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1.
Nat Commun ; 11(1): 4145, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811825

RESUMO

In haemoglobin the change from the low-spin (LS) hexacoordinated haem to the high spin (HS, S = 2) pentacoordinated domed deoxy-myoglobin (deoxyMb) form upon ligand detachment from the haem and the reverse process upon ligand binding are what ultimately drives the respiratory function. Here we probe them in the case of Myoglobin-NO (MbNO) using element- and spin-sensitive femtosecond Fe Kα and Kß X-ray emission spectroscopy at an X-ray free-electron laser (FEL). We find that the change from the LS (S = 1/2) MbNO to the HS haem occurs in ~800 fs, and that it proceeds via an intermediate (S = 1) spin state. We also show that upon NO recombination, the return to the planar MbNO ground state is an electronic relaxation from HS to LS taking place in ~30 ps. Thus, the entire ligand dissociation-recombination cycle in MbNO is a spin cross-over followed by a reverse spin cross-over process.


Assuntos
Heme/química , Hemoglobinas/química , Mioglobina/química , Heme/metabolismo , Hemoglobinas/metabolismo , Cinética , Ligantes , Modelos Moleculares , Mioglobina/metabolismo , Espectrometria por Raios X
2.
PLoS Comput Biol ; 16(7): e1008024, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609716

RESUMO

Vitamin B12 (or cobalamin) is an enzymatic cofactor essential both for mammals and bacteria. However, cobalamin can be synthesized only by few microorganisms so most bacteria need to take it up from the environment through the TonB-dependent transport system. The first stage of cobalamin import to E. coli cells occurs through the outer-membrane receptor called BtuB. Vitamin B12 binds with high affinity to the extracellular side of the BtuB protein. BtuB forms a ß-barrel with inner luminal domain and extracellular loops. To mechanically allow for cobalamin passage, the luminal domain needs to partially unfold with the help of the inner-membrane TonB protein. However, the mechanism of cobalamin permeation is unknown. Using all-atom molecular dynamics, we simulated the transport of cobalamin through the BtuB receptor embedded in an asymmetric and heterogeneous E. coli outer-membrane. To enhance conformational sampling of the BtuB loops, we developed the Gaussian force-simulated annealing method (GF-SA) and coupled it with umbrella sampling. We found that cobalamin needs to rotate in order to permeate through BtuB. We showed that the mobility of BtuB extracellular loops is crucial for cobalamin binding and transport and resembles an induced-fit mechanism. Loop mobility depends not only on the position of cobalamin but also on the extension of luminal domain. We provided atomistic details of cobalamin transport through the BtuB receptor showing the essential role of the mobility of BtuB extracellular loops. A similar TonB-dependent transport system is used also by many other compounds, such as haem and siderophores, and importantly, can be hijacked by natural antibiotics. Our work could have implications for future delivery of antibiotics to bacteria using this transport system.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Vitamina B 12/metabolismo , Algoritmos , Antibacterianos/química , Sítios de Ligação , Biologia Computacional , Cristalografia por Raios X , Heme/química , Íons , Bicamadas Lipídicas/química , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Distribuição Normal , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Sacarose/química , Água/química
3.
Nat Commun ; 11(1): 2813, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499479

RESUMO

5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.


Assuntos
5-Aminolevulinato Sintetase/química , Regulação Enzimológica da Expressão Gênica , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Acil Coenzima A/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Protoporfiria Eritropoética/genética , Especificidade por Substrato
4.
Inorg Chem ; 59(11): 7415-7425, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32383872

RESUMO

Heme active sites are capable of oxidizing organic substrates by four electrons using molecular oxygen (heme dioxygenases), where a dioxygen (O2) adduct of heme (FeIII-O2•-) acts as the primary oxidant, in contrast to monooxygenases, where high-valent species are involved. This chemistry, although lucrative, is difficult to access using homogeneous synthetic systems. Over the past few years using a combination of self-assembly and in situ resonance Raman spectroscopy, the distribution of different reactive intermediates formed during the electrochemical reduction of oxygen has been elucidated. An FeIII-O2•- species, which is the reactive species of dioxygenase, is an intermediate in heterogeneous electrochemical O2 reduction by iron porphyrins and its population, under electrochemical conditions, may be controlled by controlling the applied potential. Iron porphyrins having different axial ligands are constructed on a self-assembled monolayer of thiols on an electrode, and these constructs can activate O2 and efficiently catalyze the dioxygenation of 3-methylindole and oxidation of a series of organic compounds having C-H bond energies between 80 and 90 kcal mol-1 at potentials where FeIII-O2•- species are formed on the electrode. Isotope effects suggest that hydrogen-atom transfer from the substrate is likely to be the rate-determining step. Axial thiolate ligands are found to be more efficient than axial imidazoles or phenolates with turnover numbers above 60000 and turnover frequencies over 60 s-1. These results highlight a new reaction engineering approach to harness O2 as a green oxidant for efficient chemical oxidation.


Assuntos
Compostos Férricos/química , Heme/química , Oxigênio/química , Superóxidos/química , Catálise , Estrutura Molecular , Oxirredução
5.
Proc Natl Acad Sci U S A ; 117(17): 9349-9355, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291342

RESUMO

Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e- stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO's hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Oxirredutases/química , Bactérias/metabolismo , Cobre/química , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Íons , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Bombas de Próton/metabolismo , Prótons , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Nucleic Acids Res ; 48(10): 5254-5267, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329781

RESUMO

Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ-heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn't extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.


Assuntos
Biotina/química , Biotinilação , DNA/química , Quadruplex G , Heme/química , RNA/química , Animais , Sequência de Bases , Biotina/análogos & derivados , Drosophila melanogaster , Ligantes , Masculino , Salmão , Espermatozoides , Tiramina/análogos & derivados , Tiramina/química
7.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 375-384, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254062

RESUMO

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Š(spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.


Assuntos
Proteínas de Transporte de Ânions/química , Proteínas de Bactérias/química , Heme/análogos & derivados , Pseudomonas aeruginosa/enzimologia , Proteínas de Transporte de Ânions/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Heme/química , Modelos Moleculares , Óperon , Multimerização Proteica , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/genética
8.
Phys Chem Chem Phys ; 22(16): 8817-8826, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32285865

RESUMO

The oxygen-dependent heme utilization degrading enzyme in Mycobacterium tuberculosis (MhuD) uniquely integrates monooxygenase and dioxygenase functions in a single active site. It cannot convert heme to biliverdin as canonical heme oxygenases but generates mycobilin without releasing carbon monoxide. Herein, by employing ONIOM calculations, we investigated the heme degradation mechanism of MhuD. Our calculations revealed that MhuD firstly follows a canonical monooxygenation mechanism to hydroxylate heme on the δ-meso carbon guided by the asparagine residue Asn7, which experiences a 21.2 kcal mol-1 energy barrier in the O-O cleavage rate-limiting step during the conversion process from ferric heme-hydroperoxy species to mycobilin. In the second degradation step, the ruffled conformation of oxoheme (oxoheme is the ferrous π radical complex formed by hydroxyheme experiencing deprotonation in the hydroxyl group and intramolecular electron transfer) imposed by the hydrophobic environment of the enzyme not only inhibits the continuing conversion of oxoheme to biliverdin but also endows the meso-carbons with radical characteristics, which turns the second degradation step to a dioxygenation reaction with 20.4 kcal mol-1 energy barrier. We further analysed the electronic structure change along the reaction process. Our calculation discovered that the ruffled structure of oxoheme is critical to the regiospecificity and even atom location selectivity, as well as the reaction mechanism of the degradation process.


Assuntos
Heme/química , Proteólise , Heme/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Mycobacterium tuberculosis/efeitos dos fármacos
9.
Biomol Concepts ; 11(1): 32-56, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32187011

RESUMO

The inefficiency of cyanide/HCN (CN) binding with heme proteins (under physiological regimes) is demonstrated with an assessment of thermodynamics, kinetics, and inhibition constants. The acute onset of toxicity and CN's mg/Kg LD50 (µM lethal concentration) suggests that the classical hemeFe binding-based inhibition rationale is untenable to account for the toxicity of CN. In vitro mechanistic probing of CN-mediated inhibition of hemeFe reductionist systems was explored as a murburn model for mitochondrial oxidative phosphorylation (mOxPhos). The effect of CN in haloperoxidase catalyzed chlorine moiety transfer to small organics was considered as an analogous probe for phosphate group transfer in mOxPhos. Similarly, inclusion of CN in peroxidase-catalase mediated one-electron oxidation of small organics was used to explore electron transfer outcomes in mOxPhos, leading to water formation. The free energy correlations from a Hammett study and IC50/Hill slopes analyses and comparison with ligands ( CO/ H 2 S/ N 3 - ) $\left( {\text{CO}}/{{{{\text{H}}_{2}}\text{S}}/{\text{N}_{3}^{\text{-}}}\;}\; \right)$ provide insights into the involvement of diffusible radicals and proton-equilibriums, explaining analogous outcomes in mOxPhos chemistry. Further, we demonstrate that superoxide (diffusible reactive oxygen species, DROS) enables in vitro ATP synthesis from ADP+phosphate, and show that this reaction is inhibited by CN. Therefore, practically instantaneous CN ion-radical interactions with DROS in matrix catalytically disrupt mOxPhos, explaining the acute lethal effect of CN.


Assuntos
Cianetos/toxicidade , Heme/química , Hemeproteínas/antagonistas & inibidores , Hemoglobinas/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Catalase/metabolismo , Catálise , Respiração Celular/efeitos dos fármacos , Respiração Celular/fisiologia , Cloreto Peroxidase/química , Cianetos/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/antagonistas & inibidores , Heme/metabolismo , Hemeproteínas/química , Hemeproteínas/metabolismo , Hemoglobinas/química , Peroxidase do Rábano Silvestre/metabolismo , Hidróxidos/química , Cinética , Ligantes , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Estirenos/química , Estirenos/farmacologia , Superóxidos/química , Termodinâmica
10.
J Phys Chem Lett ; 11(8): 2824-2829, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32202796

RESUMO

The heme ATP-binding cassette (ABC) transporter BhuUV-T of bacterial pathogen Burkholderia cenocepacia is required to transport heme across the inner cell membrane. The current hypothesis is that the binding of two ATPs to the nucleotide-binding domains of the transporter drives the initial steps of the transport cycle in which the empty transport sites are reoriented from the cytosol to the periplasm. Molecular details are missing because the structure of a key occluded intermediate remains hypothetical. Here we perform molecular simulations to analyze the free energy surface (FES) of the first step of the reorientation, namely the transition from an open inward-facing (IF) transport site to an occluded (Occ) conformation. We have modeled the latter structure in silico in a previous study. A simple annealing procedure removes residual bias originating from non-equilibrium targeted molecular dynamics. The calculated FES reveals the role of the ATPs in inducing the IF → Occ conformational change and validates the modeled Occ conformation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Burkholderia cenocepacia/metabolismo , Biologia Computacional/métodos , Heme/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Burkholderia cenocepacia/química , Heme/química , Conformação Proteica , Estrutura Secundária de Proteína
11.
Proc Natl Acad Sci U S A ; 117(12): 6484-6490, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152099

RESUMO

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation.


Assuntos
Elétrons , Metaloproteínas/química , Prótons , Ascorbato Peroxidases/química , Ascorbato Peroxidases/metabolismo , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Catálise , Heme/química , Ligação de Hidrogênio , Metaloproteínas/metabolismo , Modelos Moleculares , Difração de Nêutrons , Oxirredução
12.
Biochem J ; 477(6): 1123-1136, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32141496

RESUMO

Bacterial heme nitric oxide/oxygen (H-NOX) domains are nitric oxide (NO) or oxygen sensors. This activity is mediated through binding of the ligand to a heme cofactor. However, H-NOX from Vibrio cholerae (Vc H-NOX) can be easily purified in a heme-free state that is capable of reversibly responding to oxidation, suggesting a heme-independent function as a redox sensor. This occurs by oxidation of Cys residues at a zinc-binding site conserved in a subset of H-NOX homologs. Remarkably, zinc is not lost from the protein upon oxidation, although its ligation environment is significantly altered. Using a combination of computational and experimental approaches, we have characterized localized structural changes that accompany the formation of specific disulfide bonds between Cys residues upon oxidation. Furthermore, the larger-scale structural changes accompanying oxidation appear to mimic those changes observed upon NO binding to the heme-bound form. Thus, Vc H-NOX and its homologs may act as both redox and NO sensors by completely separate mechanisms.


Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/fisiologia , Biologia Computacional/métodos , Cristalografia por Raios X , Heme/química , Heme/genética , Óxido Nítrico/química , Óxido Nítrico/genética , Estrutura Secundária de Proteína , Vibrio cholerae/química , Vibrio cholerae/genética
13.
Molecules ; 25(3)2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32046297

RESUMO

Aromatase is an enzyme member of the cytochrome P450 superfamily coded by the CYP19A1 gene. Its main action is the conversion of androgens into estrogens, transforming androstenedione into estrone and testosterone into estradiol. This enzyme is present in several tissues and it has a key role in the maintenance of the balance of androgens and estrogens, and therefore in the regulation of the endocrine system. With regard to chemical safety and human health, azoles, which are used as agrochemicals and pharmaceuticals, are potential endocrine disruptors due to their agonist or antagonist interactions with the human aromatase enzyme. This theoretical study investigated the active agonist and antagonist properties of "chemical classes of azoles" to determine the relationships of azole interaction with CYP19A1, using stereochemical and electronic properties of the molecules through classification and multilinear regression (MLR) modeling. The antagonist activities for the same substituent on diazoles and triazoles vary with its chemical composition and its position and both heterocyclic systems require aromatic substituents. The triazoles require the spherical shape and diazoles have to be in proper proportion of the branching index and the number of ring systems for the inhibition. Considering the electronic aspects, triazole antagonist activity depends on the electrophilicity index that originates from interelectronic exchange interaction (ωHF) and the LUMO energy ( E LUMO PM 7 ), and the diazole antagonist activity originates from the penultimate orbital ( E HOMONL PM 7 ) of diazoles. The regression models for agonist activity show that it is opposed by the static charges but favored by the delocalized charges on the diazoles and thiazoles. This study proposes that the electron penetration of azoles toward heme group decides the binding behavior and stereochemistry requirement for antagonist activity against CYP19A1 enzyme.


Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/química , Azóis/farmacologia , Indutores das Enzimas do Citocromo P-450/farmacologia , Elétrons , Disruptores Endócrinos/farmacologia , Modelos Estatísticos , Aromatase/metabolismo , Inibidores da Aromatase/química , Azóis/química , Indutores das Enzimas do Citocromo P-450/química , Disruptores Endócrinos/química , Heme/química , Heme/metabolismo , Humanos , Modelos Químicos , Ligação Proteica , Teoria Quântica , Eletricidade Estática , Estereoisomerismo , Relação Estrutura-Atividade , Termodinâmica
14.
Biochemistry ; 59(9): 1038-1050, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32058707

RESUMO

The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, ß-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Proteínas de Bactérias/química , Benzoatos/metabolismo , Sítios de Ligação , Heme/química , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica/fisiologia , Rodopseudomonas/enzimologia , Rodopseudomonas/metabolismo , Especificidade por Substrato
15.
Proc Natl Acad Sci U S A ; 117(9): 4741-4748, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071219

RESUMO

Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial. Here we captured the CO migration processes in the tense (T), relaxed (R), and second relaxed (R2) quaternary structures of human hemoglobin by crystallography using a high-repetition pulsed laser technique at cryogenic temperatures. We found that in each quaternary structure, the photodissociated CO molecules migrate along distinct pathways in the α and ß subunits by hopping between the internal cavities with correlated side chain motions of large nonpolar residues, such as α14Trp(A12), α105Leu(G12), ß15Trp(A12), and ß71Phe(E15). We also observe electron density evidence for the distal histidine [α58/ß63His(E7)] swing-out motion regardless of the quaternary structure, although less evident in α subunits than in ß subunits, suggesting that some CO molecules have escaped directly through the E7 gate. Remarkably, in T-state Fe(II)-Ni(II) hybrid hemoglobins in which either the α or ß subunits contain Ni(II) heme that cannot bind CO, the photodissociated CO molecules not only dock at the cavities in the original Fe(II) subunit, but also escape from the protein matrix and enter the cavities in the adjacent Ni(II) subunit even at 95 K, demonstrating the high gas permeability and porosity of the hemoglobin molecule. Our results provide a comprehensive picture of ligand movements in hemoglobin and highlight the relevance of cavities, nonpolar residues, and distal histidines in facilitating the ligand migration.


Assuntos
Hemoglobinas/química , Hemoglobinas/metabolismo , Monóxido de Carbono/metabolismo , Cristalografia por Raios X , Difusão , Heme/química , Histidina/química , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão
16.
Nat Commun ; 11(1): 864, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054833

RESUMO

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD+-dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of siroheme synthesis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Heme/análogos & derivados , Metiltransferases/química , Metiltransferases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Eletroquímica , Ferroquelatase/química , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/biossíntese , Heme/química , Metiltransferases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade por Substrato , Tetrapirróis/química , Tetrapirróis/metabolismo , Uroporfirinas/química , Uroporfirinas/metabolismo
17.
Nat Commun ; 11(1): 657, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005876

RESUMO

To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). We achieve submicron jets with speeds exceeding 160 m s-1, which allows for the use of megahertz XFEL repetition rates. By integrating an additional orifice, we implement a low consumption flow-focusing nozzle, which is validated by solving a hemoglobin structure. Also, aberration-free in operando X-ray microtomography is introduced to study efficient equivolumetric millisecond mixing in channels with 3D features integrated into the nozzle. Such devices can be printed in minutes by locally adjusting print resolution during fabrication. This technology has the potential to permit ultracompact devices and performance improvements through 3D flow optimization in all fields of microfluidic engineering.


Assuntos
Microfluídica/instrumentação , Impressão Tridimensional/instrumentação , Biologia Sintética/instrumentação , Heme/química , Hemoglobinas/química , Humanos , Lasers , Microfluídica/métodos , Biologia Sintética/métodos , Microtomografia por Raio-X
18.
Chem Biol Interact ; 318: 108973, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32035862

RESUMO

Carbon monoxide (CO) is an inorganic chemical compound that can bind with hemoglobin with highly toxic effects. In living organisms, it is produced endogenously during the degradation of heme by oxygenase, which occurs in three isoforms: HO-1, HO-2 and HO-3. CO can play an important role in the regulation of many physiological functions. Carbon Oxide Releasing Molecules (CORMs) are a novel group of chemical compounds capable of controlled CO release directly in tissues or organs. This release depends on concentration, pH, solvent type and temperature. The biological role and the therapeutic potential of different CORMs is not always well demonstrated. However, this mini review summarizes the various function of these compounds.


Assuntos
Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Animais , Monóxido de Carbono/toxicidade , Heme/química , Heme/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Isoenzimas
19.
J Med Chem ; 63(3): 1415-1433, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-31965799

RESUMO

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV-visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme-clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.


Assuntos
Clobetasol/metabolismo , Clobetasol/farmacologia , Inibidores do Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A/metabolismo , Heme/metabolismo , Linhagem Celular Tumoral , Clobetasol/química , Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/química , Ensaios Enzimáticos , Heme/química , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica
20.
Proc Natl Acad Sci U S A ; 117(2): 872-876, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888984

RESUMO

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.


Assuntos
Bacillus subtilis/metabolismo , Cobre/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Hidroquinonas/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Grupo dos Citocromos b/química , Transporte de Elétrons , Ligação de Hidrogênio , Modelos Moleculares , Naftóis/metabolismo , Oxirredutases , Conformação Proteica , Subunidades Proteicas/química , Bombas de Próton/química , Bombas de Próton/metabolismo , Terpenos/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K 2/química
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