a NH-deprotonated/carboxyl-deligated form having two conformers. The conformers differ in the N lone pair (NLp) orientation (either endo or exo with respect to the oxo ligand) and thus have slightly different ReO stretching frequencies. Although they can be detected by resonance Raman spectroscopy, the conformers are indistinguishable by NMR spectroscopy because NLp inversion (and hence conformer interconversion) is very fast. Interchange of the NH and NLp sites affects the NMR spectra. At pH 8.3 the signals of the dianion are sharpest because interchange is slowest. Below and above pH 8.3, the signals are broader because acid and base catalysis, respectively, increase the rate of interchange between the NH and NLp sites

Nature of Cysteine-Based Re(V)O(N₂S₂) Radiopharmaceuticals at Physiological pH Ascertained by Investigation of a New Complex with a <i>M</i><i>eso</i> N₂S₂ Ligand Having Carboxyl Groups Anti to the Oxo Group

X-ray structural characterization of a new isomer of ReO(TMECH3) revealed that it is anti-ReO(dl-TMECH3) (1). (dl-TMECH6 is meso-tetramethyl-ethylene-dicysteine prepared from racemic penicillamine (penH4), the subscript on H indicating the number of dissociable protons; anti denotes the geometric isomer having both carboxyl groups anti to the oxo ligand.) In 1, one carboxyl is deprotonated and coordinated trans to the oxo ligand, and the other is protonated and dangling. The 1H NMR spectrum (assigned by 2D methods) of 1 at pH 4 in aqueous solution revealed that the structure of 1 is the same as in the solid state except for deprotonation of the dangling carboxyl group, affording the monoanion. All chelate ring protons and methyl groups are inequivalent and give sharp signals. As the pH was raised above 7, the 1D 1H NMR signals of the monoanion broadened. Broadening was severe for the methyl and ethylene signals of the tridentate half of the monoanion, and these signals were replaced by new signals for the dianion. The changes suggested a rate process that was intermediate on the NMR time scale, such as CO2- ligation/deligation. With increasing pH the dianion signals sharpened up to pH ∼8 and then broadened up to pH ≈ 10. Finally, the spectrum at pH 10.8 showed only half the number of signals. Each signal was at the midpoint shift between two corresponding signals observed at lower pH, indicating a time averaging between the halves of the dl-TMEC ligand, but no change in protonation state. Two ReO stretching bands (923 and 933 cm-1) with a constant intensity ratio of ∼1 were observed for the dianion. These results can be explained if the dianion exists detectably only as a NH-deprotonated/carboxyl-deligated form having two conformers. The conformers differ in the N lone pair (NLp) orientation (either endo or exo with respect to the oxo ligand) and thus have slightly different ReO stretching frequencies. Although they can be detected by resonance Raman spectroscopy, the conformers are indistinguishable by NMR spectroscopy because NLp inversion (and hence conformer interconversion) is very fast. Interchange of the NH and NLp sites affects the NMR spectra. At pH 8.3 the signals of the dianion are sharpest because interchange is slowest. Below and above pH 8.3, the signals are broader because acid and base catalysis, respectively, increase the rate of interchange between the NH and NLp sites