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1.
Gene ; 712: 143945, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31279712

RESUMO

Reactive oxygen species, generated in all the aerobic organisms, can cause oxidative stress. Excessive ROS may become a source of carcinogen due to DNA damage, lipid peroxidation, cell injury, and cell death. In order to prevent these adverse effects of ROS, antioxidant enzymes have evolved in aerobic organisms. Catalase is a major antioxidant enzyme that breaks down excessive H2O2 and inhibits apoptotic cell death. Here we molecularly characterized catalase from red-lip mullet. The cDNA sequence of LhCAT consists of an ORF of 1545 bp, which encodes a 527 amino acid peptide (~60 kDa). Based on bioinformatics analysis, LhCAT possesses a domain architecture characteristic of catalases, including a catalase proximal active site signature and a catalase proximal heme-ligand signature. It also has heme and NADPH binding sites homologous to previously described catalases. Pairwise alignment with its homologs revealed that LhCAT shares 95.1% identity with Oplegnathus fasciatus catalase and 97.4% similarity with Sparus aurata catalase. An uprooted phylogenetic tree demonstrated that LhCAT resides in a clade with catalases from other teleosts and exhibits a close relationship with Oplegnathus fasciatus catalase. Among twelve tissue types, we observed the highest LhCAT mRNA expression in the liver, followed by blood. Immune challenge by Lactococcus garvieae, or Poly I:C in the blood or spleen resulted in up-regulation at 24 h post injection. We also tested the antioxidant activity of recombinant LhCAT against hydrogen peroxide and found its optimal concentration to be 12.5 µg/mL. Collectively, these data suggested that LhCAT play an important role in antioxidant defense and immune response of red-lip mullet.


Assuntos
Catalase/metabolismo , Proteínas de Peixes/metabolismo , Smegmamorpha , Adjuvantes Imunológicos , Animais , Antioxidantes/metabolismo , Catalase/genética , DNA Complementar/genética , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Heme/química , Peróxido de Hidrogênio/química , Sistema Imunitário , Ligantes , Fígado/enzimologia , Estresse Oxidativo , Filogenia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para Cima
2.
Phys Chem Chem Phys ; 21(27): 14957-14963, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31236551

RESUMO

Heme, as the cofactor and active site of Hb, enables Hb to carry out the necessary function required for O2 management for life, that is, reversible O2 binding for transport. In this paper, the microscopic mechanism of heme-associated poisoning has been elucidated from the perspective of electronic interaction by performing first-principles calculations. The results show that the functional groups (-CHO, -COOH, -NO2, -NH2) and CN exhibit a stronger affinity for heme than O2 and are more likely to occupy the O2 binding site, which results in the loss of the ability of heme to carry O2. Moreover, the addition of functional groups, CO and CN to heme at the side site can cause a pronounced enhancement toward the O2 binding characteristics of heme, which prevents heme from releasing O2 to oxygen-consuming tissues as the blood circulates. The reversible O2 binding function of heme is disrupted by the presence of these toxic ligands in the heme binding pocket, which greatly affects O2 transport in the blood. The inability of tissues to obtain O2 leads to tissue hypoxia, which is the main cause of poisoning. Based on the energy, geometry and electronic properties, the hypoxia mechanism proposed by us coincides well with experiment, and the research has the potential to provide a theoretical reference for the relevant areas of bioscience.


Assuntos
Heme/metabolismo , Ligantes , Oxigênio/metabolismo , Hipóxia Celular/efeitos dos fármacos , Heme/química , Ligação Proteica/efeitos dos fármacos , Toxinas Biológicas/metabolismo , Toxinas Biológicas/toxicidade
3.
Chem Commun (Camb) ; 55(46): 6610-6613, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31119219

RESUMO

The X-ray crystal structure of F43Y/T67R myoglobin revealed unique Tyr-heme double cross-links between Tyr43 and the heme 4-vinyl group, which represents a novel post-translational modification of heme proteins. Moreover, with the feature of a distal His-Arg pair, the designed artificial enzyme exhibited a peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase.


Assuntos
Materiais Biomiméticos/química , Heme/química , Mioglobina/química , Tirosina/química , Animais , Arginina/química , Benzotiazóis/química , Guaiacol/química , Histidina/química , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Mutação , Mioglobina/genética , Oxirredução , Peroxidases/química , Processamento de Proteína Pós-Traducional , Cachalote , Ácidos Sulfônicos/química
4.
Nat Commun ; 10(1): 2303, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127106

RESUMO

The spin state transition from low spin to high spin upon substrate addition is one of the key steps in cytochrome P450 catalysis. External perturbations such as pH and hydrogen bonding can also trigger the spin state transition of hemes through deprotonated histidine (e.g. Cytochrome c). In this work, we report the isolated 2-methylimidazole Cobalt(II) [Co(TPP)(2-MeHIm)] and [Co(TTP)(2-MeHIm)], and the corresponding 2-methylimidazolate derivatives where the N-H proton of axial 2-MeHIm is removed. Interestingly, various spectroscopies including EPR and XAFS determine a high-spin state (S = 3/2) for the imidazolate derivatives, in contrast to the low-spin state (S = 1/2) of all known imidazole analogs. DFT assisted stereoelectronic investigations are applied to understand the metal-ligand interactions, which suggest that the dramatically displaced metal center allowing a promotion eg(dπ) → b1g([Formula: see text]) is crucial for the occurrence of the spin state transition.


Assuntos
Cobalto/química , Heme/química , Prótons , Biocatálise , Cobalto/metabolismo , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Heme/análogos & derivados , Heme/metabolismo , Histidina/química , Concentração de Íons de Hidrogênio , Imidazóis/metabolismo , Ferro/química , Ligantes , Oxirredução
5.
Food Chem ; 292: 227-236, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054669

RESUMO

The role of chicken hemoglobin in lipid oxidation of washed chicken muscle exposed to high hydrostatic pressure (0, 200, 400 and 600 MPa) was examined. The observed decrease in redness was higher with elevated pressures (5.0 vs 3.7). During storage, redness decreased in samples exposed to 400 and 600 MPa. This decrease was concomitant with the progression of oxidation and the 2.5 fold decrease of soluble heme. An additional experiment was conducted to examine the effect of the hemoglobin mode of addition into pressurized muscle. The exposure at 600 MPa led to washed muscle oxidation even in the absence of hemoglobin, thus indicating that pressure treatments triggered lipid oxidation. However, the presence of native or pressurized hemoglobin into pressurized washed muscle caused more hexanal than the pressurized control without hemoglobin. Overall, results suggest that membrane disruption and the release of hemin are crucial for the onset of oxidation.


Assuntos
Hemoglobinas/química , Músculos/química , Aldeídos/análise , Animais , Galinhas/metabolismo , Heme/química , Hemina/análise , Hemina/metabolismo , Pressão Hidrostática , Peroxidação de Lipídeos , Oxirredução , Espectrofotometria
6.
Dalton Trans ; 48(21): 7451-7461, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31086893

RESUMO

A significant abundance of copper (Cu) and iron in amyloid ß (Aß) plaques, and several heme related metabolic disorders are directly correlated with Alzheimer's disease (AD), and these together with co-localization of Aß plaques with heme rich deposits in the brains of AD sufferers indicates a possible association of the said metals with the disease. Recently, the Aß peptides have been found to bind heme and Cu individually as well as simultaneously. Another significant finding relevant to this is the lower levels of nitrite and nitrate found in the brains of patients suffering from AD. In this study, a combination of absorption and electron paramagnetic resonance spectroscopy and kinetic assays have been used to study the interaction of nitrite with the metal bound Aß complexes. The data indicate that heme(III)-Cu(i)-Aß, heme(II)-Cu(i)-Aß, heme(II)-Aß and Cu(i)-Aß can reduce nitrite to nitric oxide (NO), an important biological messenger also related to AD, and thus behave as nitrite reductases. However these complexes reduce nitrite at different rates with heme(III)-Cu(i)-Aß being the fastest following an inner sphere electron transfer mechanism. The rest of the metal-Aß adducts follow an outer sphere electron transfer mechanism during nitrite reduction. Protonation from the Arg5 residue triggering the N-O bond heterolysis in heme(III) bound nitrite with a simultaneous electron transfer from the Cu(i) center to produce NO is the rate determining step, indicating a proton transfer followed by electron transfer (PTET) mechanism.


Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Heme/química , Nitrito Redutases/química , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Humanos , Ferro/química , Cinética , Óxido Nítrico/química , Nitritos/química , Oxirredução , Placa Amiloide/química
7.
Dalton Trans ; 48(16): 5408-5416, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30946408

RESUMO

HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes oxygen-dependent degradation of heme via a similar catalytic mechanism to mammalian heme oxygenase. However, HutZ oxidizes the ß- or δ-meso position of heme at a ∼1 : 1 ratio distinct from heme oxygenase, which initiates the degradation of heme solely at the α-meso position. His63 is a residue that potentially forms hydrogen bond with the heme 7-propionate group. To establish the role of His63 in regioselectivity of heme degradation by HutZ and heme binding, we constructed mutants of His63. Interestingly, the H63L mutant retained a comparable level of ß- or δ-regioselectivity as wild-type HutZ. Ascorbic acid-assisted heme degradation by HutZ is pH-dependent, showing activity at pH 6.0 but not above pH 8.0. Compared to the wild-type protein, the H63L mutant was inactive, even at pH 6.0, and affinity for heme was significantly decreased in contrast with a comparable heme binding affinity at pH 8.0, as observed for the mutant of Asp132 to Val, which is located within hydrogen bonding distance of the heme axial ligand His170, but in a different protomer. In addition, the distance between heme and Trp109 increased from 16-18 Å for wild-type HutZ to 24-28 Å for the H63L mutant, indicating that protomer orientation is altered by the mutation, since Trp109 is in another subunit of the heme axial ligand. Our results collectively suggest that His63 positioned near heme does not contribute to regioselectivity of heme degradation but plays a key role in maintaining the orientation of subunits for HutZ to function of heme degradation.


Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Histonas/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Heme/química , Histonas/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Análise Espectral Raman
8.
J Biochem Mol Toxicol ; 33(7): e22325, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004546

RESUMO

Cartap hydrochloride is a mildly perilous insecticide known as "Padan" which is used largely in agricultural farms to control weevil and caterpillars. The over use of cartap causes harmful effects on human health. Since the blood may acts as a target and carrier for insecticides, the effect of these compounds on blood in mammalian toxicology is very important. Hemoglobin is a tetramer protein that play critical role in oxygen transport. The aim of this study was to analyze and compare the function and structural changes of hemoglobin in the presence of different concentrations of cartap by employing different spectroscopic techniques. The obtained results show that cartap has a high hemolytic effect which is increased with cartap concentration and reduces the thermal midpoint of hemoglobin. Fluorescence measurements reveal heme degradation at different concentrations of cartap. In consequence of theoretical and experimental results, cartap has an undesirable effect on hemoglobin structure and function.


Assuntos
Heme/química , Hemoglobinas/química , Inseticidas/química , Proteólise , Tiocarbamatos/química , Animais , Hemólise , Humanos
9.
Chem Commun (Camb) ; 55(25): 3654-3657, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30855056

RESUMO

Herein, we present a serendipitously discovered chemoselective labelling of protein N-homocysteinylation with bioorthogonal azide probes. The reaction proceeds rapidly under alkaline and heating conditions. Our experiments suggest that azides can be converted to aldehydes in situ catalyzed by heme(ii), followed by a condensation with protein N-homocysteinylation to afford stable 1,3-thiazines.


Assuntos
Azidas/química , Heme/química , Homocisteína/análogos & derivados , Proteínas/química , Animais , Biotina/química , Catálise , Bovinos , Homocisteína/química , Concentração de Íons de Hidrogênio , Mioglobina/química , Mioglobina/metabolismo , Peptídeos/análise , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Massas em Tandem , Temperatura Ambiente
10.
Dalton Trans ; 48(12): 3973-3983, 2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30834412

RESUMO

HutZ, a dimeric protein, from Vibrio cholerae is a protein that catalyzes the oxygen-dependent degradation of heme. Interestingly, the ascorbic acid-supported heme-degradation activity of HutZ depends on pH: less than 10% of heme is degraded by HutZ at pH 8.0, but nearly 90% of heme is degraded at pH 6.0. We examined here pH-dependent conformational changes in HutZ using fluorescence spectroscopy. Trp109 is estimated to be located approximately 21 Å from heme and is present in a different subunit containing a heme axial ligand. Thus, we postulated that the distance between heme and Trp109 reflects subunit-subunit orientational changes. On the basis of resonance energy transfer from Trp109 to heme, we estimated the distance between heme and Trp109 to be approximately 17 Å at pH 8.0, while the distance increased by less than 2 Å at pH 6.0. We presumed that such changes led to a decrease in electron donation from the proximal histidine, resulting in enhancement of the heme-degradation activity. To confirm this scenario, we mutated Ala31, located at the dimer interface, to valine to alter the distance through the subunit-subunit interaction. The distance between heme and Trp109 for the A31V mutant was elongated to 24-27 Å. Although resonance Raman spectra and reduction rate of heme suggested that this mutation resulted in diminished electron donation from the heme axial ligand, ascorbic acid-supported heme-degradation activity was not observed. Based on our findings, it can be proposed that the relative positioning of two protomers is important in determining the heme degradation rate by HutZ.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Heme/química , Heme/metabolismo , Vibrio cholerae/química , Proteínas de Bactérias/isolamento & purificação , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Oxirredução , Espectrometria de Fluorescência , Vibrio cholerae/metabolismo
11.
Int J Mol Sci ; 20(3)2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30736292

RESUMO

Soluble guanylate cyclase (sGC) regulates numerous physiological processes. The ß subunit Heme Nitric Oxide/Oxygen (HNOX) domain makes this protein sensitive to small gaseous ligands. The structural basis of the activation mechanism of sGC under the influence of ligands (NO, O2, CO) is poorly understood. We examine the effect of different ligands on the human sGC HNOX domain. HNOX systems with gaseous ligands were generated and explored using Molecular Dynamics (MD). The distance between heme Fe2+ and histidine in the NO-ligated HNOX (NO-HNOX) system is larger compared to the O2, CO systems. NO-HNOX rapidly adopts the conformation of the five-group metal coordination system. Loops α, ß, γ and helix-f exhibit increased mobility and different hydrogen bond networks in NO-HNOX compared to the other systems. The removal of His from the Fe coordination sphere in NO-HNOX is assisted by interaction of the imidazole ring with the surrounding residues which in turn leads to the release of signaling helix-f and activation of the sGC enzyme. Insights into the conformational dynamics of a human sGC HNOX domain, especially for regions which are functionally critical for signal transduction, are valuable in the understanding of cardiovascular diseases.


Assuntos
Heme/química , Óxido Nítrico/química , Oxigênio/química , Heme/metabolismo , Humanos , Ligações de Hidrogênio , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Ligação Proteica , Guanilil Ciclase Solúvel/química , Guanilil Ciclase Solúvel/metabolismo
12.
Proc Natl Acad Sci U S A ; 116(8): 2854-2859, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718404

RESUMO

Hemoglobin and myoglobin are oxygen-binding proteins with S = 0 heme {FeO2}8 active sites. The electronic structure of these sites has been the subject of much debate. This study utilizes Fe K-edge X-ray absorption spectroscopy (XAS) and 1s2p resonant inelastic X-ray scattering (RIXS) to study oxyhemoglobin and a related heme {FeO2}8 model compound, [(pfp)Fe(1-MeIm)(O2)] (pfp = meso-tetra(α,α,α,α-o-pivalamido-phenyl)porphyrin, or TpivPP, 1-MeIm = 1-methylimidazole) (pfpO2), which was previously analyzed using L-edge XAS. The K-edge XAS and RIXS data of pfpO2 and oxyhemoglobin are compared with the data for low-spin FeII and FeIII [Fe(tpp)(Im)2]0/+ (tpp = tetra-phenyl porphyrin) compounds, which serve as heme references. The X-ray data show that pfpO2 is similar to FeII, while oxyhemoglobin is qualitatively similar to FeIII, but with significant quantitative differences. Density-functional theory (DFT) calculations show that the difference between pfpO2 and oxyhemoglobin is due to a distal histidine H bond to O2 and the less hydrophobic environment in the protein, which lead to more backbonding into the O2 A valence bond configuration interaction multiplet model is used to analyze the RIXS data and show that pfpO2 is dominantly FeII with 6-8% FeIII character, while oxyhemoglobin has a very mixed wave function that has 50-77% FeIII character and a partially polarized Fe-O2 π-bond.


Assuntos
Ferro/química , Oxigênio/química , Oxiemoglobinas/química , Porfirinas/química , Domínio Catalítico , Compostos Férricos/química , Heme/química , Metaloporfirinas/química , Modelos Moleculares , Mioglobina/química , Espalhamento de Radiação , Espectroscopia por Absorção de Raios X , Raios X
13.
Proc Natl Acad Sci U S A ; 116(9): 3572-3577, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808749

RESUMO

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a 3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Ferro/química , Oxigênio/química , Animais , Catálise , Domínio Catalítico , Bovinos , Cobre/química , Cristalografia por Raios X , Complexo IV da Cadeia de Transporte de Elétrons/genética , Oxirredução , Conformação Proteica
14.
Proc Natl Acad Sci U S A ; 116(9): 3425-3430, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755526

RESUMO

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.


Assuntos
Grupo dos Citocromos c/genética , Transporte de Elétrons/genética , Modelos Moleculares , Shewanella/genética , Sequência de Aminoácidos/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos c/química , Citocromos/química , Citocromos/genética , Elétrons , Heme/química , Heme/genética , Oxirredução , Shewanella/química , Shewanella/patogenicidade
15.
Biochemistry ; 58(6): 489-492, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30605595

RESUMO

Mycobacterium tuberculosis heme-degrading protein MhuD degrades heme to mycobilin isomers and iron, while its closest homologues from Staphylococcus aureus, IsdG and IsdI, degrade heme to staphylobilin isomers, formaldehyde, and iron. Superposition of the structures of the heme-bound complexes reveals that the heme molecule in the MhuD active site is rotated ∼90° about the tetrapyrrole plane with respect to IsdG and IsdI active site heme molecules. Therefore, the variation in IsdG/IsdI and MhuD chromophore products may be attributed to the different heme orientations. In MhuD, two arginines, Arg22 and Arg26, stabilize the heme propionates and may account for the heme orientation. Herein, we demonstrate that the MhuD-R26S variant alters the resulting chromophore product from mycobilin to biliverdin IXα (α-BV), whereas the R22S variant does not. Surprisingly, unlike canonical heme oxygenase (HO) that also degrades heme to α-BV, the MhuD-R26S variant produces the C1 product formaldehyde rather than carbon monoxide as observed for HO. The MhuD-R26S variant is an important tool for further probing the mechanism of action of MhuD and for studying the fate of the MhuD product in mycobacterium.


Assuntos
Proteínas de Bactérias/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Mutação , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Formaldeído/metabolismo , Heme/química , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/genética , Modelos Moleculares , Conformação Proteica
16.
Inorg Chem ; 58(4): 2627-2636, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30694043

RESUMO

A heme oxygen binding behavior was described through a unique geometric and electronic comparison of zinc porphyrin complexes. In this work, a charge transfer model for saddled metalloporphyrin complexes outlined the push effects of the ring nonplanarity and axial imidazole, and the pull effect of the axial dioxygen. The origin and role of the horizontal (ring nonplanarity) push effect and its relationship to the vertical (axial ligand) push/pull effect and its contribution to dioxygen binding were considered from the perspectives of crystal structures, theoretical calculations, and bathochromic shifts. Single-point energy and molecular orbital calculations starting from crystal structures were used to obtain the electronic structures of zinc porphyrin complexes. This study not only revealed that the electronic behavior of metalloporphyrins is driven by ring nonplanarity and axial ligation but also afforded new insight into the oxygen carrier mechanism in heme.


Assuntos
Heme/química , Metaloporfirinas/química , Transporte de Elétrons , Modelos Moleculares , Conformação Molecular
17.
J Biol Chem ; 294(8): 2771-2785, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30593511

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that utilizes heme as a primary iron source within the host. Extracellular heme is sensed via a heme assimilation system (has) that encodes an extracytoplasmic function (ECF) σ factor system. Herein, using has deletion mutants, quantitative PCR analyses, and immunoblotting, we show that the activation of the σ factor HasI requires heme release from the hemophore HasAp to the outer-membrane receptor HasR. Using RT-PCR and 5'-RACE, we observed that following transcriptional activation of the co-transcribed hasRAp, it is further processed into specific mRNAs varying in stability. We noted that the processing and variation in stability of the hasAp and hasR mRNAs in response to heme provide a mechanism for differential expression from co-transcribed genes. The multiple layers of post-transcriptional regulation of the ECF signaling cascade, including the previously reported post-transcriptional regulation of HasAp by the heme metabolites biliverdin IXß and IXδ, allow fine-tuning of the cell-surface signaling system in response to extracellular heme levels. We hypothesize that the complex post-transcriptional regulation of the Has system provides P. aeruginosa an advantage in colonizing a variety of physiological niches in the host.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Pseudomonas aeruginosa/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Bactérias/química , Cristalografia por Raios X , Heme/química , Ferro/metabolismo , Conformação Proteica , RNA Mensageiro/metabolismo
18.
Nature ; 565(7737): 67-72, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30568304

RESUMO

Although abundant in organic molecules, carbon-hydrogen (C-H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C-H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp3 carbon1,2. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp3 C-H bonds through carbene C-H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C-H functionalization1,2. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C-H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp3 C-H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C-H functionalization reactions for applications in chemistry and synthetic biology.


Assuntos
Biocatálise , Carbono/química , Carbono/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hidrogênio/química , Ferro/química , Alquilação , Animais , Coenzimas/química , Coenzimas/metabolismo , Cisteína/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular Direcionada , Heme/química , Heme/metabolismo , Hidrogênio/metabolismo , Ferro/metabolismo , Masculino , Metano/análogos & derivados , Metano/química , Serina/metabolismo , Especificidade por Substrato , Vitamina B 12/química , Vitamina B 12/metabolismo
19.
FEBS J ; 286(3): 479-494, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30570222

RESUMO

A major challenge to the control and eventual eradication of Mycobacterium tuberculosis infection is this pathogen's prolonged dormancy. The heme-based oxygen sensor protein DevS (DosS) plays a key role in this phenomenon, because it is a major activator of the transcription factor DevR. When DevS is active, its histidine protein kinase region is ON and it phosphorylates and activates DevR, which can induce the transcription of the dormancy regulon genes. Here, we have investigated the mechanism by which the ligation of molecular oxygen to a heme-binding domain in DevS switches OFF its histidine protein kinase region. To shed light on the oligomerization states of this protein and possible protein-surfaces of interaction, we used analytical gel filtration, together with dynamic light scattering, fluorescence spectroscopy and chemical crosslinking. We found that DevS exists as three major species: an octamer, a tetramer and a dimer. These three states were observed for the concentration range between 0.5 and 20 µm DevS, but not below 0.1 µm. Levels of DevS in M. tuberculosis are expected to range from 5 to 26 µm. When this histidine protein kinase was OFF, the DevS was mainly tetrameric and dimeric; by contrast, when the kinase was ON, the protein was predominantly octameric. The changes in quaternary structure were rapid upon binding to the physiological signal. This finding represents a novel strategy for switching the activity of a two-component heme-based sensor. An enhanced understanding of this process might potentially lead to the design of novel regulatory agents that target the multimer interfaces for treatment of latent tuberculosis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Heme/química , Mycobacterium tuberculosis/efeitos dos fármacos , Oxigênio/farmacologia , Protamina Quinase/química , Proteínas Quinases/genética , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Tuberculose Latente/microbiologia , Tuberculose Latente/patologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Fosforilação , Protamina Quinase/genética , Protamina Quinase/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulon , Transdução de Sinais , Espectrometria de Fluorescência , Transcrição Genética/efeitos dos fármacos
20.
Inorg Chem ; 58(1): 152-164, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30576115

RESUMO

A variety of heme derivatives are pervasive in nature, having different architectures that are complementary to their function. Herein, we report the synthesis of a series of iron porphyrinoids, which bear electron-withdrawing groups and/or are saturated at the ß-pyrrolic position, mimicking the structural variation of naturally occurring hemes. The effects of the aforementioned factors were systematically studied using a combination of electrochemistry, spectroscopy, and theoretical calculations with the carbon monoxide (CO) and nitric oxide (NO) adducts of these iron porphyinoids. The reduction potentials of iron porphyrinoids vary over several hundreds of millivolts, and the X-O (X = C, N) vibrations of the adducts vary over 10-15 cm-1. Density functional theory calculations indicate that the presence of electron-withdrawing groups and saturation of the pyrrole ring lowers the π*-acceptor orbital energies of the macrocycle, which, in turn, attenuates the bonding of iron to CO and NO. A hypothesis has been presented as to why cytochrome c containing nitrite reductases and cytochrome cd1 containing nitrite reductases follow different mechanistic pathways of nitrite reduction. This study also helps to rationalize the choice of heme a3 and not the most abundant heme b cofactor in cytochrome c oxidase.


Assuntos
Heme/análogos & derivados , Ferro/química , Metaloporfirinas/química , Monóxido de Carbono/química , Complexos de Coordenação/química , Teoria da Densidade Funcional , Heme/química , Metaloporfirinas/síntese química , Modelos Químicos , Óxido Nítrico/química , Oxirredução
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