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1.
Nat Commun ; 11(1): 5448, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116131

RESUMO

Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diameter) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the production of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen production of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.


Assuntos
Proteínas de Bactérias/metabolismo , Reatores Biológicos , Hidrogênio/metabolismo , Organelas/metabolismo , Proteínas de Bactérias/genética , Biocatálise , Bioengenharia , Reatores Biológicos/microbiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Halothiobacillus/genética , Halothiobacillus/metabolismo , Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Organelas/genética , Organelas/ultraestrutura , Fotossíntese , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(34): 20520-20529, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32796105

RESUMO

As paradigms for proton-coupled electron transfer in enzymes and benchmarks for a fully renewable H2 technology, [FeFe]-hydrogenases behave as highly reversible electrocatalysts when immobilized on an electrode, operating in both catalytic directions with minimal overpotential requirement. Using the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1) we have conducted site-directed mutagenesis and protein film electrochemistry to determine how efficient catalysis depends on the long-range coupling of electron and proton transfer steps. Importantly, the electron and proton transfer pathways in [FeFe]-hydrogenases are well separated from each other in space. Variants with conservative substitutions (glutamate to aspartate) in either of two positions in the proton-transfer pathway retain significant activity and reveal the consequences of slowing down proton transfer for both catalytic directions over a wide range of pH and potential values. Proton reduction in the variants is impaired mainly by limiting the turnover rate, which drops sharply as the pH is raised, showing that proton capture from bulk solvent becomes critical. In contrast, hydrogen oxidation is affected in two ways: by limiting the turnover rate and by a large overpotential requirement that increases as the pH is raised, consistent with the accumulation of a reduced and protonated intermediate. A unique observation having fundamental significance is made under conditions where the variants still retain sufficient catalytic activity in both directions: An inflection appears as the catalytic current switches direction at the 2H+/H2 thermodynamic potential, clearly signaling a departure from electrocatalytic reversibility as electron and proton transfers begin to be decoupled.


Assuntos
Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Chlamydomonas reinhardtii , Clostridium , Transporte de Elétrons , Hidrogenase/genética , Proteínas com Ferro-Enxofre/genética , Mutagênese Sítio-Dirigida , Prótons
3.
Met Ions Life Sci ; 202020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32851832

RESUMO

Enzymes relying on the interplay of nickel, iron, and sulfur in their active sites are used by prokaryotes to catalyze reactions driving the global carbon and hydrogen cycles. The three enzymes, [NiFe] hydrogenases, Ni,Fe-containing carbon monoxide dehydrogenases and acetyl-CoA synthases share an ancient origin possibly derived from abiotic processes. Although their active sites have different compositions and assemble Ni, Fe, and S in different ways and for different purposes, they share a central role of Ni in substrate binding and activation, with sulfur linking the Ni ion to one or more Fe ions, which, although indispensable for function, supports the catalytic process in less understood ways. The review gives a short overview on the properties of the three individual enzymes highlighting their parallels and differences.


Assuntos
Níquel/metabolismo , Sítios de Ligação , Domínio Catalítico , Hidrogenase/metabolismo , Ferro/metabolismo , Proteínas com Ferro-Enxofre , Enxofre
4.
Microb Cell Fact ; 19(1): 65, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156284

RESUMO

BACKGROUND: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H2) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2-consumption on H2 photoproduction yields in the heterocyte-forming, N2-fixing cyanobacterium Nostoc PCC7120. RESULTS: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses. CONCLUSION: We conclude that the overproduced Flv3B protein might have enhanced O2-consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrogênio/metabolismo , Nostoc/metabolismo , Oxigênio/metabolismo , Proteínas de Bactérias/genética , Genes Homeobox , Hidrogenase/genética , Hidrogenase/metabolismo , Engenharia Metabólica , Nitrogênio/metabolismo , Fixação de Nitrogênio , Nostoc/genética , Fotossíntese
5.
Nat Commun ; 11(1): 920, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060304

RESUMO

Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as hydrogenases for their use in fuel cells. However, the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day. Here, we employ operando confocal microscopy to elucidate the deactivation processes. The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst. We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] hydrogenase to over one week, approaching the experimental anaerobic half-life. Altogether, our data support the theory that redox films make the hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress.


Assuntos
Proteínas de Bactérias/química , Desulfovibrio vulgaris/enzimologia , Peróxido de Hidrogênio/química , Hidrogenase/química , Oxigênio/química , Proteínas de Bactérias/metabolismo , Desulfovibrio vulgaris/química , Peróxido de Hidrogênio/metabolismo , Hidrogenase/metabolismo , Cinética , Oxirredução , Oxigênio/metabolismo
6.
ISME J ; 14(5): 1223-1232, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32042101

RESUMO

The trace amounts (0.53 ppmv) of atmospheric hydrogen gas (H2) can be utilized by microorganisms to persist during dormancy. This process is catalyzed by certain Actinobacteria, Acidobacteria, and Chloroflexi, and is estimated to convert 75 × 1012 g H2 annually, which is half of the total atmospheric H2. This rapid atmospheric H2 turnover is hypothesized to be catalyzed by high-affinity [NiFe] hydrogenases. However, apparent high-affinity H2 oxidation has only been shown in whole cells, rather than for the purified enzyme. Here, we show that the membrane-associated hydrogenase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV possesses a high apparent affinity (Km(app) = 140 nM) for H2 and that methanotrophs can oxidize subatmospheric H2. Our findings add to the evidence that the group 1h [NiFe] hydrogenase is accountable for atmospheric H2 oxidation and that it therefore could be a strong controlling factor in the global H2 cycle. We show that the isolated enzyme possesses a lower affinity (Km = 300 nM) for H2 than the membrane-associated enzyme. Hence, the membrane association seems essential for a high affinity for H2. The enzyme is extremely thermostable and remains folded up to 95 °C. Strain SolV is the only known organism in which the group 1h [NiFe] hydrogenase is responsible for rapid growth on H2 as sole energy source as well as oxidation of subatmospheric H2. The ability to conserve energy from H2 could increase fitness of verrucomicrobial methanotrophs in geothermal ecosystems with varying CH4 fluxes. We propose that H2 oxidation can enhance growth of methanotrophs in aerated methane-driven ecosystems. Group 1h [NiFe] hydrogenases could therefore contribute to mitigation of global warming, since CH4 is an important and extremely potent greenhouse gas.


Assuntos
Verrucomicrobia/fisiologia , Ecossistema , Hidrogênio , Hidrogenase/metabolismo , Metano , Oxirredução , Verrucomicrobia/metabolismo
7.
ISME J ; 14(5): 1125-1140, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31996786

RESUMO

Coupling microbial electrosynthesis to renewable energy sources can provide a promising future technology for carbon dioxide conversion. However, this technology suffers from a limited number of suitable biocatalysts, resulting in a narrow product range. Here, we present the characterization of the first thermoacidophilic electroautotrophic community using chronoamperometric, metagenomic, and 13C-labeling analyses. The cathodic biofilm showed current consumption of up to -80 µA cm-2 over a period of 90 days (-350 mV vs. SHE). Metagenomic analyses identified members of the genera Moorella, Desulfofundulus, Thermodesulfitimonas, Sulfolobus, and Acidianus as potential primary producers of the biofilm, potentially thriving via an interspecies sulfur cycle. Hydrogenases seem to be key for cathodic electron uptake. An isolation campaign led to a pure culture of a Knallgas bacterium from this community. Growth of this organism on cathodes led to increasing reductive currents over time. Transcriptomic analyses revealed a distinct gene expression profile of cells grown at a cathode. Moreover, pressurizable flow cells combined with optical coherence tomography allowed an in situ observation of cathodic biofilm growth. Autotrophic growth was confirmed via isotope analysis. As a natural polyhydroxybutyrate (PHB) producer, this novel species, Kyrpidia spormannii, coupled the production of PHB to CO2 fixation on cathode surfaces.


Assuntos
Bacillales/fisiologia , Biofilmes/crescimento & desenvolvimento , Extremófilos/fisiologia , Processos Autotróficos , Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Eletrodos , Extremófilos/metabolismo , Hidrogenase/metabolismo
8.
Inorg Chem ; 59(2): 968-971, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31891256

RESUMO

A dithiolate/hydride bridged Fe-Ni complex, [(CN)(CO)2FeII(µ-pdt)(µ-H)NiII(CN)(PCy3)]- (2, pdt = propane-1,3-dithiolate) has been synthesized by the reaction of [(CN)2(CO)2FeII(pdt)]2- with [NiII(Cl)(H)(PCy3)2] as a synthetic analogue of the Ni-R state of the active site of the [Ni-Fe] hydrogenase. X-ray crystallography of this model complex suggests that the hydride unsymmetrically binds to Ni and Fe similar to natural [Ni-Fe] hydrogenases.


Assuntos
Monóxido de Carbono/química , Complexos de Coordenação/química , Cianetos/química , Hidrogenase/química , Tolueno/análogos & derivados , Monóxido de Carbono/metabolismo , Domínio Catalítico , Complexos de Coordenação/metabolismo , Cianetos/metabolismo , Hidrogenase/metabolismo , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Conformação Molecular , Níquel/química , Níquel/metabolismo , Tolueno/química , Tolueno/metabolismo
9.
Appl Environ Microbiol ; 86(6)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31924613

RESUMO

To date, NAD(P)H, ferredoxin, and coenzyme F420 have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoded hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pulldown assays. Electrons derived from H2 oxidation by the frhAGB-encoded hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was 7-fold less efficient than when NADPH was the electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoded hydrogenase utilizing a protein as an electron acceptor.IMPORTANCE This study has importance in that TrxR can use H2 as an electron donor with the aid of the frhAGB-encoded hydrogenase as well as NAD(P)H in T. onnurineus NA1. Further studies are needed to explore the physiological significance of this protein. This study also has importance as a significant step toward understanding the functionality of the frhAGB-encoded hydrogenase in a nonmethanogen; the hydrogenase can transfer electrons derived from oxidation of H2 to a protein target by direct contact without the involvement of an electron carrier, which is distinct from the mechanism of its homologs, F420-reducing hydrogenases of methanogens.


Assuntos
Proteínas Arqueais/metabolismo , Elétrons , Hidrogenase/metabolismo , Thermococcus/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Transporte de Elétrons , Oxirredução
10.
Int J Mol Sci ; 21(1)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31935923

RESUMO

The hyperthermo-piezophilic archaeon Palaeococcus pacificus DY20341T, isolated from East Pacific hydrothermal sediments, can utilize elemental sulfur as a terminal acceptor to simulate growth. To gain insight into sulfur metabolism, we performed a genomic and transcriptional analysis of Pa. pacificus DY20341T with/without elemental sulfur as an electron acceptor. In the 2001 protein-coding sequences of the genome, transcriptomic analysis showed that 108 genes increased (by up to 75.1 fold) and 336 genes decreased (by up to 13.9 fold) in the presence of elemental sulfur. Palaeococcus pacificus cultured with elemental sulfur promoted the following: the induction of membrane-bound hydrogenase (MBX), NADH:polysulfide oxidoreductase (NPSOR), NAD(P)H sulfur oxidoreductase (Nsr), sulfide dehydrogenase (SuDH), connected to the sulfur-reducing process, the upregulation of iron and nickel/cobalt transfer, iron-sulfur cluster-carrying proteins (NBP35), and some iron-sulfur cluster-containing proteins (SipA, SAM, CobQ, etc.). The accumulation of metal ions might further impact on regulators, e.g., SurR and TrmB. For growth in proteinous media without elemental sulfur, cells promoted flagelin, peptide/amino acids transporters, and maltose/sugar transporters to upregulate protein and starch/sugar utilization processes and riboflavin and thiamin biosynthesis. This indicates how strain DY20341T can adapt to different living conditions with/without elemental sulfur in the hydrothermal fields.


Assuntos
Adaptação Fisiológica , Fontes Hidrotermais/microbiologia , Enxofre/metabolismo , Thermococcaceae/metabolismo , Transcriptoma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrogenase/genética , Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/metabolismo , Oceanos e Mares , Thermococcaceae/genética
11.
Amino Acids ; 52(2): 287-299, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31621031

RESUMO

Branched-chain polyamine (BCPA) synthase (BpsA), encoded by the bpsA gene, is responsible for the biosynthesis of BCPA in the hyperthermophilic archaeon Thermococcus kodakarensis, which produces N4-bis(aminopropyl)spermidine and spermidine. Here, next-generation DNA sequencing and liquid chromatography-mass spectrometry (LC-MS) were used to perform transcriptomic and proteomic analyses of a T. kodakarensis strain (DBP1) lacking bpsA. Subsequently, the contributions of BCPA to gene transcription (or transcript stabilization) and translation (or protein stabilization) were analyzed. Compared with those in the wild-type strain (KU216) cultivated at 90 °C, the transcript levels of 424 and 21 genes were up- and downregulated in the DBP1 strain, respectively. The expression levels of 12 frequently-used tRNAs were lower in DBP1 cells than KU216 cells, suggesting that BCPA affects translation efficiency in T. kodakarensis. LC-MS analyses of cells grown at 90 °C detected 50 proteins in KU216 cells only, 109 proteins in DBP1 cells only, and 499 proteins in both strains. Notably, the transcript levels of some genes did not correlate with those of the proteins. RNA-seq and RT-qPCR analyses of ten proteins that were detected in KU216 cells only, including three flagellin-related proteins (FlaB2-4) and cytosolic NiFe-hydrogenase subunit alpha (HyhL), revealed that the corresponding transcripts were expressed at higher levels in DBP1 cells than KU216 cells. Electron microscopy analyses showed that flagella formation was disrupted in DBP1 cells at 90 °C, and western blotting confirmed that HyhL expression was eliminated in the DBP1 strain. These results suggest that BCPA plays a regulatory role in gene expression in T. kodakarensis.


Assuntos
Poliaminas/metabolismo , Thermococcus/genética , Thermococcus/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea , Temperatura Alta , Hidrogenase/genética , Hidrogenase/metabolismo , Poliaminas/química , Thermococcus/crescimento & desenvolvimento
12.
Cell Mol Life Sci ; 77(8): 1461-1481, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31630229

RESUMO

The reversible interconversion of molecular hydrogen and protons is one of the most ancient microbial metabolic reactions and catalyzed by hydrogenases. A widespread yet largely enigmatic group comprises multisubunit [NiFe] hydrogenases, that directly couple H2 metabolism to the electrochemical ion gradient across the membranes of bacteria and of archaea. These complexes are collectively referred to as energy-converting hydrogenases (Ech), as they reversibly transform redox energy into physicochemical energy. Redox energy is typically provided by a low potential electron donor such as reduced ferredoxin to fuel H2 evolution and the establishment of a transmembrane electrochemical ion gradient ([Formula: see text]). The [Formula: see text] is then utilized by an ATP synthase for energy conservation by generating ATP. This review describes the modular structure/function of Ech complexes, focuses on insights into the energy-converting mechanisms, describes the evolutionary context and delves into the implications of relying on an Ech complex as respiratory enzyme for microbial metabolism.


Assuntos
Archaea/enzimologia , Bactérias/enzimologia , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Trifosfato de Adenosina/metabolismo , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Metabolismo Energético , Oxirredução
13.
Photosynth Res ; 143(2): 155-163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31673863

RESUMO

Photosystem I complexes from the menB deletion mutant of Synechocystis sp. PCC 6803 were previously wired to a Pt nanoparticle via a molecular wire consisting of 15-(3-methyl-1,4-naphthoquinone-2-yl)]pentadecyl sulfide. In the presence of a sacrificial electron donor and an electron transport mediator, the PS I-NQ(CH2)15S-Pt nanoconstruct generated dihydrogen at a rate of 44.3 µmol of H2 mg Chl-1 h-1 during illumination at pH 8.3. The menB deletion strain contains an interruption in the biosynthetic pathway of phylloquinone, which results in the presence of a displaceable plastoquinone-9 in the A1A/A1B sites. The synthesized quinone contains a headgroup identical to the native phylloquinone along with a 15-carbon long tail that is terminated in a thiol. The thiol on the molecular wire is used to bind the Pt nanoparticle. In this short communication, we replaced the Pt nanoparticle with an [FeFe]H2ase variant from Clostridium acetobutylicum that contains an exposed iron on the distal [4Fe-4S] cluster afforded by mutating the surface exposed Cys97 residue to Gly. The thiol on the molecular wire is then used to coordinate the corner iron atom of the iron-sulfur cluster. When all three components are combined and illuminated in the presence of a sacrificial electron donor and an electron transport mediator, the PS I-NQ(CH2)15S-[FeFe]H2ase nanoconstruct generated dihydrogen at a rate of 50.3 ± 9.96 µmol of H2 mg Chl-1 h-1 during illumination at pH 8.3. This successful in vitro experiment sets the stage for assembling a PS I-NQ(CH2)15S-[FeFe]H2ase nanoconstruct in vivo in the menB mutant of Synechocystis sp. PCC 6803.


Assuntos
Hidrogênio/metabolismo , Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Clostridium acetobutylicum/enzimologia , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Complexo de Proteína do Fotossistema I/química , Quinonas/química , Synechocystis/metabolismo
14.
Chemistry ; 26(13): 2859-2868, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-31743487

RESUMO

Inspired by the sulfur-rich environment found in active hydrogenase enzymes, a Ni-based proton reduction catalyst with pentadentate N2 S3 ligand was synthesised. When coupled with [Ru(bpy)3 ]2+ (bpy=2,2'-bipyridine) as photosensitiser and ascorbate as electron donor in a 1:1 mixture of dimethylacetamide and aqueous ascorbic acid/ascorbate buffer, the catalyst showed improved photocatalytic activity compared with a homologous counterpart bearing a tetradentate N2 S2 ligand. The mechanistic pathway of photoinduced hydrogen evolution was comprehensively analysed through optical transient absorption and time-resolved X-ray absorption spectroscopy, which revealed important electronic and structural changes in the catalytic system during photoirradiation. The NiII catalyst undergoes a photoinduced metal-centred reduction to form a NiI intermediate with distorted square-bipyramidal geometry. Further kinetic analyses revealed differences in charge-separation dynamics between the pentadentate and tetradentate forms.


Assuntos
Complexos de Coordenação/química , Hidrogenase/química , Rênio/química , Enxofre/química , Catálise , Hidrogenase/metabolismo , Ligantes , Prótons , Espectroscopia por Absorção de Raios X
15.
Proc Natl Acad Sci U S A ; 117(2): 1167-1173, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879356

RESUMO

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of [Formula: see text], but it can also be composed of Na+ Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:[Formula: see text] oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is [Formula: see text]-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden-Meyerhof-Parnas or the pentose-phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for [Formula: see text] bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.


Assuntos
Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Clostridiales/metabolismo , Metabolismo Energético/fisiologia , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Clostridiales/enzimologia , Clostridiales/genética , Clostridiales/crescimento & desenvolvimento , Metabolismo Energético/genética , Ferredoxinas/metabolismo , Hidrogenase/metabolismo , Transporte de Íons , Oxirredução , Oxirredutases/metabolismo , Sódio/metabolismo
16.
Biochim Biophys Acta Bioenerg ; 1861(1): 148087, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669490

RESUMO

Electron bifurcating, [FeFe]-hydrogenases are recently described members of the hydrogenase family and catalyze a combination of exergonic and endergonic electron exchanges between three carriers (2 ferredoxinred- + NAD(P)H + 3 H+ = 2 ferredoxinox + NAD(P)+ + 2 H2). A thermodynamic analysis of the bifurcating, [FeFe]-hydrogenase reaction, using electron path-independent variables, quantified potential biological roles of the reaction without requiring enzyme details. The bifurcating [FeFe]-hydrogenase reaction, like all bifurcating reactions, can be written as a sum of two non-bifurcating reactions. Therefore, the thermodynamic properties of the bifurcating reaction can never exceed the properties of the individual, non-bifurcating, reactions. The bifurcating [FeFe]-hydrogenase reaction has three competitive properties: 1) enabling NAD(P)H-driven proton reduction at pH2 higher than the concurrent operation of the two, non-bifurcating reactions, 2) oxidation of NAD(P)H and ferredoxin simultaneously in a 1:1 ratio, both are produced during typical glucose fermentations, and 3) enhanced energy conservation (~10 kJ mol-1 H2) relative to concurrent operation of the two, non-bifurcating reactions. Our analysis demonstrated ferredoxin E°' largely determines the sensitivity of the bifurcating reaction to pH2, modulation of the reduced/oxidized electron carrier ratios contributed less to equilibria shifts. Hydrogenase thermodynamics data were integrated with typical and non-typical glycolysis pathways to evaluate achieving the 'Thauer limit' (4 H2 per glucose) as a function of temperature and pH2. For instance, the bifurcating [FeFe]-hydrogenase reaction permits the Thauer limit at 60 °C if pH 2 ≤ ~10 mbar. The results also predict Archaea, expressing a non-typical glycolysis pathway, would not benefit from a bifurcating [FeFe]-hydrogenase reaction; interestingly, no Archaea have been observed experimentally with a [FeFe]-hydrogenase enzyme.


Assuntos
Proteínas de Bactérias , Hidrogênio , Hidrogenase , Proteínas com Ferro-Enxofre , Thermotoga maritima/enzimologia , Anaerobiose/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrogênio/química , Hidrogênio/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/metabolismo , Oxirredução , Termodinâmica
17.
Photosynth Res ; 143(2): 193-203, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31641988

RESUMO

Biohybrid artificial photosynthesis aims to combine the advantages of biological specificity with a range of synthetic nanomaterials to create innovative semi-synthetic systems for solar-to-chemical conversion. Biological systems utilize highly efficient molecular catalysts for reduction-oxidation reactions. They can operate with minimal overpotentials while selectively channeling reductant energy into specific transformation chemistries and product forming pathways. Nanomaterials can be synthesized to have efficient light-absorption capacity and tuneability of charge separation by manipulation of surface chemistries and bulk compositions. These complementary aspects have been combined in a variety of ways, for example, where biological light-harvesting complexes function as antenna for nanoparticle catalysts or where nanoparticles function as light capture, charge separation components for coupling to chemical conversion by redox enzymes and whole cells. The synthetic diversity that is possible with biohybrids is still being explored. The progress arising from creative approaches is generating new model systems to inspire scale-up technologies and generate understanding of the fundamental mechanisms that control energy conversion at the molecular scale. These efforts are leading to discoveries of essential design principles that can enable the development of scalable artificial photosynthesis systems.


Assuntos
Nanoestruturas/química , Fotossíntese , Biologia Sintética , Catálise , Hidrogenase/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Tilacoides/metabolismo
18.
Microb Cell Fact ; 18(1): 201, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31739794

RESUMO

BACKGROUND: The chemolithoautotrophic ß-proteobacterium Ralstonia eutropha H16 (Cupriavidus necator) is one of the most studied model organisms for growth on H2 and CO2. R. eutropha H16 is also a biologically significant bacterium capable of synthesizing O2-tolerant [NiFe]-hydrogenases (Hyds), which can be used as anode biocatalysts in enzyme fuel cells. For heterotrophic growth of R. eutropha, various sources of organic carbon and energy can be used. RESULTS: Growth, bioenergetic properties, and oxidation-reduction potential (ORP) kinetics were investigated during cultivation of R. eutropha H16 on fructose and glycerol or lignocellulose-containing brewery spent grain hydrolysate (BSGH). BSGH was used as carbon and energy source by R. eutropha H16, and the activities of the membrane-bound hydrogenase (MBH) and cytoplasmic, soluble hydrogenase (SH) were measured in different growth phases. Growth of R. eutropha H16 on optimized BSGH medium yielded ~ 0.7 g cell dry weight L-1 with 3.50 ± 0.02 (SH) and 2.3 ± 0.03 (MBH) U (mg protein)-1 activities. Upon growth on fructose and glycerol, a pH drop from 7.0 to 6.7 and a concomitant decrease of ORP was observed. During growth on BSGH, in contrast, the pH and ORP stayed constant. The growth rate was slightly stimulated through addition of 1 mM K3[Fe(CN)6], whereas temporarily reduced growth was observed upon addition of 3 mM dithiothreitol. The overall and N,N'-dicyclohexylcarbodiimide-sensitive ATPase activities of membrane vesicles were ~ 4- and ~ 2.5-fold lower, respectively, upon growth on fructose and glycerol (FGN) compared with only fructose utilization (FN). Compared to FN, ORP was lower upon bacterial growth on FGN, GFN, and BSGH. CONCLUSIONS: Our results suggest that reductive conditions and low ATPase activity might be signals for energy depletion, which, in turn, leads to increased hydrogenase biosynthesis to overcome this unfavorable situation. Addition of fructose or microelements have no, or a negative, influence on hydrogenase activity. Organic wastes (glycerol, BSGH) are promising carbon and energy sources for the formation of biomass harboring significant amounts of the biotechnologically relevant hydrogenases MBH and SH. The results are valuable for using microbial cells as producers of hydrogenase enzymes as catalysts in enzymatic fuel cells.


Assuntos
Proteínas de Bactérias/metabolismo , Cupriavidus necator/enzimologia , Cupriavidus necator/crescimento & desenvolvimento , Hidrogenase/biossíntese , Biocatálise , Biodegradação Ambiental , Glicerol/metabolismo , Processos Heterotróficos , Hidrogenase/metabolismo , Oxirredução , Resíduos
19.
Acc Chem Res ; 52(11): 3120-3131, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31675209

RESUMO

Achieving a unified understanding of the mechanism of a multicenter redox enzyme such as [NiFe] hydrogenase is complicated by difficulties in reconciling information obtained by using different techniques and on samples in different physical forms. Measurements of the activity of the enzyme, and of factors which perturb activity, are generally carried out using biochemical assays in solution or with electrode-immobilized enzymes using protein film electrochemistry (PFE). Conversely, spectroscopy aimed at reporting on features of the metalloclusters in the enzyme, such as electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy (XAS), is often conducted on frozen samples and is thus difficult to relate to catalytically relevant states as information about turnover and activity has been lost. To complicate matters further, most of our knowledge of the atomic-level structure of metalloenzymes comes from X-ray diffraction studies in the solid, crystalline state, which are again difficult to link to turnover conditions. Taking [NiFe] hydrogenases as our case study, we show here how it is possible to apply infrared (IR) spectroscopic sampling approaches to unite direct spectroscopic study with catalytic turnover. Using a method we have named protein film IR electrochemistry (PFIRE), we reveal the steady-state distribution of intermediates during catalysis and identify catalytic "bottlenecks" introduced by site-directed mutagenesis. We also show that it is possible to study dynamic transitions between active site states of enzymes in single crystals, uniting solid state and solution spectroscopic information. In all of these cases, the spectroscopic data complement and enhance interpretation of purely activity-based measurements by providing direct chemical insight that is otherwise hidden. The [NiFe] hydrogenases possess a bimetallic [NiFe] active site, coordinated by CO and CN- ligands, linked to the protein via bridging and terminal cysteine sulfur ligands, as well as an electron relay chain of iron sulfur clusters. Infrared spectroscopy is ideal for probing hydrogenases because the CO and CN- ligands are strong IR absorbers, but the suite of IR-based approaches we describe here will be equally valuable in studying substrate- or intermediate-bound states of other metalloenzymes where key mechanistic questions remain open, such as nitrogenase, formate dehydrogenase, or carbon monoxide dehydrogenase. We therefore hope that this Account will encourage future studies which unify information from different techniques across bioinorganic chemistry.


Assuntos
Hidrogenase , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/química , Hidrogenase/metabolismo , Fenômenos Mecânicos , Conformação Proteica , Espectrofotometria Infravermelho , Espectroscopia por Absorção de Raios X
20.
J Am Chem Soc ; 141(43): 17394-17403, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31580662

RESUMO

Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, our understanding of proton transfer is poor. Previously, residues were identified forming a hydrogen-bonding network between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ infrared difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon photoreduction. While proton transfer appears to be impaired in the oxidized state (Hox), the presented data support continuous proton transfer in the reduced state (Hred). Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases.


Assuntos
Hidrogenase/química , Proteínas com Ferro-Enxofre/química , Domínio Catalítico , Chlamydomonas reinhardtii/enzimologia , Ácido Glutâmico/química , Ligação de Hidrogênio , Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Prótons , Serina/química , Espectroscopia de Infravermelho com Transformada de Fourier
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