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1.
Proc Natl Acad Sci U S A ; 117(45): 28036-28045, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33106422

RESUMO

Photosynthetic O2 evolution is catalyzed by the Mn4CaO5 cluster of the water oxidation complex of the photosystem II (PSII) complex. The photooxidative self-assembly of the Mn4CaO5 cluster, termed photoactivation, utilizes the same highly oxidizing species that drive the water oxidation in order to drive the incorporation of Mn2+ into the high-valence Mn4CaO5 cluster. This multistep process proceeds with low quantum efficiency, involves a molecular rearrangement between light-activated steps, and is prone to photoinactivation and misassembly. A sensitive polarographic technique was used to track the assembly process under flash illumination as a function of the constituent Mn2+ and Ca2+ ions in genetically engineered membranes of the cyanobacterium Synechocystis sp. PCC6803 to elucidate the action of Ca2+ and peripheral proteins. We show that the protein scaffolding organizing this process is allosterically modulated by the assembly protein Psb27, which together with Ca2+ stabilizes the intermediates of photoactivation, a feature especially evident at long intervals between photoactivating flashes. The results indicate three critical metal-binding sites: two Mn and one Ca, with occupation of the Ca site by Ca2+ critical for the suppression of photoinactivation. The long-observed competition between Mn2+ and Ca2+ occurs at the second Mn site, and its occupation by competing Ca2+ slows the rearrangement. The relatively low overall quantum efficiency of photoactivation is explained by the requirement of correct occupancy of these metal-binding sites coupled to a slow restructuring of the protein ligation environment, which are jointly necessary for the photooxidative trapping of the first stable assembly intermediate.


Assuntos
Cálcio/metabolismo , Oxigênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Água/metabolismo , Manganês/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema II/química , Conformação Proteica , Synechocystis/metabolismo , Tilacoides/química , Tilacoides/metabolismo
2.
Nat Commun ; 11(1): 4557, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917908

RESUMO

Why metalloenzymes often show dramatic changes in their catalytic activity when subjected to chemically similar but non-native metal substitutions is a long-standing puzzle. Here, we report on the catalytic roles of metal ions in a model metalloenzyme system, human carbonic anhydrase II (CA II). Through a comparative study on the intermediate states of the zinc-bound native CA II and non-native metal-substituted CA IIs, we demonstrate that the characteristic metal ion coordination geometries (tetrahedral for Zn2+, tetrahedral to octahedral conversion for Co2+, octahedral for Ni2+, and trigonal bipyramidal for Cu2+) directly modulate the catalytic efficacy. In addition, we reveal that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring water network in the active site. Our study provides evidence that the metal ions in metalloenzymes have a crucial impact on the catalytic mechanism beyond their primary chemical properties.


Assuntos
Anidrases Carbônicas/química , Íons/química , Metaloproteínas/química , Metais/química , Sítios de Ligação , Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Anidrases Carbônicas/metabolismo , Catálise , Domínio Catalítico , Cobalto/química , Cobre/química , Cristalografia por Raios X , Humanos , Íons/metabolismo , Cinética , Metaloproteínas/metabolismo , Metais/metabolismo , Modelos Moleculares , Níquel/química , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Zinco/química
3.
Inorg Chem ; 59(16): 11248-11252, 2020 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-32799485

RESUMO

Titanium is one of the most abundant elements on Earth but is commonly thought to have no role in biology because of its propensity to hydrolyze. Nature stabilizes hard Lewis acidic metals from hydrolysis using a variety of mechanisms, providing inspiration for how titanium can be stabilized using biological ligands. The well-characterized Due Ferri single-chain (DFsc) de novo designed protein was developed to bind and stabilize iron and provides a binding site with hard Lewis basic residues able to bind two metal ions. We demonstrate that the DFsc scaffold stably binds 2 equiv of titanium and protects them from unwanted hydrolysis. The Ti4+-DFsc protein complex was tested for its ability to hydrolytically cleave DNA, where it was seen to linearize plasmid DNA in an overnight reaction. Ti4+-DFsc is thus the first example of a functional, soluble titanium-protein complex.


Assuntos
DNA/química , Metaloproteínas/química , Titânio/química , Domínio Catalítico , Hidrólise , Estudo de Prova de Conceito
4.
Inorg Chem ; 59(16): 11514-11527, 2020 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-32799489

RESUMO

The nitrogenase enzymes are responsible for all biological nitrogen reduction. How this is accomplished at the atomic level, however, has still not been established. The molybdenum-dependent nitrogenase has been extensively studied and is the most active catalyst for dinitrogen reduction of the nitrogenase enzymes. The vanadium-dependent form, on the other hand, displays different reactivity, being capable of CO and CO2 reduction to hydrocarbons. Only recently did a crystal structure of the VFe protein of vanadium nitrogenase become available, paving the way for detailed theoretical studies of the iron-vanadium cofactor (FeVco) within the protein matrix. The crystal structure revealed a bridging 4-atom ligand between two Fe atoms, proposed to be either a CO32- or NO3- ligand. Using a quantum mechanics/molecular mechanics model of the VFe protein, starting from the 1.35 Å crystal structure, we have systematically explored multiple computational models for FeVco, considering either a CO32- or NO3- ligand, three different redox states, and multiple broken-symmetry states. We find that only a [VFe7S8C(CO3)]2- model for FeVco reproduces the crystal structure of FeVco well, as seen in a comparison of the Fe-Fe and V-Fe distances in the computed models. Furthermore, a broken-symmetry solution with Fe2, Fe3, and Fe5 spin-down (BS7-235) is energetically preferred. The electronic structure of the [VFe7S8C(CO3)]2- BS7-235 model is compared to our [MoFe7S9C]- BS7-235 model of FeMoco via localized orbital analysis and is discussed in terms of local oxidation states and different degrees of delocalization. As previously found from Fe X-ray absorption spectroscopy studies, the Fe part of FeVco is reduced compared to FeMoco, and the calculations reveal Fe5 as locally ferrous. This suggests resting-state FeVco to be analogous to an unprotonated E1 state of FeMoco. Furthermore, V-Fe interactions in FeVco are not as strong compared to Mo-Fe interactions in FeMoco. These clear differences in the electronic structures of otherwise similar cofactors suggest an explanation for distinct differences in reactivity.


Assuntos
Metaloproteínas/química , Nitrogenase/química , Teoria Quântica , Catálise , Cristalografia por Raios X , Ligantes , Conformação Proteica
5.
Nat Struct Mol Biol ; 27(8): 735-742, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32632277

RESUMO

The cytosolic iron-sulfur (Fe-S) assembly (CIA) pathway is required for the insertion of Fe-S clusters into cytosolic and nuclear client proteins, including many DNA replication and repair factors. The molecular mechanisms of client protein recognition and Fe-S cluster transfer remain unknown. Here, we report crystal structures of the CIA targeting complex (CTC), revealing that its CIAO2B subunit is centrally located and bridges CIAO1 and the client adaptor protein MMS19. Cryo-EM reconstructions of human CTC bound either to the DNA replication factor primase or to the DNA helicase DNA2, combined with biochemical, biophysical and yeast complementation assays, reveal an evolutionarily conserved, bipartite client recognition mode facilitated by CIAO1 and the structural flexibility of the MMS19 subunit. Unexpectedly, the primase Fe-S cluster is located ~70 Å away from the CTC reactive cysteine, implicating conformational dynamics of the CTC or additional maturation factors in the mechanism of Fe-S cluster transfer.


Assuntos
Proteínas com Ferro-Enxofre/metabolismo , Metalochaperonas/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Drosophila , Células HEK293 , Humanos , Proteínas com Ferro-Enxofre/química , Metalochaperonas/química , Metaloproteínas/química , Camundongos , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/química
6.
Nat Commun ; 11(1): 2738, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483131

RESUMO

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal's redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.


Assuntos
Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Metaloproteínas/metabolismo , Staphylococcus aureus/enzimologia , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Evolução Molecular , Ferro/química , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Manganês/química , Metaloproteínas/química , Metaloproteínas/genética , Mutação , Oxirredução , Filogenia , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/genética , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxidos/metabolismo
7.
Environ Microbiol ; 22(6): 2007-2026, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32239579

RESUMO

The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5'-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years.


Assuntos
Coenzimas/biossíntese , Escherichia coli/metabolismo , Metaloproteínas/biossíntese , Coenzimas/química , Metaloproteínas/química , Molibdênio/metabolismo , Compostos Organofosforados , Pteridinas/química , Pterinas/química
8.
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
9.
Sci Rep ; 10(1): 3774, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111964

RESUMO

Hydrogen has the potential to play an important role in decarbonising our energy systems. Crucial to achieving this is the ability to produce clean sources of hydrogen using renewable energy sources. Currently platinum is commonly used as a hydrogen evolution catalyst, however, the scarcity and expense of platinum is driving the need to develop non-platinum-based catalysts. Here we report a protein-based hydrogen evolution catalyst based on a recombinant silk protein from honeybees and a metal macrocycle, cobalt protoporphyrin (CoPPIX). We enhanced the hydrogen evolution activity three fold compared to the unmodified silk protein by varying the coordinating ligands to the metal centre. Finally, to demonstrate the use of our biological catalyst, we built a proton exchange membrane (PEM) water electrolysis cell using CoPPIX-silk as the hydrogen evolution catalyst that is able to produce hydrogen with a 98% Faradaic efficiency. This represents an exciting advance towards allowing protein-based catalysts to be used in electrolysis cells.


Assuntos
Abelhas/química , Hidrogênio/química , Proteínas de Insetos/química , Metaloproteínas/química , Protoporfirinas/química , Seda/química , Animais , Abelhas/genética , Catálise , Proteínas de Insetos/genética , Metaloproteínas/genética , Engenharia de Proteínas , Protoporfirinas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Seda/genética
10.
Artigo em Inglês | MEDLINE | ID: mdl-32161212

RESUMO

In order to harness the functionality of metals, nature has evolved over billions of years to utilize metalloproteins as key components in numerous cellular processes. Despite this, transition metals such as ruthenium, palladium, iridium, and gold are largely absent from naturally occurring metalloproteins, likely due to their scarcity as precious metals. To mimic the evolutionary process of nature, the field of artificial metalloenzymes (ArMs) was born as a way to benefit from the unique chemoselectivity and orthogonality of transition metals in a biological setting. In its current state, numerous examples have successfully incorporated transition metals into a variety of protein scaffolds. Using these ArMs, many examples of new-to-nature reactions have been carried out, some of which have shown substantial biocompatibility. Given the rapid rate at which this field is growing, this review aims to highlight some important studies that have begun to take the next step within this field; namely the development of ArM-centered drug therapies or biotechnological tools.


Assuntos
Materiais Biomiméticos/química , Coenzimas/química , Metaloproteínas/química , Metais/química , Biocatálise , Técnicas Biossensoriais , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Estereoisomerismo
11.
Biochemistry ; 59(12): 1289-1297, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32167292

RESUMO

Cobalt-mimochrome VI*a (CoMC6*a) is a synthetic mini-protein that catalyzes aqueous proton reduction to hydrogen (H2). In buffered water, there are multiple possible proton donors, complicating the elucidation of the mechanism. We have found that the buffer pKa and sterics have significant effects on activity, evaluated via cyclic voltammetry (CV). Protonated buffer is proposed to act as the primary proton donor to the catalyst, specifically through the protonated amine of the buffers that were tested. At a constant pH of 6.5, catalytic H2 evolution in the presence of buffer acids with pKa values ranging from 5.8 to 11.6 was investigated, giving rise to a potential-pKa relationship that can be divided into two regions. For acids with pKa values of ≤8.7, the half-wave catalytic potential (Eh) changes as a function of pKa with a slope of -128 mV/pKa unit, and for acids with pKa of ≥8.7, Eh changes as a function of pKa with a slope of -39 mV/pKa unit. In addition, a series of buffer acids were synthesized to explore the influence of steric bulk around the acidic proton on catalysis. The catalytic current in CV shows a significant decrease in the presence of the sterically hindered buffer acids compared to those of their parent compounds, also consistent with the added buffer acid acting as the primary proton donor to the catalyst and showing that acid structure in addition to pKa impacts activity. These results demonstrate that buffer acidity and structure are important considerations when optimizing and evaluating systems for proton-dependent catalysis in water.


Assuntos
Cobalto/química , Deuteroporfirinas/química , Hidrogênio/química , Metaloproteínas/química , Prótons , Tampões (Química) , Catálise , Concentração de Íons de Hidrogênio , Água/química
12.
Acta Crystallogr D Struct Biol ; 76(Pt 2): 176-192, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32038048

RESUMO

Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X-ray absorption edges, which shift to higher energy for progressively more positive valence numbers. Indeed, X-ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X-ray sources at synchrotron facilities with energy resolutions in the range 2-3 × 10-4 (ΔE/E). This paper tests whether X-ray free-electron laser (XFEL) pulses, which have a broader bandpass (ΔE/E = 3 × 10-3) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength-dependent anomalous scattering factors contributed by two differently charged iron centers in the 110-amino-acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X-ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.


Assuntos
Cristalografia por Raios X/métodos , Lasers , Metaloproteínas/química , Animais , Teorema de Bayes , Interpretação Estatística de Dados , Ferredoxinas/química , Humanos , Síncrotrons
13.
Nat Commun ; 11(1): 862, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054841

RESUMO

Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.


Assuntos
Materiais Revestidos Biocompatíveis/química , Metaloproteínas/química , Mytilus edulis/química , Estruturas Animais/anatomia & histologia , Estruturas Animais/química , Estruturas Animais/ultraestrutura , Animais , Di-Hidroxifenilalanina/química , Imageamento Tridimensional , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mytilus edulis/anatomia & histologia , Mytilus edulis/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Vesículas Secretórias/química , Vesículas Secretórias/ultraestrutura
14.
Photosynth Res ; 143(3): 301-314, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31933173

RESUMO

Auracyanin (Ac) is a blue copper protein that mediates the electron transfer between Alternative Complex III (ACIII) and downstream electron acceptors in both fort chains of filamentous anoxygenic phototrophs. Here, we extracted and purified the air-oxidized RfxAc from the photoheterotrophically grown Roseiflexus castenholzii, and we illustrated the structural basis underlying its electron transferring features. Spectroscopic and enzymatic analyses demonstrated the reduction of air-oxidized RfxAc by the ACIII upon oxidation of menaquinol-4 and menaquinol-7. Crystal structures of the air-oxidized and Na-dithionite-reduced RfxAc at 2.2 and 2.0 Å resolutions, respectively, showed that the copper ions are coordinated by His77, His146, Cys141, and Met151 in minor different geometries. The Cu1-Sδ bond length increase of Met151, and the electron density Fourier differences at Cu1 and His77 demonstrated their essential roles in the dithionite-induced reduction. Structural comparisons further revealed that the RfxAc contains a Chloroflexus aurantiacus Ac-A-like copper binding pocket and a hydrophobic patch surrounding the exposed edge of His146 imidazole, as well as an Ac-B-like Ser- and Thr-rich polar patch located at a different site on the surface. These spectroscopic and structural features allow RfxAc to mediate electron transfers between the ACIII and redox partners different from those of Ac-A and Ac-B. These results provide a structural basis for further investigating the electron transfer and energy transformation mechanism of bacterial photosynthesis, and the diversity and evolution of electron transport chains.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chloroflexi/metabolismo , Cobre/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Fotossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Cobre/química , Ditionita/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Metaloproteínas/isolamento & purificação , Modelos Moleculares , Naftóis/farmacologia , Oxirredução , Fotossíntese/efeitos dos fármacos , Solventes/química , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
15.
Nat Commun ; 11(1): 316, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949281

RESUMO

Here we propose an experimental setup based on operando X-ray absorption spectroscopy (XAS) to understand why copper-containing oxidoreductase enzymes show exceptional performance as catalysts for the oxygen reduction reaction (ORR). An electrode based on carbon nanoparticles organized in mesoporous structures with bilirubin oxidase (BOD) was developed to be used in a home-made operando XAS electrochemical cell, and we probed the electron transfer under ORR regime. In the presence of molecular oxygen, the BOD cofactor containing 4 copper ions require an overpotential about 150 mV to be reduced as compared to that in the absence of oxygen. A second electron transfer step, which occurs faster than the cofactor reduction, suggests that the cooper ions act as a tridimensional redox active electronic bridges for the electron transfer reaction.


Assuntos
Cobre/química , Transporte de Elétrons , Elétrons , Metaloproteínas/química , Oxirredutases/química , Espectroscopia por Absorção de Raios X/métodos , Catálise , Eletrodos , Modelos Químicos , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxigênio/química , Saccharomycetales
16.
Inorg Chem ; 59(1): 790-800, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31829577

RESUMO

Investigation of the diverse evolutionary developed mechanisms enabling bacteria to maintain homeostasis and to be resistant to lead is crucial for the discovery of novel strategies for isolation of this highly toxic metal and its subsequent elimination from contaminated environments. The metalloregulatory protein pbrR and its homologues that were identified in the Cupriavidus metallidurans CH34 chromosome are the only characterized natural metalloproteins that have a special affinity toward Pb(II) and that bind it with at least a 1000-fold selectivity over other heavy metals. The X-ray structures of apo and Pb(II)-bound pbrR have been recently reported. In the present study, the binding of Pb(II) at pbrR was investigated by means of multiscale computational modeling. Molecular dynamics simulations substantiated how conformations amenable for the Pb(II) complexation through the tris-cysteine motif are formed from the antiparallel coiled-coil packing interaction of two dimerization helices of two pbrR monomers, and the phase space of apo-pbrR has been extensively sampled. Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on metal-bound structures of pbrR also allowed us to determine the most probable protonation state for the lead binding motif and evaluate the structural features mostly affecting the Pb(II) coordination in this protein. In agreement with available experimental data, we found that pbrR may control its Pb(II) affinity, probably, by conformational changes that affect the distance between Cys78' and Cys122 and their protonation states, thus being able to switch on the Pb(II) sequestration/release-prone states in response to external stimuli. The protein structure enveloping the metal binding motif favors the thiol-thiolate-thiolate protonation state of Pb(II)-pbrR, thus probably enhancing the binding selectivity for Pb(II), compared to other metal ions.


Assuntos
Cupriavidus/química , Chumbo/análise , Metaloproteínas/química , Simulação de Dinâmica Molecular , Teoria Quântica
17.
Inorg Chem ; 59(1): 214-225, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31814403

RESUMO

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.


Assuntos
Coenzimas/química , Formiato Desidrogenases/química , Metaloproteínas/química , Pteridinas/química , Rhodobacter capsulatus/enzimologia , Ânions/química , Ânions/metabolismo , Sítios de Ligação , Coenzimas/metabolismo , Formiato Desidrogenases/isolamento & purificação , Formiato Desidrogenases/metabolismo , Metaloproteínas/metabolismo , Oxirredução , Pteridinas/metabolismo , Espectroscopia por Absorção de Raios X
18.
Chemistry ; 26(1): 249-258, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31710732

RESUMO

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper-binding scaffolds: H3 (His3 ), H4 (His4 ), H2 DH (His3 Asp with two His and one Asp in the same plane) and H3 D (His3 Asp with three His in the same plane) by using the scaffold of the de novo protein GRα3 D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good CuII -bound structural mimics of Cu-only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.


Assuntos
Cobre/química , Metaloproteínas/química , Peptídeos/química , Sequência de Aminoácidos , Cobre/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Ensaios Enzimáticos , Metaloproteínas/metabolismo , Peptídeos/metabolismo , Estabilidade Proteica , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Temperatura , Termodinâmica
19.
Biochim Biophys Acta Gen Subj ; 1864(2): 129466, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31678142

RESUMO

BACKGROUND: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. SCOPE OF REVIEW: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. MAJOR CONCLUSIONS: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. GENERAL SIGNIFICANCE: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Lasers , Substâncias Macromoleculares/química , Metaloproteínas/química , Animais , Carboidratos/química , Bovinos , Cristalografia por Raios X , Cianobactérias , Dimerização , Ligantes , Conformação Molecular , Oxirredutases/química , Fotólise , Complexo de Proteína do Fotossistema II/química , Plantas/enzimologia , Raios X
20.
Biochim Biophys Acta Bioenerg ; 1861(1): 148118, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734195

RESUMO

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH-. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.


Assuntos
Aldeído Oxirredutases/química , Bradyrhizobiaceae/enzimologia , Metaloproteínas/química , Complexos Multienzimáticos/química , Aldeído Oxirredutases/metabolismo , Catálise , Domínio Catalítico , Cobalto/química , Cobalto/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Metaloproteínas/metabolismo , Molibdênio/química , Molibdênio/metabolismo , Complexos Multienzimáticos/metabolismo
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