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1.
Extremophiles ; 25(5-6): 513-526, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34647163

RESUMO

Thermoanaerobacter kivui is an acetogenic model organism that reduces CO2 with electrons derived from H2 or CO, or from organic substrates in the Wood-Ljugdahl pathway (WLP). For the calculation of ATP yields, it is necessary to know the electron carriers involved in coupling of the oxidative and reductive parts of metabolism. Analyses of key catabolic oxidoreductases in cell-free extract (CFE) or with purified enzymes revealed the physiological electron carriers involved. The glyceraldehyde-3-phosphate dehydrogenase (GA3P-DH) assayed in CFE was NAD+-specific, NADP+ was used with less than 4% and ferredoxin (Fd) was not used. The methylene-THF dehydrogenase was NADP+-specific, NAD+ or Fd were not used. A Nfn-type transhydrogenase that catalyzes reduced Fd-dependent reduction of NADP+ with NADH as electron donor was also identified in CFE. The electron carriers used by the potential electron-bifurcating hydrogenase (HydABC) could not be unambiguously determined in CFE for technical reasons. Therefore, the enzyme was produced homologously in T. kivui and purified by affinity chromatography. HydABC contained 33.9 ± 4.5 mol Fe/mol of protein and FMN; it reduced NADP+ but not NAD+. The methylene-THF reductase (MetFV) was also produced homologously in T. kivui and purified by affinity chromatography. MetFV contained 7.2 ± 0.4 mol Fe/mol of protein and FMN; the complex did neither use NADPH nor NADH as reductant but only reduced Fd. In sum, these analysis allowed us to propose a scheme for entire electron flow and bioenergetics in T. kivui.


Assuntos
Elétrons , Hidrogenase , Processos Autotróficos , Hidrogenase/metabolismo , NAD/metabolismo , NADP , Oxirredução , Thermoanaerobacter/metabolismo
2.
Metab Eng ; 68: 199-209, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34673236

RESUMO

Molecular hydrogen (H2) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H2 production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H2 syntheses rely mainly on light, water, and CO2 and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H2-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O2) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O2-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O2-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O2 sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O2 tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH+ to utilize H2 as sole electron source to fix CO2. Syn_ReSH+ also was able to produce H2 under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H2 production.


Assuntos
Hidrogenase , Synechocystis , Hidrogênio , Hidrogenase/genética , Oxigênio , Fotossíntese , Synechocystis/genética , Synechocystis/metabolismo
3.
Nat Commun ; 12(1): 5281, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489402

RESUMO

The archaeal phylum Woesearchaeota, within the DPANN superphylum, includes phylogenetically diverse microorganisms that inhabit various environments. Their biology is poorly understood due to the lack of cultured isolates. Here, we analyze datasets of Woesearchaeota 16S rRNA gene sequences and metagenome-assembled genomes to infer global distribution patterns, ecological preferences and metabolic capabilities. Phylogenomic analyses indicate that the phylum can be classified into ten subgroups, termed A-J. While a symbiotic lifestyle is predicted for most, some members of subgroup J might be host-independent. The genomes of several Woesearchaeota, including subgroup J, encode putative [FeFe] hydrogenases (known to be important for fermentation in other organisms), suggesting that these archaea might be anaerobic fermentative heterotrophs.


Assuntos
Archaea/genética , Proteínas Arqueais/genética , Genoma Arqueal , Hidrogenase/genética , RNA Arqueal/genética , RNA Ribossômico 16S/genética , Sequência de Aminoácidos , Anaerobiose/genética , Archaea/classificação , Archaea/enzimologia , Proteínas Arqueais/metabolismo , Evolução Biológica , Fermentação , Processos Heterotróficos/genética , Hidrogenase/metabolismo , Metagenoma , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
4.
Biochemistry ; 60(40): 3016-3026, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34569243

RESUMO

The [FeFe] hydrogenase catalyzes the redox interconversion of protons and H2 with a Fe-S "H-cluster" employing CO, CN, and azadithiolate ligands to two Fe centers. The biosynthesis of the H-cluster is a highly interesting reaction carried out by a set of Fe-S maturase enzymes called HydE, HydF, and HydG. HydG, a member of the radical S-adenosylmethionine (rSAM) family, converts tyrosine, cysteine, and Fe(II) into an organometallic Fe(II)(CO)2(CN)cysteine "synthon", which serves as the substrate for HydE. Although key aspects of the HydG mechanism have been experimentally determined via isotope-sensitive spectroscopic methods, other important mechanistic questions have eluded experimental determination. Here, we use computational quantum chemistry to refine the mechanism of the HydG catalytic reaction. We utilize quantum mechanics/molecular mechanics simulations to investigate the reactions at the canonical Fe-S cluster, where a radical cleavage of the tyrosine substrate takes place and proceeds through a relay of radical intermediates to form HCN and a COO•- radical anion. We then carry out a broken-symmetry density functional theory study of the reactions at the unusual five-iron auxiliary Fe-S cluster, where two equivalents of CN- and COOH• coordinate to the fifth "dangler iron" in a series of substitution and redox reactions that yield the synthon as the final product for further processing by HydE.


Assuntos
Proteínas de Bactérias/química , Complexos de Coordenação/química , Cisteína/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Biocatálise , Ferro/química , Ligantes , Modelos Químicos , Teoria Quântica , Thermoanaerobacter/enzimologia , Tirosina/química
5.
Nat Commun ; 12(1): 5092, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429430

RESUMO

Development of a versatile, sustainable and efficient photosynthesis system that integrates intricate catalytic networks and energy modules at the same location is of considerable future value to energy transformation. In the present study, we develop a coenzyme-mediated supramolecular host-guest semibiological system that combines artificial and enzymatic catalysis for photocatalytic hydrogen evolution from alcohol dehydrogenation. This approach involves modification of the microenvironment of a dithiolene-embedded metal-organic cage to trap an organic dye and NADH molecule simultaneously, serving as a hydrogenase analogue to induce effective proton reduction inside the artificial host. This abiotic photocatalytic system is further embedded into the pocket of the alcohol dehydrogenase to couple enzymatic alcohol dehydrogenation. This host-guest approach allows in situ regeneration of NAD+/NADH couple to transfer protons and electrons between the two catalytic cycles, thereby paving a unique avenue for a synergic combination of abiotic and biotic synthetic sequences for photocatalytic fuel and chemical transformation.


Assuntos
Etanol/química , Fotossíntese/fisiologia , Luz Solar , Catálise , Corantes , Elétrons , Hidrogênio/química , Hidrogenase/química , Cinética , Ligantes , Simulação de Acoplamento Molecular
6.
Angew Chem Int Ed Engl ; 60(38): 20744-20747, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34324230

RESUMO

Azadithiolate, a cofactor found in all [FeFe]-hydrogenases, is shown to undergo acid-catalyzed rearrangement. Fe2 [(SCH2 )2 NH](CO)6 self-condenses to give Fe6 [(SCH2 )3 N]2 (CO)17 . The reaction, which is driven by loss of NH4 + , illustrates the exchange of the amine group. X-ray crystallography reveals that three Fe2 (SR)2 (CO)x butterfly subunits interconnected by the aminotrithiolate [N(CH2 S)3 ]3- . Mechanistic studies reveal that Fe2 [(SCH2 )2 NR](CO)6 participate in a range of amine exchange reactions, enabling new methodologies for modifying the adt cofactor. Ru2 [(SCH2 )2 NH](CO)6 also rearranges, but proceeds further to give derivatives with Ru-alkyl bonds Ru6 [(SCH2 )3 N][(SCH2 )2 NCH2 ]S(CO)17 and [Ru2 [(SCH2 )2 NCH2 ](CO)5 ]2 S.


Assuntos
Compostos Aza/metabolismo , Complexos de Coordenação/metabolismo , Hidrogenase/metabolismo , Rubídio/metabolismo , Tolueno/análogos & derivados , Compostos Aza/química , Complexos de Coordenação/química , Modelos Moleculares , Estrutura Molecular , Rubídio/química , Tolueno/química , Tolueno/metabolismo
7.
Elife ; 102021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34085924

RESUMO

The composition of gut-associated microbial communities changes during intestinal inflammation, including an expansion of Enterobacteriaceae populations. The mechanisms underlying microbiota changes during inflammation are incompletely understood. Here, we analyzed previously published metagenomic datasets with a focus on microbial hydrogen metabolism. The bacterial genomes in the inflamed murine gut and in patients with inflammatory bowel disease contained more genes encoding predicted hydrogen-utilizing hydrogenases compared to communities found under non-inflamed conditions. To validate these findings, we investigated hydrogen metabolism of Escherichia coli, a representative Enterobacteriaceae, in mouse models of colitis. E. coli mutants lacking hydrogenase-1 and hydrogenase-2 displayed decreased fitness during colonization of the inflamed cecum and colon. Utilization of molecular hydrogen was in part dependent on respiration of inflammation-derived electron acceptors. This work highlights the contribution of hydrogenases to alterations of the gut microbiota in the context of non-infectious colitis.


Assuntos
Ceco/microbiologia , Colite/induzido quimicamente , Colite/microbiologia , Colo/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/metabolismo , Microbioma Gastrointestinal , Hidrogênio/metabolismo , Animais , Ceco/metabolismo , Ceco/patologia , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Bases de Dados Genéticas , Sulfato de Dextrana , Modelos Animais de Doenças , Disbiose , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Humanos , Hidrogenase/genética , Hidrogenase/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Metagenoma , Metagenômica , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piroxicam
8.
J Hazard Mater ; 417: 126090, 2021 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-34020357

RESUMO

Due to the excellent hydrogen affinity and high conductivity, palladium nanoparticles (Pd NPs) were considered as a potential strategy to regulate bacterial electron transfer and energy metabolism. Herein, Citrobacter freundii JH, capable of in-situ biosynthesizing Pd(0) NPs, was employed to promote Pt(IV) reduction. The results showed that the Pt(IV) reduction to Pt(II) was accomplished mainly via the flavins-mediated extracellular electron transfer (EET) process, while Pt(II) reduction to Pt(0) was limit step, and proceeded via two intracellular respiratory chains, including FDH/Hases-based short chain (S-chain) and typical CoQ-involved long respiratory chain (L-chain). Noteworthily, the incorporation of Pd(0) NPs mainly diverted the electrons to S-chain (as high as 71.7%-73.4%) by improving the hydrogenases (Hases) activity. Furthermore, Pd(0) NPs could stimulate the secreting of flavins and the combination between flavins and cytochrome c (c-Cyt), which converted electron transfer manner of L-chain. Additionally, Pd(0) NPs might also act as alternative proton channels to improve the energy metabolism. These findings provided significant insights into the promotion by Pd(0) NPs in terms of electron generation, electron consumption and proton translocation.


Assuntos
Hidrogenase , Nanopartículas Metálicas , Transporte de Elétrons , Elétrons , Paládio
9.
Chem Commun (Camb) ; 57(33): 3952-3974, 2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33885698

RESUMO

Quantum chemical approaches today are a powerful tool to study the properties and reactivity of metalloenzymes. In the field of solar fuels research these involve predominantly photosystem II and hydrogenases, which catalyze water oxidation and hydrogen evolution, as well as related biomimetic and bio-inspired models. Theoretical methods are extensively used to better comprehend the nature of catalytic intermediates, establish important structure-function and structure-property correlations, elucidate functional principles, and uncover the catalytic activity of these complex systems by unravelling the key steps of their reaction mechanism. Computations in the field of water oxidation and hydrogen evolution are used as predictive tools to elucidate structures, explain and synthesize complex experimental observations from advanced spectroscopic techniques, rationalize reactivity on the basis of atomistic models and electronic structure, and guide the design of new synthetic targets. This feature article covers recent advances in the application of quantum chemical methods for understanding the nature of catalytic intermediates and the mechanism by which photosystem II and hydrogenases achieve their function, and points at essential questions that remain only partly answered and at challenges that will have to be met by future advances and applications of quantum and computational chemistry.


Assuntos
Materiais Biomiméticos/química , Complexos de Coordenação/química , Hidrogênio/química , Hidrogenase/química , Metaloproteínas/química , Complexo de Proteína do Fotossistema II/química , Catálise , Modelos Químicos , Oxirredução , Fotossíntese , Energia Solar , Relação Estrutura-Atividade , Luz Solar , Água
10.
J Biol Chem ; 296: 100710, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33930466

RESUMO

Pyrococcus furiosus is a hyperthermophilic anaerobic archaeon whose metabolism depends on whether elemental sulfur is (+S0) or is not (-S0) included in growth medium. Under +S0 conditions, expression of respiratory hydrogenase declines while respiratory membrane-bound sulfane reductase and the putative iron-storage protein IssA increase. Our objective was to investigate the iron content of WT and ΔIssA cells under these growth conditions using Mössbauer spectroscopy. WT-S0 cells contained ∼1 mM Fe, with ∼85% present as two spectroscopically distinct forms of S = 0 [Fe4S4]2+ clusters; the remainder was mainly high-spin FeII. WT+S0 cells contained 5 to 9 mM Fe, with 75 to 90% present as magnetically ordered thioferrate-like (TFL) iron nanoparticles. TFL iron was similar to chemically defined thioferrates; both consisted of FeIII ions coordinated by an S4 environment, and both exhibited strong coupling between particles causing high applied fields to have little spectral effect. At high temperatures with magnetic hyperfine interactions abolished, TFL iron exhibited two doublets overlapping those of [Fe4S4]2+ clusters in -S0 cells. This coincidence arose because of similar coordination environments of TFL iron and cluster iron. The TFL structure was more heterogeneous in the presence of IssA. Presented data suggest that IssA may coordinate insoluble iron sulfides as TFL iron, formed as a byproduct of anaerobic sulfur respiration under high iron conditions, which thereby reduces its toxicity to the cell. This was the first Mössbauer characterization of the ironome of an archaeon, and it illustrates differences relative to the iron content of better-studied bacteria such as Escherichia coli.


Assuntos
Ferro/metabolismo , Pyrococcus furiosus/metabolismo , Enxofre/metabolismo , Hidrogenase/metabolismo , Oxirredução
11.
Parasitol Res ; 120(6): 2189-2198, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33855618

RESUMO

Recent phylogenetic and morphologic studies of Trichomonas spp. suggests that there are more than 3 species that infect the upper alimentary tract of wild birds, which include T. gallinae, T. stableri, and T. gypaetinii. In this study, investigations were conducted on the prevalence of trichomonads in the upper alimentary tract of 12 Steller's sea eagles (Haliaeetus pelagicus) and 18 white-tailed sea eagles (H. albicilla). All birds were rescued from the wild and kept at a rehabilitation facility in Hokkaido, Japan, for variable durations and did not show any symptoms of trichomonosis. The ITS1-5.8SrRNA-ITS2 (ITS) genomic region of Trichomonas spp. was detected from 29 samples by PCR, and flagellates were confirmed from 4 samples by culture. Morphologic observations and measurement recordings were conducted under a light microscope on trophozoites obtained from the cultured isolates. Genomic sequences of the ITS, 18S ribosomal RNA (18S rRNA), Fe-hydrogenase, and RNA polymerase II largest subunit (Rpb1) regions were determined by direct sequencing, and phylogenetic analyses were conducted with previously published sequences of Trichomonas spp. All isolates were concluded as T. gypaetinii based on morphologic and molecular characterizations of the ITS and 18S rRNA genes. This is the first study to isolate T. gypaetinii from Haliaeetus eagles and further provide novel sequences of the Fe-hydrogenase and Rpb1 genes of T. gypaetinii. Both genomic regions also confirmed that T. gypaetinii belong to independent clusters from other Trichomonas spp.


Assuntos
Doenças das Aves/parasitologia , Águias/parasitologia , Tricomoníase/veterinária , Animais , Animais Selvagens/parasitologia , Doenças das Aves/epidemiologia , Águias/genética , Feminino , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Japão/epidemiologia , Masculino , Reação em Cadeia da Polimerase/veterinária , Trichomonas , Tricomoníase/epidemiologia , Tricomoníase/parasitologia
12.
ACS Nano ; 15(4): 6530-6539, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33844499

RESUMO

Molecular oxygen (O2) is a highly reactive oxidizing agent and is harmful to many biological and industrial systems. Although O2 often interacts via metals or reducing agents, a binding mechanism involving an organic supramolecular structure has not been described to date. In this work, the prominent dipeptide hydrogelator fluorenylmethyloxycarbonyl-diphenylalanine is shown to encage O2 and significantly limit its diffusion and penetration through the hydrogel. Molecular dynamics simulations suggested that the O2 binding mechanism is governed by pockets formed between the aromatic rings in the supramolecular structure of the gel, which bind O2 through hydrophobic interactions. This phenomenon is harnessed to maintain the activity of the O2-hypersensitive enzyme [FeFe]-hydrogenase, which holds promising potential for utilizing hydrogen gas for sustainable energy applications. Hydrogenase encapsulation within the gel allows hydrogen production following exposure to ambient O2. This phenomenon may lead to utilization of this low molecular weight gelator in a wide range of O2-sensitive applications.


Assuntos
Hidrogenase , Oxigênio , Hidrogéis , Hidrogênio , Peptídeos
13.
Physiol Plant ; 173(2): 555-567, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33860946

RESUMO

Photosynthetic production of molecular hydrogen (H2 ) by cyanobacteria and green algae is a potential source of renewable energy. These organisms are capable of water biophotolysis by taking advantage of photosynthetic apparatus that links water oxidation at Photosystem II and reduction of protons to H2 downstream of Photosystem I. Although the process has a theoretical potential to displace fossil fuels, photosynthetic H2 production in its current state is not yet efficient enough for industrial applications due to a number of physiological, biochemical, and engineering barriers. This article presents a short overview of the metabolic pathways and enzymes involved in H2 photoproduction in cyanobacteria and green algae and our present understanding of the mechanisms of this process. We also summarize recent advances in engineering photosynthetic cell factories capable of overcoming the major barriers to efficient and sustainable H2 production.


Assuntos
Clorófitas , Hidrogenase , Clorófitas/genética , Clorófitas/metabolismo , Hidrogênio , Hidrogenase/genética , Hidrogenase/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo
14.
Chemosphere ; 278: 130485, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33839391

RESUMO

Photocatalytic systems comprising a hydrogenase-type catalyst and CdX (X = S, Se, Te) chalcogenide quantum dot (QD) photosensitizers show extraordinary hydrogen production rates under visible light excitation. What remains unknown is the mechanism of energy conversion in these systems. Here, we have explored this question by comparing the performance of two QD sensitizers, CdSe and CdTe, in photocatalytic systems featuring aqueous suspensions of a [Fe2 (µ-1,2-benzenedithiolate) CO6] catalyst and an ascorbic acid sacrificial agent. Overall, the hydrogen production yield for CdSe-sensitized reactions QDs was found to be 13 times greater than that of CdTe counterparts. According to emission quenching experiments, an enhanced performance of CdSe sensitizers reflected a greater rate of electron transfer from the ascorbic acid (kAsc). The observed difference in the QD-ascorbic acid charge transfer rates between the two QD materials was consistent with respective driving forces for these systems.


Assuntos
Compostos de Cádmio , Hidrogenase , Pontos Quânticos , Compostos de Selênio , Biomimética , Hidrogênio , Fármacos Fotossensibilizantes , Telúrio
15.
Angew Chem Int Ed Engl ; 60(26): 14488-14497, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33871139

RESUMO

An artificial amyloid-based redox hydrogel was designed for mediating electron transfer between a [NiFeSe] hydrogenase and an electrode. Starting from a mutated prion-forming domain of fungal protein HET-s, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized. This protein was able to self-assemble into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing the hydrogenase under mild conditions at the surface of carbon electrodes were obtained by a simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H2 were fabricated that afforded catalytic current densities of up to 270 µA cm-2 , with an overpotential of 0.33 V, under quiescent conditions at 45 °C.


Assuntos
Amiloide/metabolismo , Hidrogéis/metabolismo , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Amiloide/química , Biocatálise , Eletrodos , Transporte de Elétrons , Hidrogéis/química , Hidrogênio/química , Hidrogenase/química , Modelos Moleculares , Oxirredução , Tamanho da Partícula
16.
Angew Chem Int Ed Engl ; 60(25): 13824-13828, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-33721401

RESUMO

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene-reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.


Assuntos
Alcenos/metabolismo , Escherichia coli/enzimologia , Flavinas/metabolismo , Hidrogenase/metabolismo , Oxirredutases/metabolismo , Alcenos/química , Biocatálise , Flavinas/química , Hidrogenase/química , Hidrogenação , Estrutura Molecular , Oxirredução , Oxirredutases/química
17.
Biochim Biophys Acta Bioenerg ; 1862(6): 148401, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33684340

RESUMO

The concomitant presence of two distinctive polypeptide modules, which we have chosen to denominate as the "Y-junction" and the "flavin" module, is observed in 3D structures of enzymes as functionally diverse as complex I, NAD(P)-dependent [NiFe]-hydrogenases and NAD(P)-dependent formate dehydrogenases. Amino acid sequence conservation furthermore suggests that both modules are also part of NAD(P)-dependent [FeFe]-hydrogenases for which no 3D structure model is available yet. The flavin module harbours the site of interaction with the substrate NAD(P) which exchanges two electrons with a strictly conserved flavin moiety. The Y-junction module typically contains four iron-sulphur centres arranged to form a Y-shaped electron transfer conduit and mediates electron transfer between the flavin module and the catalytic units of the respective enzymes. The Y-junction module represents an electron transfer hub with three potential electron entry/exit sites. The pattern of specific redox centres present both in the Y-junction and the flavin module is correlated to present knowledge of these enzymes' functional properties. We have searched publicly accessible genomes for gene clusters containing both the Y-junction and the flavin module to assemble a comprehensive picture of the diversity of enzymes harbouring this dyad of modules and to reconstruct their phylogenetic relationships. These analyses indicate the presence of the dyad already in the last universal common ancestor and the emergence of complex I's EFG-module out of a subgroup of NAD(P)- dependent formate dehydrogenases.


Assuntos
Proteínas de Bactérias/metabolismo , Elétrons , Flavinas/metabolismo , Formiato Desidrogenases/metabolismo , Hidrogenase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte de Elétrons , Flavinas/química , Formiato Desidrogenases/química , Formiato Desidrogenases/genética , Hidrogenase/química , Hidrogenase/genética , Filogenia
18.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33753519

RESUMO

In [NiFe]-hydrogenases, the active-site Ni is coordinated by four cysteine-S ligands (Cys; C), two of which are bridging to the Fe(CO)(CN)2 fragment. Substitution of a single Cys residue by selenocysteine (Sec; U) occurs occasionally in nature. Using a recent method for site-specific Sec incorporation into proteins, each of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function. Steady-state solution activity of each Sec-substituted enzyme (on a per-milligram basis) is lowered, although this may reflect the unquantified presence of recalcitrant inactive/immature/misfolded forms. Protein film electrochemistry, however, reveals detailed kinetic data that are independent of absolute activities. Like native Hyd-1, the variants have low apparent K MH2 values, do not produce H2 at pH 6, and display the same onset overpotential for H2 oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O2 tolerance (apparent K MH2 and V max [solution] values relative to native Hyd-1 of 0.13 and 0.04, respectively) implying the importance of a selenium atom in the position cis to the site where exogenous ligands (H-, H2, O2) bind. Observation of the same unusual electrocatalytic signature seen earlier for the proton transfer-defective E28Q variant highlights the direct role of the chalcogen atom (S/Se) at position 576 close to E28, with the caveat that Se is less effective than S in facilitating proton transfer away from the Ni during H2 oxidation by this enzyme.


Assuntos
Cisteína/química , Proteínas de Escherichia coli/química , Hidrogenase/química , Oxigênio/química , Selenocisteína/química , Substituição de Aminoácidos , Biocatálise , Cisteína/genética , Proteínas de Escherichia coli/genética , Hidrogenase/genética , Selenocisteína/genética
19.
Angew Chem Int Ed Engl ; 60(29): 15854-15862, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33783938

RESUMO

To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O2 -tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for 57 Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses.


Assuntos
Hidrogenase/química , Hidrogenase/metabolismo , Solventes/química , Domínio Catalítico , Cristalografia por Raios X , Liofilização , Modelos Moleculares , Oxirredução
20.
ChemSusChem ; 14(10): 2237-2246, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33787007

RESUMO

Hydrogenase enzymes produce H2 gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo-designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H2 photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Through combining these results, a fine balance is found between solution acidity and the electron transfer steps. This balance is critical to maximize the production of NiI -peptide and protonation of the NiII -H- intermediate (Ni-R) by a Cys (pKa ≈6.4) to produce H2 .


Assuntos
Materiais Biomiméticos/química , Cisteína/química , Desenho de Fármacos , Hidrogenase/metabolismo , Processos Fotoquímicos , Prótons
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