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1.
mBio ; 13(5): e0215022, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36106730

RESUMO

Sideroxydans species are important chemolithoautotrophic Fe(II)-oxidizing bacteria in freshwater environments and play a role in biogeochemical cycling of multiple elements. Due to difficulties in laboratory cultivation and genetic intractability, the electron transport proteins required for the growth and survival of this organism remain understudied. In Sideroxydans lithotrophicus ES-1, it is proposed that the Mto pathway transfers electrons from extracellular Fe(II) oxidation across the periplasm to an inner membrane NapC/NirT family protein encoded by Slit_2495 to reduce the quinone pool. Based on sequence similarity, Slit_2495 has been putatively called CymA, a NapC/NirT family protein which in Shewanella oneidensis MR-1 oxidizes the quinol pool during anaerobic respiration of a wide range of substrates. However, our phylogenetic analysis using the alignment of different NapC/NirT family proteins shows that Slit_2495 clusters closer to NirT sequences than to CymA. We propose the name ImoA (inner membrane oxidoreductase) for Slit_2495. Our data demonstrate that ImoA can oxidize quinol pools in the inner membrane and is able to functionally replace CymA in S. oneidensis. The ability of ImoA to oxidize quinol in vivo as opposed to its proposed function of reducing quinone raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus. IMPORTANCE Fe(II)-oxidizing bacteria play an important role in biogeochemical cycles. At circumneutral pH, these organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside the cell, potentially through a porin-cytochrome complex in the outer membrane encoded by the Mto pathway. Electrons from Fe(II) oxidation would then be transported to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. Directly demonstrating the functionality of genes in neutrophilic iron oxidizers is challenging due to the absence of robust genetic methods. Here, we heterologously expressed a NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to reduce the quinone pool. ImoA functionally replaced the inner membrane c-type cytochrome CymA in the Fe(III)-reducing bacterium Shewanella oneidensis. We suggest that ImoA may function primarily to oxidize quinol in S. lithotrophicus.


Assuntos
Grupo dos Citocromos c , Shewanella , Grupo dos Citocromos c/química , Hidroquinonas/metabolismo , Compostos Férricos/metabolismo , Filogenia , Shewanella/genética , Shewanella/metabolismo , Oxirredução , Transporte de Elétrons , Compostos Ferrosos/metabolismo , Quinonas/metabolismo , Porinas/metabolismo , Oxirredutases/metabolismo , Ferro/metabolismo
2.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077365

RESUMO

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called "intrinsically disordered" nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.


Assuntos
Heme , Tiocianatos , Grupo dos Citocromos c , Ectothiorhodospiraceae , Escherichia coli/metabolismo , Heme/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução , Oxirredutases/metabolismo
3.
Sci Rep ; 12(1): 14298, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-35995915

RESUMO

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivax gelatinosus and Rhodobacter sphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria.


Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética , Rhodobacter sphaeroides , Grupo dos Citocromos c/genética , Citocromos/metabolismo , Transporte de Elétrons , Cinética , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo
4.
Int J Mol Sci ; 23(3)2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-35162964

RESUMO

The branched aerobic respiratory chain in Bacillus cereus comprises three terminal oxidases: cytochromes aa3, caa3, and bd. Cytochrome caa3 requires heme A for activity, which is produced from heme O by heme A synthase (CtaA). In this study, we deleted the ctaA gene in B. cereus AH187 strain, this deletion resulted in loss of cytochrome caa3 activity. Proteomics data indicated that B. cereus grown in glucose-containing medium compensates for the loss of cytochrome caa3 activity by remodeling its respiratory metabolism. This remodeling involves up-regulation of cytochrome aa3 and several proteins involved in redox stress response-to circumvent sub-optimal respiratory metabolism. CtaA deletion changed the surface-composition of B. cereus, affecting its motility, autoaggregation phenotype, and the kinetics of biofilm formation. Strikingly, proteome remodeling made the ctaA mutant more resistant to cold and exogenous oxidative stresses compared to its parent strain. Consequently, we hypothesized that ctaA inactivation could improve B. cereus fitness in a nutrient-limited environment.


Assuntos
Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Grupo dos Citocromos b/genética , Grupo dos Citocromos c/metabolismo , Citocromos a3/metabolismo , Citocromos a/metabolismo , Deleção de Genes , Proteínas de Membrana/genética , Bacillus cereus/genética , Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/análogos & derivados , Heme/metabolismo , Estresse Oxidativo , Fenótipo , Proteômica , Transdução de Sinais
5.
J Am Chem Soc ; 144(6): 2590-2602, 2022 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-35107997

RESUMO

The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N-H carbene insertion reaction catalyzed by engineered variants of cytochrome c552 from Hydrogenobacter thermophilus. Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched ß-trifluoromethyl-ß-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N-H carbenoid insertion with an acceptor-acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.


Assuntos
Aminas/síntese química , Grupo dos Citocromos c/química , Hidrocarbonetos Fluorados/síntese química , Aminas/metabolismo , Compostos Azo/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Evolução Molecular Direcionada , Heme/química , Mutação , Ligação Proteica , Engenharia de Proteínas , Estereoisomerismo
6.
Int J Mol Sci ; 22(23)2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34884428

RESUMO

Cytochrome c3 (uranyl reductase) from Desulfovibrio vulgaris can reduce uranium in bacterial cells and in cell-free systems. This gene was introduced in tobacco under control of the RbcS promoter, and the resulting transgenic plants accumulated uranium when grown on a uranyl ion containing medium. The uptaken uranium was detected by EM in chloroplasts. In the presence of uranyl ions in sublethal concentration, the transgenic plants grew phenotypically normal while the control plants' development was impaired. The data on uranium oxidation state in the transgenic plants and the possible uses of uranium hyperaccumulation by plants for environmental cleanup are discussed.


Assuntos
Grupo dos Citocromos c/genética , Desulfovibrio vulgaris/metabolismo , Tabaco/crescimento & desenvolvimento , Compostos de Urânio/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Cloroplastos , Grupo dos Citocromos c/metabolismo , Engenharia Genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Tabaco/genética , Tabaco/metabolismo
7.
J Inorg Biochem ; 224: 111564, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34418715

RESUMO

Bacteria have evolved several outstanding strategies to resist to compounds or factors that compromise their survival. The first line of defense of the cell against environmental stresses is the membrane with fatty acids as fundamental building blocks of phospholipids. In this review, we focus on a periplasmic heme enzyme that catalyzes the cis-trans isomerization of unsaturated fatty acids to trigger a decrease in the fluidity of the membrane in order to rapidly counteract the danger. We particularly detailed the occurrence of such cis-trans isomerase in Nature, the different stresses that are at the origin of the double bond isomerization, the first steps in the elucidation of the mechanism of this peculiar metalloenzyme and some aspects of its regulation.


Assuntos
Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Ácidos Graxos Insaturados/metabolismo , Estresse Fisiológico , cis-trans-Isomerases/metabolismo , Heme/metabolismo , Isomerismo , Periplasma/metabolismo , Fosfolipídeos/metabolismo , Temperatura
8.
J Microbiol Biotechnol ; 31(8): 1154-1162, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34226414

RESUMO

The transcriptional capacities of target genes are strongly influenced by promoters, whereas few studies have focused on the development of robust, high-performance and cross-species promoters for wide application in different bacteria. In this work, four novel promoters (Pk.rtufB, Pk.r1, Pk.r2, and Pk.r3) were predicted from Ketogulonicigenium robustum and their inconsistency in the -10 and -35 region nucleotide sequences indicated they were different promoters. Their activities were evaluated by using green fluorescent protein (gfp) as a reporter in different species of bacteria, including K. vulgare SPU B805, Pseudomonas putida KT2440, Paracoccus denitrificans PD1222, Bacillus licheniformis and Raoultella ornithinolytica, due to their importance in metabolic engineering. Our results showed that the four promoters had different activities, with Pk.r1 showing the strongest activity in almost all of the experimental bacteria. By comparison with the commonly used promoters of E. coli (tufB, lac, lacUV5), K. vulgare (Psdh, Psndh) and P. putida KT2440 (JE111411), the four promoters showed significant differences due to only 12.62% nucleotide similarities, and relatively higher ability in regulating target gene expression. Further validation experiments confirmed their ability in initiating the target minCD cassette because of the shape changes under the promoter regulation. The overexpression of sorbose dehydrogenase and cytochrome c551 by Pk.r1 and Pk.r2 resulted in a 22.75% enhancement of 2-KGA yield, indicating their potential for practical application in metabolic engineering. This study demonstrates an example of applying bioinformatics to find new biological components for gene operation and provides four novel promoters with broad suitability, which enriches the usable range of promoters to realize accurate regulation in different genetic backgrounds.


Assuntos
Engenharia Metabólica , Regiões Promotoras Genéticas/genética , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Reprodutibilidade dos Testes , Rhodobacteraceae/genética
9.
Biochem J ; 478(14): 2927-2944, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34240737

RESUMO

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Cellvibrio/enzimologia , Grupo dos Citocromos c/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Celulose/metabolismo , Cellvibrio/genética , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Peróxido de Hidrogênio/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , Oligossacarídeos/metabolismo , Oxirredução , Oxigênio/metabolismo , Domínios Proteicos , Espectrofotometria/métodos , Especificidade por Substrato
10.
J Microbiol Methods ; 187: 106260, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34090997

RESUMO

Nitric oxide (NO) is a reactive gas that participates in many physiological as well as pathogenic processes in higher eukaryotic organisms. Inflammatory responses elicit higher levels of this molecule. Nevertheless, there are many technical challenges to accurately measure the amount of NO produced. Previously, a method using whole-cell extracts from Escherichia coli was able to generate the conversion of nitrate into nitrite to measure the amount of nitrate or indirectly the NO present in a sample using the Griess reaction. Here we present an improvement to this method, by using E. coli whole-cell extracts lacking one of the two nitrite reductases, rendered a more precise measurement when coupled with the Griess reaction than our previous report. Alternatively, osmotic stress showed to downregulate the expression of both nitrate reductases, which can be an alternative for indirect nitrate and NO reduction. The results presented here show an easy method for nitrate and NO reduction to nitrite and avoid the reconversion to nitrate, also as an alternative for other analytical methods that are based on cadmium, purified nitrate reductase enzyme, or salicylic methods to reduce NO. This method can be widely used for measuring NO production in living organisms, soil, and other relevant microbiological samples.


Assuntos
Escherichia coli/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/análise , Nitritos/análise , Animais , Grupo dos Citocromos c/genética , Escherichia coli/genética , Ativação de Macrófagos , Macrófagos/imunologia , Camundongos , Mutação , Nitrato Redutase/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Oxirredução , Células RAW 264.7 , Sensibilidade e Especificidade
11.
Biochemistry ; 60(23): 1853-1867, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34061493

RESUMO

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.


Assuntos
Grupo dos Citocromos c/metabolismo , Citocromos a1/metabolismo , Citocromos c1/metabolismo , Geobacter/metabolismo , Nitrato Redutases/metabolismo , Domínio Catalítico , Cristalografia por Raios X/métodos , Grupo dos Citocromos c/química , Citocromos a1/química , Citocromos c1/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Elétrons , Geobacter/química , Heme/química , Heme/metabolismo , Modelos Moleculares , Nitrato Redutases/química , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Oxirredução , Conformação Proteica
12.
J Biosci ; 462021.
Artigo em Inglês | MEDLINE | ID: mdl-33952726

RESUMO

Among all the proteins of Periplasmic C type Cytochrome family obtained from cytochrome C7 found in Geobacter sulfurreducens, only the Periplasmic C type Cytochrome A (PPCA) protein can recognize the deoxycholate (DXCA), while its other paralogs do not, as observed from the crystal structures. Though some existing works have used graph-theoretic approaches to realize the 3-D structural properties of proteins, its usage in the rationalisation of the physiochemical behavior of proteins has been very limited. To understand the driving force towards the recognition of DXCA exclusively by PPCA among its paralogs, in this work, we propose two graph theoretic models based on the combinatorial properties, namely, base-pair-type and impact, of the nucleotide bases and the amino acid residues, respectively. Combinatorial analysis of the binding sequences using the proposed base-pair type based graph theoretic model reveals the differential behaviour of PPCA among its other paralogs. Further, to investigate the underlying chemical phenomenon, another graph theoretic model has been developed based on impact. Analysis of the results obtained from impact-based model clearly indicates towards the helix formation of PPCA which is essential for the recognition of DXCA, making PPCA a completely different entity from its paralogs.


Assuntos
Grupo dos Citocromos c/química , Ácido Desoxicólico/química , Geobacter/química , Modelos Químicos , Proteínas de Bactérias/química , Conformação Proteica
13.
Biochemistry (Mosc) ; 86(3): 361-369, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33838635

RESUMO

Flavocytochrome c sulfide dehydrogenase (FCC) is one of the central enzymes of the respiratory chain in sulfur-oxidizing bacteria. FCC catalyzes oxidation of sulfide and polysulfide ions to elemental sulfur accompanied by electron transfer to cytochrome c. The catalytically active form of the enzyme is a non-covalently linked heterodimer composed of flavin- and heme-binding subunits. The Thioalkalivibrio paradoxus ARh1 genome contains five copies of genes encoding homologous FCCs with an amino acid sequence identity from 36 to 54%. When growing on thiocyanate or thiosulfate as the main energy source, the bacterium synthesizes products of different copies of FCC genes. In this work, we isolated and characterized FCC synthesized during the growth of Tv. paradoxus on thiocyanate. FCC was shown to oxidize exclusively sulfide but not other reduced sulfur compounds, such as thiosulfate, sulfite, tetrathionate, and sulfur, and it also does not catalyze the reverse reaction of sulfur reduction to sulfide. Kinetic parameters of the sulfide oxidation reaction are characterized.


Assuntos
Grupo dos Citocromos c/metabolismo , Ectothiorhodospiraceae/enzimologia , Oxirredutases/metabolismo , Sulfetos/metabolismo , Tiocianatos/metabolismo , Ectothiorhodospiraceae/metabolismo , Transporte de Elétrons , Cinética , Especificidade por Substrato
14.
Mol Biol Evol ; 38(8): 3445-3458, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-33878189

RESUMO

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favors the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selection, however, the intimate collaboration between mitochondria- and nuclear-encoded gene products offers theoretical potential for compensatory adaptive changes. In plants, cytoplasmic male sterilities are known examples of nucleo-mitochondrial coadaptation situations in which nuclear-encoded restorer of fertility (Rf) genes evolve to counteract the effect of mitochondria-encoded cytoplasmic male sterility (CMS) genes and restore fertility. Most cloned Rfs belong to a small monophyletic group, comprising 26 pentatricopeptide repeat genes in Arabidopsis, called Rf-like (RFL). In this analysis, we explored the functional diversity of RFL genes in Arabidopsis and found that the RFL8 gene is not related to CMS suppression but essential for plant embryo development. In vitro-rescued rfl8 plantlets are deficient in the production of the mitochondrial heme-lyase complex. A complete ensemble of molecular and genetic analyses allowed us to demonstrate that the RFL8 gene has been selected to permit the translation of the mitochondrial ccmFN2 gene encoding a heme-lyase complex subunit which derives from the split of the ccmFN gene, specifically in Brassicaceae plants. This study represents thus a clear case of nuclear compensation to a lineage-specific mitochondrial genomic rearrangement in plants and demonstrates that RFL genes can be selected in response to other mitochondrial deviancies than CMS suppression.


Assuntos
Arabidopsis/genética , Genoma Mitocondrial , Seleção Genética , Arabidopsis/embriologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Grupo dos Citocromos c/metabolismo , Desenvolvimento Embrionário , Biossíntese de Proteínas , Splicing de RNA
15.
Bioelectrochemistry ; 140: 107818, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33905959

RESUMO

The highly efficient bioelectrodes based on single layer graphene (SLG) functionalized with pyrene self-assembled monolayer and novel cytochromec553(cytc553)peptide linker variants were rationally designed to optimize the direct electron transfer (DET) between SLG and the heme group of cyt. Through a combination of photoelectrochemical and quantum mechanical (QM/MM) approaches we show that the specific amino acid sequence of a short peptide genetically inserted between the cytc553holoprotein and thesurface anchoring C-terminal His6-tag plays a crucial role in ensuring the optimal orientation and distance of the heme group with respect to the SLG surface. Consequently, efficient DET occurring between graphene and cyt c553 leads to a 20-fold enhancement of the cathodic photocurrent output compared to the previously reported devices of a similar type. The QM/MM modeling implies that a perpendicular or parallel orientation of the heme group with respect to the SLG surface is detrimental to DET, whereas the tilted orientation favors the cathodic photocurrent generation. Our work confirms the possibility of fine-tuning the electronic communication within complex bio-organic nanoarchitectures and interfaces due to optimization of the tilt angle of the heme group, its distance from the SLG surface and optimal HOMO/LUMO levels of the interacting redox centers.


Assuntos
Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Grafite/química , Heme , Mutação , Sequência de Aminoácidos , Eletrodos , Transporte de Elétrons
16.
Mol Omics ; 17(2): 288-295, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33554980

RESUMO

Shewanella has been widely investigated for its metabolic versatility and use of a large number of extracellular electron acceptors. Many c-type cytochromes are responsible for this diversity, mainly in condition-specific fashions. By using genome-scale mutant fitness data, we studied which genes (particularly c-type cytochromes) were used to coordinate various electron transfer processes in the present work. First, by integrating fitness profiles with protein-protein interaction (PPI) networks, we showed that the genes with a high total fitness value were generally more important in PPI networks than those with low fitness values. Then, we identified genes that are important across many experiments, and further fitness analysis confirmed five versatile c-type cytochromes: ScyA (SO0264), PetC (SO0610), CcoP (SO2361), CcoO (SO2363) and CytcB (SO4666), which are considered to be crucial in most experimental conditions. Finally, we demonstrated a mediating role in the periplasm for the less-reported CytcB by combining protein structure, subcellular localization and disordered region analysis. Comparative genome analysis further revealed that it is distinctive in Shewanella species. Collectively, these results suggest that periplasmic electron transfer processes are more diverse and flexible than previously reported, giving insight for further experimental studies of Shewanella oneidensis MR-1.


Assuntos
Grupo dos Citocromos c/genética , Transporte de Elétrons/genética , Periplasma/genética , Shewanella/genética , Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos c/classificação , Regulação Bacteriana da Expressão Gênica/genética , Mapas de Interação de Proteínas/genética
17.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34556577

RESUMO

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.


Assuntos
Grupo dos Citocromos c/metabolismo , Citocromos/metabolismo , Elétrons , Heme/metabolismo , Histidina/metabolismo , Metionina/metabolismo , Shewanella/metabolismo , Grupo dos Citocromos c/química , Citocromos/química , Transporte de Elétrons , Heme/química , Histidina/química , Metionina/química , Simulação de Dinâmica Molecular , Nanofios , Oxirredução
18.
Bioresour Technol ; 320(Pt A): 124290, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33129092

RESUMO

While cell membrane composition is critical for the function of membrane proteins, membrane modification has not been used to control the rate of extracellular electron transfer (EET) via the outer membrane protein complexes. Here, the rate of electron flow via the cell-surface redox protein, MtrC, was highly enhanced upon change in the outer membrane composition in Shewanella oneidensis MR-1. The MR-1 strain was pre-cultured at 4 °C and 30 °C to initiate differentiation of membrane composition. The whole-cell difference electrochemical assay of wild-type and mutant strains lacking MtrC suggested that the rate of EET via MtrC increased approximately 18 times at 4 °C than 30 °C. Circular dichroism spectroscopy showed that the molar exciton coupling coefficient for inter-heme interaction in MtrC increased in MR-1 at 4 °C than 30 °C. Results suggest that membrane modification may be a novel strategy for improving the efficiency of EET-based technologies.


Assuntos
Grupo dos Citocromos c , Shewanella , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons , Elétrons , Oxirredução , Shewanella/genética , Shewanella/metabolismo
19.
Metallomics ; 12(12): 2084-2097, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33226040

RESUMO

Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu2+ LMCT transitions.


Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/metabolismo , Grupo dos Citocromos c/metabolismo , Nitrito Redutases/metabolismo , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Clonagem Molecular , Cobre/metabolismo , Grupo dos Citocromos c/genética , Nitrito Redutases/genética , Oxirredução , Regulação para Cima
20.
Sci Rep ; 10(1): 16484, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020497

RESUMO

The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.


Assuntos
Grupo dos Citocromos c/genética , Técnicas de Diagnóstico Molecular/métodos , Nitrito Redutases/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Pseudomonas aeruginosa/genética , DNA/genética , Primers do DNA/genética , Sensibilidade e Especificidade
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