Near-UV Circular Dichroism and UV Resonance Raman Spectra of Tryptophan Residues as a Structural Marker of Proteins

Near-UV circular dichroism (CD) and UV resonance Raman (UVRR) spectra of l-tryptophan (Trp), its derivatives, and indole-C₃ derivatives were investigated to utilize Trp signals of proteins as a structural marker. CD spectra of Trp are classified into four types: Free l-Trp gives type II (around 270 nm, <i>L</i>_a transition), while l-Trp in proteins generally yields type I (around 280–290 nm, <i>L</i>_b transition) often with vibronic structures. All the indole-C₃ derivatives except for l-Trp gave no CD bands for <i>L</i>_a and <i>L</i>_b transitions, indicating that the asymmetric carbon (C_α) connected through C₃–C_β is essential to appearance of CD. We demonstrate here that the type of CD spectra is determined by a condition of the amino group of Trp; it was changed from type II to type I by the modification of the amino group. In contrast, the modification of the carboxyl group of l-Trp had little effects on a CD spectrum. The 229 nm excited UVRR spectra were almost the same between l-Trp and indole-C₃ derivatives. Comparison of CD and UVRR spectra of Trp residues in proteins suggested that mainly the W17 (possibly together with W16) mode contributes to the characteristic vibronic coupling of <i>L</i>_b transition. Both UVRR and CD spectra of l-Trp were influenced by protonation of amino and/or carboxyl groups, but those changes were distinguished from hydrogen bonding effects at N₁H of indole. It is likely that these protonations are communicated to indole through σ-bonds containing C_α and thus influence both chirality of <i>L</i>_a and <i>L</i>_b transitions and properties of the <i>B</i>_b excited state

Effects of Proton Motive Force on the Structure and Dynamics of Bovine Cytochrome <i>c</i> Oxidase in Phospholipid Vesicles

A conventional method for reconstituting cytochrome <i>c</i> oxidase (CcO) into phospholipid vesicles (COV) has been modified to permit resonance Raman (RR) analysis in the presence and absence of proton motive force (Δμ_H⁺). The COV has an average diameter of 20 nm and contains one CcO molecule within a unified orientation with Cu_A located outside the COV. The process of generation of Δμ_H⁺ across the membrane was monitored spectrophotometrically with rhodamine123 dye. The COV exhibits a respiratory control ratio (RCR) value of >30 and is tolerant to RR measurements with 10 mW laser illumination for 60 min at 441.6 nm. Structural perturbations at the heme sites caused by incorporation into vesicles were clarified by spectral comparisons between solubilized CcO and COV. Absorption spectroscopy revealed that the rate of electron transfer from cytochrome <i>c</i> to O₂ is reduced significantly more in the presence of Δμ_H⁺ than in its absence. RR spectroscopic measurements indicate that CcO in COV in the “respiratory-controlled” state adopts a mixed-valence state (heme <i>a</i>²⁺ and heme <i>a</i>₃³⁺). This study establishes a supramolecular model system for experimentally examining the energy conversion protein machinery in the presence of Δμ_H⁺

Reversible O–O Bond Scission of Peroxodiiron(III) to High-Spin Oxodiiron(IV) in Dioxygen Activation of a Diiron Center with a Bis-tpa Dinucleating Ligand as a Soluble Methane Monooxygenase Model

The conversion of peroxodiiron­(III) to high-spin <i>S</i> = 2 oxodiiron­(IV) via reversible O–O bond scission in a diiron complex with a bis-tpa dinucleating ligand, 6-hpa, has been characterized by elemental analysis; kinetic measurements for alkene epoxidation; cold-spray ionization mass spectrometry; and electronic absorption, Mössbauer, and resonance Raman spectroscopy to gain insight into the O₂ activation mechanism of soluble methane monooxygenases. This is the first synthetic example of a high-spin <i>S</i> = 2 oxodiiron­(IV) species that oxidizes alkenes to epoxides efficiently. The bistability of the peroxodiiron­(III) and high-spin <i>S</i> = 2 oxodiiron­(IV) moieties is the key feature for the reversible O–O bond scission

Interrelationship among Fe–His Bond Strengths, Oxygen Affinities, and Intersubunit Hydrogen Bonding Changes upon Ligand Binding in the β Subunit of Human Hemoglobin: The Alkaline Bohr Effect

Regulation of the oxygen affinity of human adult hemoglobin (Hb A) at high pH, known as the alkaline Bohr effect, is essential for its physiological function. In this study, structural mechanisms of the alkaline Bohr effect and pH-dependent O₂ affinity changes were investigated via ¹H nuclear magnetic resonance and visible and UV resonance Raman spectra of mutant Hbs, Hb M Iwate (αH87Y) and Hb M Boston (αH58Y). It was found that even though the binding of O₂ to the α subunits is forbidden in the mutant Hbs, the O₂ affinity was higher at alkaline pH than at neutral pH, and concomitantly, the Fe–His stretching frequency of the β subunits was shifted to higher values. Thus, it was confirmed for the β subunits that the stronger the Fe–His bond, the higher the O₂ affinity. It was found in this study that the quaternary structure of α­(Fe³⁺)­β­(Fe²⁺-CO) of the mutant Hb is closer to T than to the ordinary R at neutral pH. The retained Aspβ94–Hisβ146 hydrogen bond makes the extent of proton release smaller upon ligand binding from Hisβ146, known as one of residues contributing to the alkaline Bohr effect. For these T structures, the Aspα94–Trpβ37 hydrogen bond in the hinge region and the Tyrα42–Aspβ99 hydrogen bond in the switch region of the α₁–β₂ interface are maintained but elongated at alkaline pH. Thus, a decrease in tension in the Fe–His bond of the β subunits at alkaline pH causes a substantial increase in the change in global structure upon binding of CO to the β subunit

Heterogeneity between Two α Subunits of α₂β₂ Human Hemoglobin and O₂ Binding Properties: Raman, ¹H Nuclear Magnetic Resonance, and Terahertz Spectra

Following a previous detailed investigation of the β subunit of α₂β₂ human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α­(Fe²⁺-deoxy/O₂)­β­(Fe³⁺) hemoglobin M (Hb M) in which O₂ cannot bind to the β subunit: Hb M Hyde Park (β92His → Tyr), Hb M Saskatoon (β63His → Tyr), and Hb M Milwaukee (β67Val → Glu). In contrast with the β subunit that exhibited a clear correlation between O₂ affinity and Fe²⁺–His stretching frequencies, the Fe²⁺–His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α₂β₂ tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5–30 cm^–1 region of Hb M Milwaukee, α­(Fe²⁺-deoxy)­β­(Fe³⁺). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α­(Fe²⁺-deoxy)­β­(Co²⁺) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe–His frequencies were hardly changed with a large alteration of O₂ affinity by pH change, suggesting no correlation of frequency with O₂ affinity for the α subunit. Instead, a new Fe²⁺–His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O₂ affinity increased. The high-frequency counterpart was also observed for a partially O₂-bound form, α­(Fe²⁺-deoxy)­α­(Fe²⁺-O₂)­β­(Fe³⁺)­β­(Fe³⁺), of the present Hb M, consistent with our previous finding that binding of O₂ to one α subunit of T structure α₂β₂ tetramer changes the other α subunit to the R structure

Synthesis, Characterization, and Reactivity of Hypochloritoiron(III) Porphyrin Complexes

A hypochloritoiron­(III) porphyrin species has been proposed as a key intermediate in an antimicrobial defense system in neutrophils and in heme-catalyzed chlorination reactions. We report herein the preparation, spectroscopic characterization, and reactivity of the bis­(hypochlorito)­iron­(III) porphyrin complex [(TPFP)­Fe^III(OCl)₂]⁻ (<b>1</b>) and the imidazole–hypochloritoiron complexes (TPFP)­Fe^III(OCl)­(1-R-Im) [R = CH₃ (<b>2</b>), H (<b>3</b>), CH₂CO₂H (<b>4</b>)], in which TPFP is 5,10,15,20-tetrakis­(penta­fluoro­phenyl)­porphyrinate. The structures of <b>1</b>–<b>4</b> were confirmed by absorption, ²H and ¹⁹F NMR, EPR, and resonance Raman spectroscopy and electrospray ionization mass spectrometry at low temperature. The reactions of <b>1</b> and <b>2</b> with various organic substrates show that <b>1</b> and <b>2</b> are capable of chlorination, sulfoxidation, and epoxidation reactions and that <b>1</b> is much more reactive with these substrates than <b>2</b>

Effect of the Axial Ligand on the Reactivity of the Oxoiron(IV) Porphyrin π-Cation Radical Complex: Higher Stabilization of the Product State Relative to the Reactant State

The proximal heme axial ligand plays an important role in tuning the reactivity of oxoiron­(IV) porphyrin π-cation radical species (compound I) in enzymatic and catalytic oxygenation reactions. To reveal the essence of the axial ligand effect on the reactivity, we investigated it from a thermodynamic viewpoint. Compound I model complexes, (TMP^+•)­Fe^IVO­(L) (where TMP is 5,10,15,20-tetramesitylporphyrin and TMP^+• is its π-cation radical), can be provided with altered reactivity by changing the identity of the axial ligand, but the reactivity is not correlated with spectroscopic data (ν­(FeO), redox potential, and so on) of (TMP^+•)­Fe^IVO­(L). Surprisingly, a clear correlation was found between the reactivity of (TMP^+•)­Fe^IVO­(L) and the Fe^II/Fe^III redox potential of (TMP)­Fe^IIIL, the final reaction product. This suggests that the thermodynamic stability of (TMP)­Fe^IIIL is involved in the mechanism of the axial ligand effect. Axial ligand-exchange experiments and theoretical calculations demonstrate a linear free-energy relationship, in which the axial ligand modulates the reaction free energy by changing the thermodynamic stability of (TMP)­Fe^III(L) to a greater extent than (TMP^+•)­Fe^IVO­(L). The linear free energy relationship could be found for a wide range of anionic axial ligands and for various types of reactions, such as epoxidation, demethylation, and hydrogen abstraction reactions. The essence of the axial ligand effect is neither the electron donor ability of the axial ligand nor the electron affinity of compound I, but the binding ability of the axial ligand (the stabilization by the axial ligand). An axial ligand that binds more strongly makes (TMP)­Fe^III(L) more stable and (TMP^+•)­Fe^IVO­(L) more reactive. All results indicate that the axial ligand controls the reactivity of compound I (the stability of the transition state) by the stability of the ground state of the final reaction product and not by compound I itself

Geometric Control of Nuclearity in Copper(I)/Dioxygen Chemistry

Copper­(I) complexes supported by a series of N₃-tridentate ligands bearing a rigid cyclic diamine framework such as 1,5-diazacyclooctane (<b>L8</b>, eight-membered ring), 1,4-diazacycloheptane (<b>L7</b>, seven-membered ring), or 1,4-diazacyclohexane (<b>L6</b>, six-membered ring) with a common 2-(2-pyridyl)­ethyl side arm were synthesized and their reactivity toward O₂ were compared. The copper­(I) complex of <b>L8</b> preferentially provided a mononuclear copper­(II) end-on superoxide complex <b>S</b> as reported previously [Itoh, S., et al.<i> J. Am. Chem. Soc.</i> <b>2009</b>, 131, 2788–2789], whereas a copper­(I) complex of <b>L7</b> gave a bis­(μ-oxido)­dicopper­(III) complex <b>O</b> at a low temperature (−85 °C) in acetone. On the other hand, no such active-oxygen complex was detected in the oxygenation reaction of the copper­(I) complex of <b>L6</b> under the same conditions. In addition, O₂-reactivity of the copper­(I) complex supported by an acyclic version of the tridentate ligand (<b>LA</b>, PyCH₂CH₂N­(CH₃)­CH₂CH₂CH₂N­(CH₃)₂; Py = 2-pyridyl) was examined to obtain a mixture of a (μ–η²:η²-peroxido)­dicopper­(II) complex ^<b>S</b><b>P</b> and a bis­(μ-oxido)­dicopper­(III) complex <b>O</b>. Careful inspection of the crystal structures of copper­(I) and copper­(II) complexes and the redox potentials of copper­(I) complexes has revealed important geometric effects of the supporting ligands on controlling nuclearity of the generated copper active-oxygen complexes

Redox Properties of a Mononuclear Copper(II)-Superoxide Complex

Redox properties of a mononuclear copper­(II) superoxide complex, (L)­Cu^II–OO^•, supported by a tridentate ligand (L = 1-(2-phenethyl)-5-[2-(2-pyridyl)­ethyl]-1,5-diazacyclooctane) have been examined as a model compound of the putative reactive intermediate of peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) (Kunishita et al. <i>J. Am. Chem. Soc.</i> <b>2009</b>, <i>131</i>, 2788–2789; <i>Inorg. Chem.</i> <b>2012</b>, <i>51</i>, 9465–9480). On the basis of the reactivity toward a series of one-electron reductants, the reduction potential of (L)­Cu^II–OO^• was estimated to be 0.19 ± 0.07 V vs SCE in acetone at 298 K (cf. Tahsini et al.<i> Chem.Eur. J.</i> <b>2012</b>, <i>18</i>, 1084–1093). In the reaction of TEMPO-H (2,2,6,6-tetramethylpiperidine-<i>N</i>-hydroxide), a simple HAT (hydrogen atom transfer) reaction took place to give the corresponding hydroperoxide complex LCu^II–OOH, whereas the reaction with phenol derivatives (^XArOH) gave the corresponding phenolate adducts, LCu^II–O^XAr, presumably via an acid–base reaction between the superoxide ligand and the phenols. The reaction of (L)­Cu^II–OO^• with a series of triphenylphosphine derivatives gave the corresponding triphenylphosphine oxides via an electrophilic ionic substitution mechanism with a Hammett ρ value as −4.3, whereas the reaction with thioanisole (sulfide) only gave a copper­(I) complex. These reactivities of (L)­Cu^II–OO^• are different from those of a similar end-on superoxide copper­(II) complex supported by a tetradentate TMG₃tren ligand (1,1,1-Tris­{2-[<i>N</i>^<i>2</i>-(1,1,3,3-tetramethylguanidino)]­ethyl}­amine (Maiti et al.<i> Angew. Chem., Int. Ed.</i> <b>2008</b>, <i>47</i>, 82–85)

Pilot Quasi-Randomized Controlled Study of Herbal Medicine Hochuekkito as an Adjunct to Conventional Treatment for Progressed Pulmonary <i>Mycobacterium avium</i> Complex Disease

<div><p>Introduction</p><p>Hochuekkito, a traditional herbal medicine, is occasionally prescribed in Japan to treat patients with a poor general condition. We aimed to examine whether this medicine was beneficial and tolerable for patients with progressed pulmonary <i>Mycobacterium avium</i> complex (MAC) disease.</p><p>Methods</p><p>This pilot open-label quasi-randomized controlled trial enrolled 18 patients with progressed pulmonary MAC disease who had initiated antimycobacterial treatment over one year ago but were persistently culture-positive or intolerant. All patients continued their baseline treatment regimens with (n = 9) or without (n = 9) oral Hochuekkito for 24 weeks.</p><p>Results</p><p>Baseline characteristics were generally similar between the groups. Most patients were elderly (median age 70 years), female, had a low body mass index (<20 kg/m²), and a long-term disease duration (median approximately 8 years). After the 24-week treatment period, no patient achieved sputum conversion. Although the number of colonies in sputum tended to increase in the control group, it generally remained stable in the Hochuekkito group. Radiological disease control was frequently observed in the Hochuekkito group than the control group (8/9 vs. 3/9; p = 0.05). Patients in the Hochuekkito group tended to experience increase in body weight and serum albumin level compared with those in the control group (median body weight change: +0.4 kg vs. −0.8 kg; median albumin change: +0.2 g/dl vs. ±0.0 g/dl). No severe adverse events occurred.</p><p>Conclusions</p><p>Hochuekkito could be an effective, feasible adjunct to conventional therapy for patients with progressed pulmonary MAC disease. Future study is needed to explore this possibility.</p><p>Trial Registration</p><p>UMIN Clinical Trials Registry <a href="https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000011622&language=E" target="_blank">UMIN000009920</a></p></div