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1.
Crit Rev Biochem Mol Biol ; 56(6): 640-668, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34428995

RESUMEN

Aerobic respiration is a key energy-producing pathway in many prokaryotes and virtually all eukaryotes. The final step of aerobic respiration is most commonly catalyzed by heme-copper oxidases embedded in the cytoplasmic or mitochondrial membrane. The majority of these terminal oxidases contain a prenylated heme (typically heme a or occasionally heme o) in the active site. In addition, many heme-copper oxidases, including mitochondrial cytochrome c oxidases, possess a second heme a cofactor. Despite the critical role of heme a in the electron transport chain, the details of the mechanism by which heme b, the prototypical cellular heme, is converted to heme o and then to heme a remain poorly understood. Recent structural investigations, however, have helped clarify some elements of heme a biosynthesis. In this review, we discuss the insight gained from these advances. In particular, we present a new structural model of heme o synthase (HOS) based on distance restraints from inferred coevolutionary relationships and refined by molecular dynamics simulations that are in good agreement with the experimentally determined structures of HOS homologs. We also analyze the two structures of heme a synthase (HAS) that have recently been solved by other groups. For both HOS and HAS, we discuss the proposed catalytic mechanisms and highlight how new insights into the heme-binding site locations shed light on previously obtained biochemical data. Finally, we explore the implications of the new structural data in the broader context of heme trafficking in the heme a biosynthetic pathway and heme-copper oxidase assembly.


Asunto(s)
Transferasas Alquil y Aril/metabolismo , Proteínas Bacterianas/metabolismo , Hemo/análogos & derivados , Animales , Archaea/metabolismo , Bacterias/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Eucariontes/metabolismo , Hemo/biosíntesis , Hemo/metabolismo , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Transporte de Proteínas
2.
Arch Biochem Biophys ; 744: 109665, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37348627

RESUMEN

In eukaryotes and many aerobic prokaryotes, the final step of aerobic respiration is catalyzed by an aa3-type cytochrome c oxidase, which requires a modified heme cofactor, heme a. The conversion of heme b, the prototypical cellular heme, to heme o and ultimately to heme a requires two modifications, the latter of which is conversion of a methyl group to an aldehyde, catalyzed by heme a synthase (HAS). The N- and C-terminal halves of HAS share homology, and each half contains a heme-binding site. Previous reports indicate that the C-terminal site is occupied by a heme b cofactor. The N-terminal site may function as the substrate (heme o) binding site, although this has not been confirmed experimentally. Here, we assess the role of conserved residues from the N- and C-terminal heme-binding sites in HAS from prokaryotic (Shewanella oneidensis) and eukaryotic (Saccharomyces cerevisiae) species - SoHAS/CtaA and ScHAS/Cox15, respectively. A glutamate within the N-terminal site is found to be critical for activity in both types of HAS, consistent with the hypothesis that a carbocation forms transiently during catalysis. In contrast, the residue occupying the analogous C-terminal position is dispensable for enzyme activity. In SoHAS, the C-terminal heme ligands are critical for stability, while in ScHAS, substitutions in either heme-binding site have little effect on global structure. In both species, in vivo accumulation of heme o requires the presence of an inactive HAS variant, highlighting a potential regulatory role for HAS in heme o biosynthesis.


Asunto(s)
Ácido Glutámico , Proteínas de Saccharomyces cerevisiae , Ácido Glutámico/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Ferroquelatasa , Hemo/metabolismo
3.
Biochemistry ; 60(23): 1853-1867, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-34061493

RESUMEN

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.


Asunto(s)
Grupo Citocromo c/metabolismo , Citocromos a1/metabolismo , Citocromos c1/metabolismo , Geobacter/metabolismo , Nitrato Reductasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X/métodos , Grupo Citocromo c/química , Citocromos a1/química , Citocromos c1/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Electrones , Geobacter/química , Hemo/química , Hemo/metabolismo , Modelos Moleculares , Nitrato Reductasas/química , Nitrito Reductasas/química , Nitrito Reductasas/metabolismo , Oxidación-Reducción , Conformación Proteica
4.
J Biol Chem ; 295(33): 11455-11465, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32518164

RESUMEN

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.


Asunto(s)
Proteínas Bacterianas/metabolismo , Citocromos a1/metabolismo , Citocromos c1/metabolismo , Geobacter/metabolismo , Nitrato Reductasas/metabolismo , Compuestos de Amonio/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Citocromos a1/química , Citocromos a1/genética , Citocromos c1/química , Citocromos c1/genética , Geobacter/química , Geobacter/genética , Cinética , Modelos Moleculares , Nitrato Reductasas/química , Nitrato Reductasas/genética , Nitratos/metabolismo , Filogenia , Conformación Proteica
5.
J Am Chem Soc ; 142(8): 4037-4050, 2020 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-32017546

RESUMEN

We present here detailed mechanistic studies of electrocatalytic hydrogenation (ECH) in aqueous solution over skeletal nickel cathodes to probe the various paths of reductive catalytic C-O bond cleavage among functionalized aryl ethers relevant to energy science. Heterogeneous catalytic hydrogenolysis of aryl ethers is important both in hydrodeoxygenation of fossil fuels and in upgrading of lignin from biomass. The presence or absence of simple functionalities such as carbonyl, hydroxyl, methyl, or methoxyl groups is known to cause dramatic shifts in reactivity and cleavage selectivity between sp3 C-O and sp2 C-O bonds. Specifically, reported hydrogenolysis studies with Ni and other catalysts have hinted at different cleavage mechanisms for the C-O ether bonds in α-keto and α-hydroxy ß-O-4 type aryl ether linkages of lignin. Our new rate, selectivity, and isotopic labeling results from ECH reactions confirm that these aryl ethers undergo C-O cleavage via distinct paths. For the simple 2-phenoxy-1-phenylethane or its alcohol congener, 2-phenoxy-1-phenylethanol, the benzylic site is activated via Ni C-H insertion, followed by beta elimination of the phenoxide leaving group. But in the case of the ketone, 2-phenoxyacetophenone, the polarized carbonyl π system apparently binds directly with the electron rich Ni cathode surface without breaking the aromaticity of the neighboring phenyl ring, leading to rapid cleavage. Substituent steric and electronic perturbations across a broad range of ß-O-4 type ethers create a hierarchy of cleavage rates that supports these mechanistic ideas while offering guidance to allow rational design of the catalytic method. On the basis of the new insights, the usage of cosolvent acetone is shown to enable control of product selectivity.

7.
J Biol Chem ; 293(42): 16426-16439, 2018 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-30181213

RESUMEN

The heme a molecule is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. Heme a is synthesized from heme o by a multi-spanning inner membrane protein, heme a synthase (Cox15 in the yeast Saccharomyces cerevisiae). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. To improve our understanding of heme a insertion into cytochrome c oxidase, here we investigated the protein-protein interactions that involve Cox15 in S. cerevisiae In addition to observing Cox15 in homooligomeric complexes, we found that a portion of Cox15 also associates with the mitochondrial respiratory supercomplexes. When supercomplex formation was abolished, as in the case of stalled cytochrome bc1 or cytochrome c oxidase assembly, Cox15 maintained an interaction with select proteins from both respiratory complexes. In the case of stalled cytochrome bc1 assembly, Cox15 interacted with the late-assembling cytochrome c oxidase subunit, Cox13. When cytochrome c oxidase assembly was stalled, Cox15 unexpectedly maintained its interaction with the cytochrome bc1 protein, Cor1. Our results indicate that Cox15 and Cor1 continue to interact in the cytochrome bc1 dimer even in the absence of supercomplexes or when the supercomplexes are destabilized. These findings reveal that Cox15 not only associates with respiratory supercomplexes, but also interacts with the cytochrome bc1 dimer even in the absence of cytochrome c oxidase.


Asunto(s)
Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Deficiencia de Citocromo-c Oxidasa , Hemo/análogos & derivados , Saccharomyces cerevisiae
9.
Biochemistry ; 55(22): 3165-73, 2016 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-27186945

RESUMEN

The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process.


Asunto(s)
Aminoácidos/química , Proteínas Bacterianas/metabolismo , Clostridium/enzimología , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Protones , Aminoácidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Hidrogenasas/química , Hidrogenasas/genética , Transporte Iónico , Hierro/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética
10.
J Am Chem Soc ; 138(16): 5262-70, 2016 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-26704697

RESUMEN

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.


Asunto(s)
Proteínas Hierro-Azufre/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cisteína/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Oxidación-Reducción
11.
Rapid Commun Mass Spectrom ; 30(2): 285-92, 2016 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-27071219

RESUMEN

RATIONALE: Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling. METHODS: We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. RESULTS: All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27­0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41­0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing a more complete picture of the reactions catalyzed by hydrogenases. CONCLUSIONS: The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.


Asunto(s)
Hidrógeno/química , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Fraccionamiento Químico , Chlamydomonas reinhardtii/enzimología , Clostridium/enzimología , Deuterio/química , Shewanella/enzimología
12.
PLoS Genet ; 8(11): e1003064, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23166516

RESUMEN

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.


Asunto(s)
Genoma , Anotación de Secuencia Molecular , Estramenopilos/genética , Secuencia de Bases , Genómica , Nitrógeno/administración & dosificación , Nitrógeno/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN/métodos , Especificidad de la Especie , Estramenopilos/crecimiento & desarrollo , Transformación Genética
13.
Environ Sci Technol ; 48(18): 10707-15, 2014 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-25121461

RESUMEN

Nitrous oxide (N2O) is a potent greenhouse gas with a 100-year global warming potential approximately 300 times that of CO2. Because microbes account for over 75% of the N2O released in the U.S., understanding the biochemical processes by which N2O is produced is critical to our efforts to mitigate climate change. In the current study, we used gas chromatography-isotope ratio mass spectrometry (GC-IRMS) to measure the δ(15)N, δ(18)O, δ(15)N(α), and δ(15)N(ß) of N2O generated by purified fungal nitric oxide reductase (P450nor) from Histoplasma capsulatum. The isotope values were used to calculate site preference (SP) values (difference in δ(15)N between the central (α) and terminal (ß) N atoms in N2O), enrichment factors (ε), and kinetic isotope effects (KIEs). Both oxygen and N(α) displayed normal isotope effects during enzymatic NO reduction with ε values of -25.7‰ (KIE = 1.0264) and -12.6‰ (KIE = 1.0127), respectively. However, bulk nitrogen (average δ(15)N of N(α) and N(ß)) and N(ß) exhibited inverse isotope effects with ε values of 14.0‰ (KIE = 0.9862) and 36.1‰ (KIE = 0.9651), respectively. The observed inverse isotope effect in δ(15)N(ß) is consistent with reversible binding of the first NO in the P450nor reaction mechanism. In contrast to the constant SP observed during NO reduction in microbial cultures, the site preference measured for purified H. capsulatum P450nor was not constant, increasing from ∼ 15‰ to ∼ 29‰ during the course of the reaction. This indicates that SP for microbial cultures can vary depending on the growth conditions, which may complicate source tracing during microbial denitrification.


Asunto(s)
Histoplasma/enzimología , Marcaje Isotópico , Óxido Nitroso/metabolismo , Oxidorreductasas/metabolismo , Fraccionamiento Químico , Cinética , Óxido Nítrico/metabolismo , Isótopos de Nitrógeno , Isótopos de Oxígeno
14.
J Inorg Biochem ; 256: 112542, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38631103

RESUMEN

Cytochrome c nitrite reductase, NrfA, is a soluble, periplasmic pentaheme cytochrome responsible for the reduction of nitrite to ammonium in the Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway, a vital reaction in the global nitrogen cycle. NrfA catalyzes this six-electron and eight-proton reduction of nitrite at a single active site with the help of its quinol oxidase partners. In this review, we summarize the latest progress in elucidating the reaction mechanism of ammonia production, including new findings about the active site architecture of NrfA, as well as recent results that elucidate electron transfer and storage in the pentaheme scaffold of this enzyme.


Asunto(s)
Compuestos de Amonio , Nitratos , Oxidación-Reducción , Nitratos/metabolismo , Nitratos/química , Compuestos de Amonio/metabolismo , Citocromos c1/metabolismo , Citocromos c1/química , Nitrato Reductasas/metabolismo , Nitrato Reductasas/química , Dominio Catalítico , Transporte de Electrón , Nitritos/metabolismo , Citocromos a1
15.
Appl Environ Microbiol ; 79(4): 1183-90, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23220958

RESUMEN

Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (µLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.


Asunto(s)
Beggiatoa/enzimología , Beggiatoa/metabolismo , Citocromos/metabolismo , Pigmentos Biológicos/metabolismo , Cromatografía Líquida de Alta Presión , Citocromos/aislamiento & purificación , Sedimentos Geológicos/microbiología , México , Nitrito Reductasas/aislamiento & purificación , Nitrito Reductasas/metabolismo , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masas en Tándem
16.
Biotechnol Bioeng ; 110(4): 1078-86, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23192283

RESUMEN

Copper(II) 2,2'-bipyridine (Cu(II) (bpy))-catalyzed alkaline hydrogen peroxide (AHP) pretreatment was performed on three biomass feedstocks including alkali pre-extracted switchgrass, silver birch, and a hybrid poplar cultivar. This catalytic approach was found to improve the subsequent enzymatic hydrolysis of plant cell wall polysaccharides to monosaccharides for all biomass types at alkaline pH relative to uncatalyzed pretreatment. The hybrid poplar exhibited the most significant improvement in enzymatic hydrolysis with monomeric sugar release and conversions more than doubling from 30% to 61% glucan conversion, while lignin solubilization was increased from 36.6% to 50.2% and hemicellulose solubilization was increased from 14.9% to 32.7%. It was found that Cu(II) (bpy)-catalyzed AHP pretreatment of cellulose resulted in significantly more depolymerization than uncatalyzed AHP pretreatment (78.4% vs. 49.4% decrease in estimated degree of polymerization) and that carboxyl content the cellulose was significantly increased as well (fivefold increase vs. twofold increase). Together, these results indicate that Cu(II) (bpy)-catalyzed AHP pretreatment represents a promising route to biomass deconstruction for bioenergy applications.


Asunto(s)
Álcalis/química , Cobre/química , Peróxido de Hidrógeno/química , Biomasa , Catálisis , Concentración de Iones de Hidrógeno , Hidrólisis , Viscosidad
17.
J Biol Chem ; 286(44): 38341-38347, 2011 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-21900241

RESUMEN

[FeFe]-Hydrogenases are complex metalloproteins that catalyze the reversible reduction of protons to molecular hydrogen utilizing a unique diiron subcluster bridged to a [4Fe4S] subcluster. Extensive studies have concentrated on the nature and catalytic activity of the active site, yet relatively little information is available concerning the mechanism of proton transport that is required for this activity. Previously, structural characterization of [FeFe]-hydrogenase from Clostridium pasteurianum indicated a potential proton transport pathway involving four residues (Cys-299, Glu-279, Ser-319, and Glu-282) that connect the active site to the enzyme surface. Here, we demonstrate that substitution of any of these residues resulted in a drastic reduction in hydrogenase activity relative to the native enzyme, supporting the importance of these residues in catalysis. Inhibition studies of native and amino acid-substituted enzymes revealed that Zn(2+) specifically blocked proton transfer by binding to Glu-282, confirming the role of this residue in the identified pathway. In addition, all four of these residues are strictly conserved, suggesting that they may form a proton transport pathway that is common to all [FeFe]-hydrogenases.


Asunto(s)
Clostridium/enzimología , Hidrogenasas/química , Hierro/química , Secuencia de Aminoácidos , Transporte Biológico , Catálisis , Ácido Glutámico/química , Proteínas Hierro-Azufre/química , Cinética , Conformación Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Protones , Homología de Secuencia de Aminoácido , Zinc/química
18.
Appl Environ Microbiol ; 78(24): 8579-86, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23023750

RESUMEN

H(2) generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H(2) from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O(2) sensitive. Certain filamentous cyanobacteria protect nitrogenase from O(2) by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter P(hetN). Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O(2).


Asunto(s)
Anabaena/genética , Hidrogenasas/genética , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Shewanella/enzimología , Aerobiosis , Expresión Génica , Hidrogenasas/aislamiento & purificación , Proteínas Hierro-Azufre/aislamiento & purificación , Operón , Regiones Promotoras Genéticas , Proteínas Recombinantes/aislamiento & purificación , Shewanella/genética
19.
Rapid Commun Mass Spectrom ; 26(1): 61-8, 2012 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22215579

RESUMEN

Hydrogenases catalyze the reversible formation of H(2), and they are key enzymes in the biological cycling of H(2). H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H(2) production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H(2) evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom-designed high-throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H(2). Using our new approach, we determined that the fractionation factor for H(2) production by the [NiFe]-hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H(2) evolution. Potential applications of this newly developed method are discussed.


Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Hidrógeno/análisis , Hidrogenasas/metabolismo , Desulfovibrio/enzimología , Deuterio , Hidrógeno/química , Hidrógeno/metabolismo , Límite de Detección , Protones
20.
Biotechnol Biofuels Bioprod ; 15(1): 45, 2022 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-35509012

RESUMEN

BACKGROUND: A lignocellulose-to-biofuel biorefinery process that enables multiple product streams is recognized as a promising strategy to improve the economics of this biorefinery and to accelerate technology commercialization. We recently identified an innovative pretreatment technology that enables of the production of sugars at high yields while simultaneously generating a high-quality lignin stream that has been demonstrated as both a promising renewable polyol replacement for polyurethane applications and is highly susceptible to depolymerization into monomers. This technology comprises a two-stage pretreatment approach that includes an alkaline pre-extraction followed by a metal-catalyzed alkaline-oxidative pretreatment. Our recent work demonstrated that H2O2 and O2 act synergistically as co-oxidants during the alkaline-oxidative pretreatment and could significantly reduce the pretreatment chemical input while maintaining high sugar yields (~ 95% glucose and ~ 100% xylose of initial sugar composition), high lignin yields (~ 75% of initial lignin), and improvements in lignin usage. RESULTS: This study considers the economic impact of these advances and provides strategies that could lead to additional economic improvements for future commercialization. The results of the technoeconomic analysis (TEA) demonstrated that adding O2 as a co-oxidant at 50 psig for the alkaline-oxidative pretreatment and reducing the raw material input reduced the minimum fuel selling price from $1.08/L to $0.85/L, assuming recoverable lignin is used as a polyol replacement. If additional lignin can be recovered and sold as more valuable monomers, the minimum fuel selling price (MFSP) can be further reduced to $0.73/L. CONCLUSIONS: The present work demonstrated that high sugar and lignin yields combined with low raw material inputs and increasing the value of lignin could greatly increase the economic viability of a poplar-based biorefinery. Continued research on integrating sugar production with lignin valorization is thus warranted to confirm this economic potential as the technology matures.

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