Reductive Activation of Dioxygen by a Myoglobin Reconstituted with a Flavohemin

We successfully converted myoglobin, an oxygen-storage hemoprotein, into an oxygen-activating hemoprotein like cytochrome P450s by replacing the native hemin with the artificially created flavohemin. The reconstituted myoglobin, rMb(1), was chacterized by ESI-TOF-mass, UV−vis, and fluorescence spectra. The 1H NMR spectrum of cyanomet rMb(1) indicates that two hemin conformers are present in a ratio of 1:1. Upon the addition of NADH to the buffer solution of rMb(1) in the presence of SOD and catalase, the oxymyoglobin was rapidly formed. As compared with the formation of the oxygenated native myoglobin in the presence of 10-N-(acetylaminoethyl)isoalloxazine, the rate constant of the oxyheme formation in rMb(1) is 6 times larger. This is because the flavin covalently linked to the terminal heme propionate functions as an effective mediator of an electron transfer from NADH to the hemin in rMb(1). Furthermore, rMb(1) shows the deformylation activity, when 2-phenylpropionaldehyde (2-PPA) was employed as a substrate. This result indicates that the oxyheme is reductively activated to Fe(III)-peroxoanion (Fe(III)-O22-). The result in this report is the first example of the activation of dioxygen by myoglobin. This study shows the utility of the replacement of the native hemin with a chemically modified one for the functionalization of myoglobin

Reaction Pathway and Free Energy Profile for Conversion of π‑Conjugation Modes in Porphyrin Isomer

Porphycene is a structural isomer of porphyrin with 18π-conjugated aromatic character. Porphycene modified with trifluoromethyl (CF₃) groups in the periphery of the framework readily affords the isolable 20π-conjugated antiaromatic form through a reaction with a proton-donating reductant. The 20π-conjugated form can be characterized by not only a variety of spectroscopies in solutions but also X-ray crystallography. This paper focuses on the free energy profile in the conversion of the 18π-conjugated porphycene into the 20π-conjugated form. From the results of kinetics, electrochemical measurements, and acid/base titrations, the 20π-conjugated CF₃ porphycene is formed by a concerted proton–electron transfer (CPET) from a hydroquinone reagent to the 18π-conjugated form. The hydrogen-atom affinity of the 18π-conjugated CF₃ porphycene (for two hydrogen atoms) was calculated to be −490 kJ mol^–1, indicating that the N–H bonds in the 20π-conjugated form are rather easily cleaved. This reflects the antiaromatic characteristics of the 20π-conjugated porphycene. We propose that the kinetic and thermochemical analysis using redox potentials and p<i>K</i>_a data is applicable for determining the reaction pathway in conversion of aromatic/antiaromatic mode of π-conjugated macrocycles as well as popular investigations for oxidations of organic molecules

A Structural Isomer of Nonaromatic Porphyrin: Preparation of 20π-Conjugated Porphycene Based on Electronic Perturbation

A porphycene having four CF3 groups at the β-pyrrolic positions affords a stable 20π-conjugated form in the presence of a 2H+−2e- donor due to the high redox potential of the tetrapyrrole ring framework. No visible band in the UV−vis spectrum and the highly ruffled structure determined by X-ray crystallography support its nonaromatic character. The ethylene bridge moiety in the 20π-conjugated framework displayed the olefinic reactivities

Site-Specific Modification of Proteins through N‑Terminal Azide Labeling and a Chelation-Assisted CuAAC Reaction

Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The N-terminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol

Artificial Protein−Protein Complexation between a Reconstituted Myoglobin and Cytochrome <i>c</i>

Artificial prosthetic porphyrins, 1·Fe and 1·Zn, in which two isophthalamide units having four carboxylates were bound to the terminal of each peripheral propionate side chain in protoporphyrin IX, were inserted into horse heart apomyoglobin to give novel myoglobins, rMb(1·Fe) and rMb(1·Zn), respectively. The resultant reconstituted myoglobins were designed to bind cationic cytochrome c on the protein surface via electrostatic interaction. The isoelectric point for rMb(1·Fe) was determined to be 5.5, which is about 2 pH units lower than that of native myoglobin. The pI value suggests that eight carboxylates of prosthetic group are located on the surface of the myoglobin. A construction of a myoglobin−cytochrome c complex was probed by paramagnetic 1H NMR and flash photolysis studies. The behavior of 1H NMR paramagnetic shifts in the rMb(1·FeCN)cytochrome c complex is comparable with that in the native pairing of cytochrome ccytochrome c peroxidase. Laser flash photolysis shows that a long-range ET from photoexcited rMb(1·Zn) to cytochrome c occurs within the protein−protein complex. The time-dependence of the transient spectra at 460 nm identified as the triplet excited state of rMb(1·Zn) leads to rate constant of forward ET and affinity of the protein−protein complex; kintra = (2.2 ± 0.1) × 103 s-1 and Ka = (6.5 ± 3.0) × 104 M-1 at 10 mM ionic strength and kintra = (2.3 ± 0.2) × 103 s-1 and Ka = (1.5 ± 0.6) × 104 M-1 at 20 mM ionic strength and pH 7.0. The binding affinity for cytochrome c decreases with increasing the ionic strength, indicating that the protein−protein complex is formed by electrostatic interaction. This work demonstrates that the artificial functional groups bound to the terminal of porphyrin in the reconstituted myoglobin can act as an effective recognition domain for a protein at the surface of the myoglobin

A Structural Isomer of Nonaromatic Porphyrin: Preparation of 20π-Conjugated Porphycene Based on Electronic Perturbation

A porphycene having four CF3 groups at the β-pyrrolic positions affords a stable 20π-conjugated form in the presence of a 2H+−2e- donor due to the high redox potential of the tetrapyrrole ring framework. No visible band in the UV−vis spectrum and the highly ruffled structure determined by X-ray crystallography support its nonaromatic character. The ethylene bridge moiety in the 20π-conjugated framework displayed the olefinic reactivities

Pair recordings of AMPAR and NMDAR mediated EPSCs from neurons transfected with wild type GluN1 and GluN2 Cys cluster II mutants.

<p>A. Evoked responses recorded at −70 mV (AMPAR) or +40 mV (NMDAR) from neurons transfected with wild type GluN1 and GluN2A 4CS. Responses were evoked by stimulation of Schaffer collaterals and compared with an adjacent non-transfected neuron stimulated under the same conditions. Each dot represents a pair of neurons (n = 6 pairs). Black dot is average ± s.e. Dotted line is the unity line. <b>B.</b> Evoked responses from cells transfected with GluN2B 5CS recorded at −70 mV (AMPAR) or +40 mV (NMDAR). Responses from adjacent non-transfected neurons were recorded under the same stimulation conditions and compared with responses from transfected cells (n = 5 pairs).</p

Incorporation Index of NMDARs containing GluN2 non-palmitoylatable mutants.

<p><b>A.</b> Evoked EPSCs were recorded in CA1 pyramidal cells transfected with etag GluN1 and either wild type GluN2A (n = 9), GluN2A 3CS (n = 13), wild type GluN2B (n = 8), or GluN2B 3CS (n = 15). Evoked EPSCs were recorded at −70 mV and the Incorporation Index calculated as the ratio of the mean current from a 50 ms window 150 ms after the stimulus artifact corresponding to recombinant NMDARs (rNMDAR) normalized to the peak of the EPSC occurring within 50 ms from the stimulus artifact corresponding to endogenous AMPARs (eAMPAR). Error bars are s.e. Asterisk indicates p<0.05. Insets, example traces of EPSCS evoked in transfected cells as indicated. Vertical dotted lines indicate where measurements are taken. Scale bar = 50 ms <b>B.</b> In corporation Index measured as indicated in A from CA1 pyramidal cells transfected with etag GluN1 and either wild type GluN2A (n = 9), GluN2A 4CS (n = 15), wild type GluN2B (n = 8), or GluN2B 5CS (n = 18). Wild type values same as in A repeated here for easy comparison. Insets are example traces of EPSCs from cells transfected as indicated.</p

Porphyrinoid Chemistry in Hemoprotein Matrix: Detection and Reactivities of Iron(IV)-Oxo Species of Porphycene Incorporated into Horseradish Peroxidase

The iron porphycene with two propionates at the peripheral positions of the framework was incorporated into the heme pocket of horseradish peroxidase. In the presence of hydrogen peroxide, the ferric iron porphycene was smoothly converted into the iron(IV)-oxo porphycene π-cation radical species, which was confirmed by the appearance of a band around 800 nm in the UV−vis spectrum. The protein with the iron porphycene showed a 10-fold higher reactivity for the thioanisole oxidation when compared to the native protein. In contrast, the guaiacol oxidation proceeded with similar reaction rates in both proteins. The kinetic analyses indicated that the ferric porphycene in the protein more slowly reacts with hydrogen peroxide than the native heme, whereas the high oxidation states show higher reactivities during oxidations of an organic substrate. The formation of the iron(IV)-oxo species of porphycene and its reactivities in the hemoprotein matrix are demonstrated