RESUMO
Cytochrome c oxidase (CcO) reduces O2, pumps protons in the mitochondrial respiratory chain, and is essential for oxygen consumption in the cell. The coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2; also known as mitochondrial nuclear retrograde regulator 1 [MNRR1], Parkinson's disease 22 [PARK22] and aging-associated gene 10 protein [AAG10]) is a protein that binds to CcO from the intermembrane space and positively regulates the activity of CcO. Despite the importance of CHCHD2 in mitochondrial function, the mechanism of action of CHCHD2 and structural information regarding its binding to CcO remain unknown. Here, we utilized visible resonance Raman spectroscopy to investigate the structural changes around the hemes in CcO in the reduced and CO-bound states upon CHCHD2 binding. We found that CHCHD2 has a significant impact on the structure of CcO in the reduced state. Mapping of the heme peripheries that result in Raman spectral changes in the structure of CcO highlighted helices IX and X near the hemes as sites where CHCHD2 takes action. Part of helix IX is exposed in the intermembrane space, whereas helix X, located between both hemes, may play a key role in proton uptake to a proton-loading site in the reduced state for proton pumping. Taken together, our results suggested that CHCHD2 binds near helix IX and induces a structural change in helix X, accelerating proton uptake.
RESUMO
Resonance Raman (rR) spectroscopy has been applied to study the nature of the iron-oxo (Fe=O) moiety of oxoiron(IV) porphyrin π-cation radical complex (CompI). While the axial ligand effect on the nature of the Fe=O moiety has been studied with rR spectroscopy, the porphyrin ligand effect has not been studied well. Here, we investigated the porphyrin ligand effect on the Fe=O moiety with rR spectroscopy. The porphyrin ligand effect was modulated by the electron-withdrawing effect of the porphyrin substituent at the meso-position. This study shows that the frequency of the Fe=O stretching band, ν(Fe=O), hardly change even when the electron-withdrawing effect of the porphyrin substituent changes. This result is further supported by theoretical calculation of CompI. The natural atomic charge analysis reveals that the oxo and axial ligands work to buffer the electron-withdrawing effect of the porphyrin substituent. The electron-withdrawing porphyrin substituent shifts an electron population from the ferryl iron to the porphyrin, but the decreased electron population on the ferryl iron is compensated by the shift of the electron population from the oxo ligand and the axial ligand. The shift of the electron population makes the Fe-axial ligand bond length short, but the Fe=O bond length unchanged, resulting in the invariable ν(Fe=O) frequency.
Assuntos
Porfirinas , Ligantes , Porfirinas/química , Ferro/química , CátionsRESUMO
The peroxidase activity of cytochrome (cyt) c increases when Met80 dissociates from the heme iron, which is related to the initial cyt c membrane permeation step of apoptosis. Met80-dissociated cyt c can form an oxygenated species. Herein, resonance Raman spectra of Met80-depleted horse cyt c (M80A cyt c) were analyzed to elucidate the heme ligand properties of Met80-dissociated cyt c. The Fe-His stretching (νFe-His) mode of ferrous M80A cyt c was observed at 236 cm-1, and this frequency decreased by 1.5 cm-1 for the 15N-labeled protein. The higher νFe-His frequency of M80A cyt c than of other His-ligated heme proteins indicates strong heme coordination and the imidazolate character of His18. Peaks attributed to the Fe-O2 stretching (νFe-O2) and O-O stretching (νO-O) modes of the oxygenated species of M80A cyt c were observed at 576 and 1148 cm-1, respectively, under an 16O2 atmosphere, whereas the frequencies decreased to 544 and 1077 cm-1, respectively, under an 18O2 atmosphere. The νFe-O2 mode of Hydrogenobacter thermophilus (HT) M59A cyt c552 was observed at 580 cm-1 under an 16O2 atmosphere, whereas the frequency decreased to 553 cm-1 under an 18O2 atmosphere, indicating that relatively high νFe-O2 frequencies are characteristic of c-type cyt proteins. By comparison of the simultaneously observed νFe-O2 and νO-O frequencies of oxygenated cyt c and other oxygenated His-ligated heme proteins, the frequencies tend to have a positive linear relationship; the νFe-O2 frequency increases when the νO-O frequency increases. The imidazolate character of the heme-coordinated His and strong Fe-O and O-O bonds are characteristic of cyt c and apparently related to the peroxidase activity when Met80 dissociates from the heme iron.
Assuntos
Citocromos c , Análise Espectral Raman , Animais , Cavalos , Citocromos c/química , Heme/química , Ligantes , Ferro/química , PeroxidasesRESUMO
A novel dinitrogen-dichromium complex, [{Cr(LBn)}2(µ-N2)] (1), has been prepared from reaction of CrCl3 with a lithiated triamidoamine ligand (Li3LBn) under dinitrogen. The X-ray crystal structure analysis of 1 revealed that it is composed of two independent dimeric Cr complexes bridged by N2 in the unit cell. The bridged N-N bond lengths (1.188(4) and 1.185(7) Å) were longer than the free dinitrogen molecule. The elongations of N-N bonds in 1 were also supported by the fact that the ν(N-N) stretching vibration at 1772 cm-1 observed in toluene is smaller than the free N2. Complex 1 was identified to be a 5-coordinated high spin Cr(IV) complex by Cr K-edge XANES measurement. The 1H NMR spectrum and temperature dependent magnetic susceptibility of 1 indicated that complex 1 is in the S = 1 ground state, in which two Cr(IV) ions and unpaired electron spins of the bridging N22- ligand are strongly antiferromagnetically coupled. Reaction of complex 1 with 2.3 equiv of Na or K gave chromium complexes with N2 between the Cr ion and the respective alkali metal ion, [{CrNa(LBn)(N2)(Et2O)}2] (2) and [{CrK(LBn)(N2)}4(Et2O)2] (3), respectively. Furthermore, the complexes 2 and 3 reacted with 15-crown-5 and 18-crown-6 to form the respective crown-ether adducts, [CrNa(LBn)(N2)(15-crown-5)] (4) and [CrK(LBn)(N2)(18-crown-6)] (5). The XANES measurements of complexes 2, 3, 4, and 5 revealed that they are high spin Cr(IV) complexes like complex 1. All complexes reacted with a reducing agent and a proton source to form NH3 and/or N2H4. The yields of these products in the presence of K+ were higher than those in the presence of Na+. The electronic structures and binding properties of 1, 2, 3, 4, and 5 were evaluated and discussed based on their DFT calculations.
RESUMO
Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.
Assuntos
Antibacterianos , Heme , Antibacterianos/farmacologiaRESUMO
Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.
Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Adipócitos Marrons , Tecido Adiposo Marrom/metabolismo , Animais , Colina/metabolismo , Metilaminas , Camundongos , Infarto do Miocárdio/metabolismo , Termogênese/genéticaRESUMO
Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or ß subunit contains a ferric heme in the α2ß2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His â tyrosine [Tyr]) and M Boston (α58His â Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.
Assuntos
Hemoglobina M , Cristalografia , Teoria da Densidade Funcional , Heme/química , Hemoglobina M/química , Hemoglobina M/metabolismo , Humanos , Análise Espectral Raman , Tirosina/química , VibraçãoRESUMO
Heme compound II models bearing electron-deficient and -rich porphyrins, [FeIV(O)(TPFPP)(Cl)]- (1a) and [FeIV(O)(TMP)(Cl)]- (2a), respectively, are synthesized, spectroscopically characterized, and investigated in chemoselectivity and disproportionation reactions using cyclohexene as a mechanistic probe. Interestingly, cyclohexene oxidation by 1a occurs at the allylic C-H bonds with a high kinetic isotope effect (KIE) of 41, yielding 2-cyclohexen-1-ol product; this chemoselectivity is the same as that of nonheme iron(iv)-oxo intermediates. In contrast, as observed in heme compound I models, 2a yields cyclohexene oxide product with a KIE of 1, demonstrating a preference for C[double bond, length as m-dash]C epoxidation. The latter result is interpreted as 2a disproportionating to form [FeIV(O)(TMP+Ë)]+ (2b) and FeIII(OH)(TMP), and 2b becoming the active oxidant to conduct the cyclohexene epoxidation. In contrast to 2a, 1a does not disproportionate under the present reaction conditions. DFT calculations confirm that compound II models prefer C-H bond hydroxylation and that disproportionation of compound II models is controlled thermodynamically by the porphyrin ligands. Other aspects, such as acid and base effects on the disproportionation of compound II models, have been discussed as well.
RESUMO
A bis(µ-oxo)diiron(IV,IV) complex as a model for intermediate Q in the methane monooxygenase reaction cycle has been prepared. The precursor complex with a [FeIIIFeIV(µ-O)2] core was fully characterized by X-ray crystallography and other spectroscopic analyses and was converted to the [FeIV2(µ-O)2] complex via electrochemical oxidation at 1000 mV (vs Ag/Ag+) in acetone at 193 K. The UV-vis spectral features, Mössbauer parameters (ΔEQ = 2.079 mm/s and δ = -0.027 mm/s), and EXAFS analysis (Fe-O/N = 1.73/1.96 Å and Fe···Fe = 2.76 Å) support the structure of the low-spin (S = 1, for each Fe) [FeIV2(µ-O)2] core. The rate constants of the hydrogen abstraction reaction from 9,10-dihydroanthracene at 243 K suggest the high reactivity of these synthetic bis(µ-oxo)diiron complexes supported by simple N4 tripodal ligand.
Assuntos
OxigenasesRESUMO
Mechanistic studies are performed on the alkane hydroxylation with m-CPBA (m-chloroperbenzoic acid) catalyzed by nickel(II) complexes, NiII (L). In the oxidation of cycloalkanes, NiII (TPA) acts as an efficient catalyst with a high yield and a high alcohol selectivity. In the oxidation of adamantane, the tertiary carbon is predominantly oxidized. The reaction rate shows first-order dependence on [substrate] and [NiII (L)] but is independent on [m-CPBA]; vobs =k2 [substrate][NiII (L)]. The reaction exhibited a relatively large kinetic deuterium isotope effect (KIE) of 6.7, demonstrating that the hydrogen atom abstraction is involved in the rate-limiting step of the catalytic cycle. Furthermore, NiII (L) supported by related tetradentate ligands exhibit apparently different catalytic activity, suggesting contribution of the NiII (L) in the catalytic cycle. Based on the kinetic analysis and the significant effects of O2 and CCl4 on the product distribution pattern, possible contributions of (L)NiII -O. and the aroyloxyl radical as the reactive oxidants are discussed.
Assuntos
Alcanos , Níquel , Catálise , Clorobenzoatos , Hidroxilação , Cinética , Ligantes , OxirreduçãoRESUMO
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.
Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , DNA Catalítico/química , DNA Catalítico/metabolismo , Heme/química , Heme/metabolismo , Peroxidase/metabolismo , Biocatálise , ElétronsRESUMO
Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N2O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD+ TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm-1 upon hydride transfer from NADH to the Fe3+-NO enzyme via the dissociation of NAD+ from a transient state, with an N-O stretching of 1,330 cm-1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe3+-NHOâ¢- radical. The current findings provide conclusive evidence for the N2O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Proteínas Fúngicas/química , Fusarium/química , Óxido Nítrico/química , Óxido Nitroso/química , Oxirredutases/química , Cristalografia por Raios X/métodos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Elétrons , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Fusarium/genética , Expressão Gênica , Heme/química , Heme/metabolismo , Ferro/química , Ferro/metabolismo , NAD/química , NAD/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/química , Óxidos de Nitrogênio/metabolismo , Óxido Nitroso/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , PrótonsRESUMO
A reaction of Ni(ClO4)2·6H2O with a tripodal ligand having two di(tert-butyl)phenol moieties, H2tbuL, and 1 equivalent of triethylamine in CH2Cl2/CH3OH (1 : 1, v/v) under N2 gave a NiII-(phenol)(phenolate) complex, [Ni(HtbuL)(CH3OH)2]ClO4. The formation of the NiII-phenoxyl radical complex by O2 was observed in the reaction of this complex in the solid state. On the other hand, the NiII-phenoxyl radical complex [Ni(Me2NL)(CH3OH)2]ClO4 was obtained by the reaction of H2Me2NL having a p-(dimethylamino)phenol moiety with Ni(ClO4)2·6H2O in a similar procedure under O2, through the oxidation of the NiII-(phenol)(phenolate) complex. However, a direct redox reaction of the NiII ion could not be detected in the phenoxyl radical formation. The results of the reaction kinetics, XAS and X-ray structure analyses suggested that the O2 oxidation from the NiII-(phenol)(phenolate) complex to the NiII-phenoxyl radical complex occurs via the proton transfer-electron transfer (PT-ET) type mechanism of the phenol moiety weakly coordinated to the nickel ion.
RESUMO
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.
Assuntos
Heme/química , Ferro/química , Mioglobina/química , Oxigênio/química , Monóxido de Carbono/química , Elétrons , Estrutura Molecular , Espectroscopia de MossbauerRESUMO
Acid effects on the chemical properties of metal-oxygen intermediates have attracted much attention recently, such as the enhanced reactivity of high-valent metal(IV)-oxo species by binding proton(s) or Lewis acidic metal ion(s) in redox reactions. Herein, we report for the first time the proton effects of an iron(V)-oxo complex bearing a negatively charged tetraamido macrocyclic ligand (TAML) in oxygen atom transfer (OAT) and electron-transfer (ET) reactions. First, we synthesized and characterized a mononuclear nonheme Fe(V)-oxo TAML complex (1) and its protonated iron(V)-oxo complexes binding two and three protons, which are denoted as 2 and 3, respectively. The protons were found to bind to the TAML ligand of the Fe(V)-oxo species based on spectroscopic characterization, such as resonance Raman, extended X-ray absorption fine structure (EXAFS), and electron paramagnetic resonance (EPR) measurements, along with density functional theory (DFT) calculations. The two-protons binding constant of 1 to produce 2 and the third protonation constant of 2 to produce 3 were determined to be 8.0(7) × 108 M-2 and 10(1) M-1, respectively. The reactivities of the proton-bound iron(V)-oxo complexes were investigated in OAT and ET reactions, showing a dramatic increase in the rate of sulfoxidation of thioanisole derivatives, such as 107 times increase in reactivity when the oxidation of p-CN-thioanisole by 1 was performed in the presence of HOTf (i.e., 200 mM). The one-electron reduction potential of 2 (Ered vs SCE = 0.97 V) was significantly shifted to the positive direction, compared to that of 1 (Ered vs SCE = 0.33 V). Upon further addition of a proton to a solution of 2, a more positive shift of the Ered value was observed with a slope of 47 mV/log([HOTf]). The sulfoxidation of thioanisole derivatives by 2 was shown to proceed via ET from thioanisoles to 2 or direct OAT from 2 to thioanisoles, depending on the ET driving force.
Assuntos
Compostos de Ferro/química , Oxigênio/química , Prótons , Teoria da Densidade Funcional , Compostos de Ferro/síntese química , Conformação Molecular , OxirreduçãoRESUMO
The dinuclear copper enzyme, tyrosinase, activates O2 to form a (µ-η2 :η2 -peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X-ray crystal structures of the active tyrosinases with substrate l-tyrosine. At their catalytic sites, CuA moved toward l-tyrosine (CuA1 â CuA2), whose phenol oxygen directly coordinates to CuA2, involving the movement of CuB (CuB1 â CuB2). The crystal structures and spectroscopic analyses of the dioxygen-bound tyrosinases demonstrated that the peroxide ligand rotated, spontaneously weakening its O-O bond. Thus, the copper migration induced by the substrate-binding is accompanied by rearrangement of the bound peroxide species so as to provide one of the peroxide oxygen atoms with access to the phenol substrate's ϵâ carbon atom.
Assuntos
Cobre/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Oxigênio/metabolismo , Tirosina/metabolismo , Aspergillus oryzae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cobre/química , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Químicos , Monofenol Mono-Oxigenase/química , Oxigênio/química , Ligação Proteica , Streptomyces/enzimologia , Tirosina/químicaRESUMO
Metal complexes to promote oxidative DNA cleavage by H2O2 are desirable as anticancer drugs. A dicopper(II) complex of known p-cresol-derived methylene-tether ligand Hbcc [Cu2(bcc)]3+ did not promote DNA cleavage by H2O2. Here, we synthesized a new p-cresol-derived amide-tether one, 2,6-bis(1,4,7,10-tetrazacyclododecyl-1-carboxyamide)-p-cresol (Hbcamide). A dicopper(II) complex of the new ligand [Cu2(µ-OH)(bcamide)]2+ was structurally characterized. This complex promoted the oxidative cleavage of supercoiled plasmid pUC19 DNA (Form I) with H2O2 at pH 6.0-8.2 to give Forms II and III. The reaction was largely accelerated in a high pH region. A µ-1,1-hydroperoxo species was formed as the active species and spectroscopically identified. The amide-tether complex is more effective in cytotoxicity against HeLa cells than the methylene-tether one.
Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Cobre/farmacologia , Cresóis/farmacologia , Peróxido de Hidrogênio/farmacologia , Amidas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cobre/química , Cresóis/química , Clivagem do DNA , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Peróxido de Hidrogênio/síntese química , Peróxido de Hidrogênio/química , Ligantes , Estrutura Molecular , OxirreduçãoRESUMO
Human indoleamine 2,3-dioxygenase 1 (IDO) is a heme enzyme that catalyzes the first reaction of the main metabolic pathway of L-tryptophan (Trp) to produce N-formylkynurenin. The reaction involves cleavage of the C2=C3 bond in the Trp indole ring and insertion of two atomic oxygens from the iron-bound O2 into the indole 2 and 3 position. For establishment of the chemical mechanism of this unique enzymatic reaction, it is necessary to determine the conformation and electronic state of the substrate Trp bound to IDO. In this study, we measured the ultraviolet resonance Raman spectra of IDO in the presence of Trp to detect the vibrational modes of the substrate Trp. We compared the ultraviolet resonace Raman spectra of Trp in a ternary complex (Trp-bound cyanide enzyme) and a binary complex (Trp-bound reduced enzyme) of IDO with that of free Trp in solution and found that binding to IDO influences the conformation of Trp, resulting in similar changes in the two complexes, especially around the C3-Cß bond. However, the presence of the diatomic ligand at the heme sixth coordination site in the ternary complex significantly alters the mobility and electronic structure of Trp, most likely resulting in the C2=C3 bond cleavage in the enzymatic reaction.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/química , Triptofano/química , Heme/química , Heme/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Ligação Proteica , Conformação Proteica , Análise Espectral Raman , Triptofano/metabolismoRESUMO
Isolation and characterisation of RuIV(O) complexes were accomplished to investigate their fundamental electron transfer (ET) and proton-coupled ET (PCET) properties. Reorganisation energies (λ) in electron transfer (ET) and proton-coupled ET (PCET) from electron donors to the isolated RuIV(O) complexes have been determined for the first time to be in the range of 1.70-1.88 eV (ET) and 1.20-1.26 eV (PCET). It was suggested that the reduction of the λ values of PCET in comparison with those of ET should be due to the smaller structural change in PCET than that in ET on the basis of DFT calculations on 1 and 1e--reduced 1 in the absence and presence of TFA, respectively. In addition, the smaller λ values for the RuIV(O) complexes than those reported for FeIV(O) and MnIV(O) complexes should be due to the lack of participation of dσ orbitals in the ET and PCET reactions. This is the first example to evaluate fundamental ET and PCET properties of RuIV(O) complexes leading to further understanding of their reactivity in oxidation reactions.