RESUMO
Methane (CH4), the most abundant hydrocarbon in the atmosphere, originates largely from biogenic sources1 linked to an increasing number of organisms occurring in oxic and anoxic environments. Traditionally, biogenic CH4 has been regarded as the final product of anoxic decomposition of organic matter by methanogenic archaea. However, plants2,3, fungi4, algae5 and cyanobacteria6 can produce CH4 in the presence of oxygen. Although methanogens are known to produce CH4 enzymatically during anaerobic energy metabolism7, the requirements and pathways for CH4 production by non-methanogenic cells are poorly understood. Here, we demonstrate that CH4 formation by Bacillus subtilis and Escherichia coli is triggered by free iron and reactive oxygen species (ROS), which are generated by metabolic activity and enhanced by oxidative stress. ROS-induced methyl radicals, which are derived from organic compounds containing sulfur- or nitrogen-bonded methyl groups, are key intermediates that ultimately lead to CH4 production. We further show CH4 production by many other model organisms from the Bacteria, Archaea and Eukarya domains, including in several human cell lines. All these organisms respond to inducers of oxidative stress by enhanced CH4 formation. Our results imply that all living cells probably possess a common mechanism of CH4 formation that is based on interactions among ROS, iron and methyl donors, opening new perspectives for understanding biochemical CH4 formation and cycling.
Assuntos
Archaea , Euryarchaeota , Metano , Archaea/metabolismo , Linhagem Celular , Fenômenos Fisiológicos Celulares , Humanos , Ferro/metabolismo , Metano/química , Metano/metabolismo , Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Enxofre/metabolismoRESUMO
Nitrogenases are the only known family of enzymes that catalyze the reduction of molecular nitrogen (N2 ) to ammonia (NH3 ). The N2 reduction drives biological nitrogen fixation and the global nitrogen cycle. Besides the conversion of N2 , nitrogenases catalyze a whole range of other reductions, including the reduction of the small gaseous substrates carbon monoxide (CO) and carbon dioxide (CO2 ) to hydrocarbons. However, it remains an open question whether these 'side reactivities' play a role under environmental conditions. Nonetheless, these reactivities and particularly the formation of hydrocarbons have spurred the interest in nitrogenases for biotechnological applications. There are three different isozymes of nitrogenase: the molybdenum and the alternative vanadium and iron-only nitrogenase. The isozymes differ in their metal content, structure, and substrate-dependent activity, despite their homology. This minireview focuses on the conversion of CO and CO2 to methane and higher hydrocarbons and aims to specify the differences in activity between the three nitrogenase isozymes.
Assuntos
Dióxido de Carbono , Nitrogenase , Dióxido de Carbono/química , Monóxido de Carbono/química , Hidrocarbonetos , Isoenzimas , Nitrogênio/química , Nitrogenase/química , OxirreduçãoRESUMO
NifB, a radical SAM enzyme, catalyzes the biosynthesis of the L cluster (Fe8S9C), a structural homolog and precursor to the nitrogenase active-site M cluster ([MoFe7S9C·R-homocitrate]). Sequence analysis shows that NifB contains the CxxCxxxC motif that is typically associated with the radical SAM cluster ([Fe4S4]SAM) involved in the binding of S-adenosylmethionine (SAM). In addition, NifB houses two transient [Fe4S4] clusters (K cluster) that can be fused into an 8Fe L cluster concomitant with the incorporation of an interstitial carbide ion, which is achieved through radical SAM chemistry initiated at the [Fe4S4]SAM cluster upon its interaction with SAM. Here, we report a VTVH MCD/EPR spectroscopic study of the L cluster biosynthesis on NifB, which focuses on the initial interaction of SAM with [Fe4S4]SAM in a variant NifB protein (MaNifBSAM) containing only the [Fe4S4]SAM cluster and no K cluster. Titration of MaNifBSAM with SAM reveals that [Fe4S4]SAM exists in two forms, labeled [Formula: see text] and [Formula: see text]. It is proposed that these forms are involved in the synthesis of the L cluster. Of the two cluster types, only [Formula: see text] initially interacts with SAM, resulting in the generation of Z, an S = ½ paramagnetic [Fe4S4]SAM/SAM complex.
Assuntos
Proteínas de Bactérias/metabolismo , Dicroísmo Circular/métodos , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Bactérias/genética , Ligação Proteica , Conformação Proteica , S-Adenosilmetionina/químicaRESUMO
The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to â¼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.
Assuntos
Álcoois Benzílicos/metabolismo , Biotina/metabolismo , Ferro/metabolismo , Oxigenases de Função Mista/metabolismo , Estreptavidina/metabolismo , Álcoois Benzílicos/química , Biotina/química , Hidroxilação , Ferro/química , Oxigenases de Função Mista/química , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Estreptavidina/químicaRESUMO
Bioorthogonal uncaging reactions offer versatile tools in chemical biology. In recent years, reactions have been developed to proceed efficiently under physiological conditions. We present herein an uncaging reaction that results from ring-closing metathesis (RCM). A caged molecule, tethered to a diolefinic substrate, is released via spontaneous 1,4-elimination following RCM. Using this strategy, which we term "close-to-release", we show that drugs and fluorescent probes are uncaged with fast rates, including in the presence of mammalian cells or in the periplasm of Escherichia coli. We envision that this tool may find applications in chemical biology, bioengineering and medicine.
Assuntos
Bioquímica/métodos , Corantes Fluorescentes/química , Naftalenos/química , Aminas/química , Meios de Cultura , Cicloexenos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Corantes Fluorescentes/farmacocinética , Cromatografia Gasosa-Espectrometria de Massas , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Niacina/química , Niacina/metabolismo , Umbeliferonas/metabolismoRESUMO
The biotin-streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17â¯000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.
Assuntos
Biotina/química , Engenharia Química/métodos , Metaloproteínas/química , Compostos Organometálicos/química , Estreptavidina/química , Sítios de Ligação , Biotina/genética , Biotinilação , Catálise , Cromatografia de Afinidade , Escherichia coli/genética , Hidrogenação , Irídio/química , Cinética , Metaloproteínas/genética , Estereoisomerismo , Estreptavidina/genéticaRESUMO
The iron (Fe) proteins of molybdenum (Mo) and vanadium (V) nitrogenases mimic carbon monoxide (CO) dehydrogenase in catalyzing the interconversion between CO2 and CO under ambient conditions. Catalytic reduction of CO2 to CO is achieved in vitro and in vivo upon redox changes of the Fe-protein-associated [Fe4S4] clusters. These observations establish the Fe protein as a model for investigation of CO2 activation while suggesting its biotechnological adaptability for recycling the greenhouse gas into useful products.
Assuntos
Dióxido de Carbono/metabolismo , Oxirredutases/metabolismo , Azotobacter vinelandii/enzimologia , Biocatálise , Dióxido de Carbono/química , Oxirredução , Oxirredutases/química , Oxirredutases/isolamento & purificaçãoRESUMO
NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster-bound NifEN (designated NifEN(M)) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifEN(M) upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN(M) with an M-cluster in one αß-half and an empty cluster-binding site in the other αß-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αß-dimers of NifEN. Perhaps most importantly, NifEN(M) displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αß-dimer of NifEN(M) as a structural and functional mimic of one catalytic αß-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase.
Assuntos
Azotobacter vinelandii/química , Coenzimas/química , Molibdênio/química , Molibdoferredoxina/química , Nitrogenase/química , Subunidades Proteicas/química , Azotobacter vinelandii/enzimologia , Domínio Catalítico , Coenzimas/isolamento & purificação , Coenzimas/metabolismo , Ferro/química , Ferro/metabolismo , Quelantes de Ferro/química , Molibdênio/metabolismo , Molibdoferredoxina/isolamento & purificação , Molibdoferredoxina/metabolismo , Nitrogenase/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Ligação Proteica , Multimerização Proteica , Subunidades Proteicas/metabolismoRESUMO
Artificial metalloenzymes (ArMs), which combine an abiotic metal cofactor with a protein scaffold, catalyze various synthetically useful transformations. To complement the natural enzymes' repertoire, effective optimization protocols to improve ArM's performance are required. Here we report on our efforts to optimize the activity of an artificial transfer hydrogenase (ATHase) using Escherichia coli whole cells. For this purpose, we rely on a self-immolative quinolinium substrate which, upon reduction, releases fluorescent umbelliferone, thus allowing efficient screening. Introduction of a loop in the immediate proximity of the Ir-cofactor afforded an ArM with up to 5-fold increase in transfer hydrogenation activity compared to the wild-type ATHase using purified mutants.
Assuntos
Hidrogenase/química , Metaloproteínas/química , Engenharia de Proteínas/métodos , Compostos de Quinolínio/química , Umbeliferonas/química , Sequência de Aminoácidos , Sequência de Bases , Evolução Molecular Direcionada/métodos , Escherichia coli/metabolismo , Hidrogenase/genética , Hidrogenação , Metaloproteínas/genética , Oxirredução , Periplasma/metabolismo , Compostos de Quinolínio/síntese química , Umbeliferonas/síntese químicaRESUMO
Two reaction systems based on vanadium nitrogenase were previously shown to reduce CO2 to hydrocarbons: 1)â an enzyme-based system that used both components of Vâ nitrogenase for ATP-dependent reduction of CO2 to ≤C2 hydrocarbons; and 2)â a cofactor-based system that employed SmI2 to supply electrons to the isolated Vâ cluster for an ATP-independent reduction of CO2 to ≤C3 hydrocarbons. Here, we report ATP-independent reduction of CO2 to hydrocarbons by a reaction system comprising Eu(II) DTPA and the VFe protein of Vâ nitrogenase. Combining features of both enzyme- and cofactor-based systems, this system exhibits improved C-C coupling and a broader product profile of ≤C4 hydrocarbons. The C-C coupling does not employ CO2 -derived CO, and it is significantly enhanced in D2 O. These observations afford initial insights into the characteristics of this unique reaction and provide a potential template for future design of catalysts to recycle the greenhouse gas CO2 into useful products.
Assuntos
Azotobacter vinelandii/enzimologia , Dióxido de Carbono/metabolismo , Hidrocarbonetos/metabolismo , Nitrogenase/metabolismo , Azotobacter vinelandii/química , Azotobacter vinelandii/metabolismo , Monóxido de Carbono/metabolismo , Európio/metabolismo , Hidrocarbonetos/química , Modelos Moleculares , OxirreduçãoRESUMO
The molybdenum and vanadium nitrogenases are two homologous enzymes with distinct structural and catalytic features. Previously, it was demonstrated that the Vâ nitrogenase was nearly 700 times more active than its Mo counterpart in reducing CO to hydrocarbons. Herein, a similar discrepancy between the two nitrogenases in the reduction of CO2 is reported, with the Vâ nitrogenase being capable of reducing CO2 to CO, CD4, C2D4, and C2D6, and its Moâ counterpart only capable of reducing CO2 to CO. Furthermore, it is shown that the Vâ nitrogenase may direct the formation of CD4 in part via CO2-derived CO, but that it does not catalyze the formation of C2D4 and C2D6 along this route. The exciting observation of a Vâ nitrogenase-catalyzed C-C coupling with CO2 as the origin of the building blocks adds another interesting reaction to the catalytic repertoire of this unique enzyme system. The differential activities of the V and Moâ nitrogenases in CO2 reduction provide an important framework for systematic investigations of this reaction in the future.
Assuntos
Dióxido de Carbono/química , Molibdênio/química , Nitrogenase/química , OxirreduçãoRESUMO
Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviors of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.IMPORTANCENitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.
Assuntos
Nitrogenase , Rhodobacter capsulatus , Nitrogenase/metabolismo , Fixação de Nitrogênio/genética , Ferredoxinas/metabolismo , Proteoma/metabolismo , Ferro/metabolismo , Amônia/metabolismo , Nitrogênio/metabolismoRESUMO
Nitrogenases are best known for catalyzing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO2) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the three nitrogenase isozymes, the iron nitrogenase has the highest wild-type activity for the reduction of CO2, but the molecular architecture facilitating these activities has remained unknown. Here, we report a 2.35-Å cryogenic electron microscopy structure of the ADP·AlF3-stabilized iron nitrogenase complex from Rhodobacter capsulatus, revealing an [Fe8S9C-(R)-homocitrate] cluster in the active site. The enzyme complex suggests that the iron nitrogenase G subunit is involved in cluster stabilization and substrate channeling and confers specificity between nitrogenase reductase and catalytic component proteins. Moreover, the structure highlights a different interface between the two catalytic halves of the iron and the molybdenum nitrogenase, potentially influencing the intrasubunit 'communication' and thus the nitrogenase mechanism.
Assuntos
Dióxido de Carbono , Ferro , Ferro/metabolismo , Dióxido de Carbono/química , Oxirredução , Nitrogenase/química , Nitrogenase/metabolismo , Hidrocarbonetos/metabolismoRESUMO
Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and "Agrobacterium fabrum" C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in R. capsulatus.
Assuntos
Alphaproteobacteria , Vetores Genéticos , Plasmídeos , Plasmídeos/genética , Vetores Genéticos/genética , Alphaproteobacteria/genética , Especificidade de Hospedeiro/genéticaRESUMO
Methane is a potent greenhouse gas, which likely enabled the evolution of life by keeping the early Earth warm. Here, we demonstrate routes towards abiotic methane and ethane formation under early-earth conditions from methylated sulfur and nitrogen compounds with prebiotic origin. These compounds are demethylated in Fenton reactions governed by ferrous iron and reactive oxygen species (ROS) produced by light and heat in aqueous environments. After the emergence of life, this phenomenon would have greatly intensified in the anoxic Archean by providing methylated sulfur and nitrogen substrates. This ROS-driven Fenton chemistry can occur delocalized from serpentinization across Earth's humid realm and thereby substantially differs from previously suggested methane formation routes that are spatially restricted. Here, we report that Fenton reactions driven by light and heat release methane and ethane and might have shaped the chemical evolution of the atmosphere prior to the origin of life and beyond.
RESUMO
Artificial metalloenzymes result from anchoring a metal cofactor within a host protein. Such hybrid catalysts combine the selectivity and specificity of enzymes with the versatility of (abiotic) transition metals to catalyze new-to-nature reactions in an evolvable scaffold. With the aim of improving the localization of an arylsulfonamide-bearing iridium-pianostool catalyst within human carbonic anhydrase II (hCAII) for the enantioselective reduction of prochiral imines, we introduced a covalent linkage between the host and the guest. Herein, we show that a judiciously positioned cysteine residue reacts with a p-nitropicolinamide ligand bound to iridium to afford an additional sulfonamide covalent linkage. Three rounds of directed evolution, performed on the dually anchored cofactor, led to improved activity and selectivity for the enantioselective reduction of harmaline (up to 97% ee (R) and >350 turnovers on a preparative scale). To evaluate the substrate scope, the best hits of each generation were tested with eight substrates. X-ray analysis, carried out at various stages of the evolutionary trajectory, was used to scrutinize (i) the nature of the covalent linkage between the cofactor and the host as well as (ii) the remodeling of the substrate-binding pocket.
RESUMO
Artificial metalloenzymes combine a synthetic metallocofactor with a protein scaffold and can catalyze abiotic reactions in vivo. Herein, we report on our efforts to valorize human carbonic anhydrase II as a scaffold for whole-cell transfer hydrogenation. Two platforms were tested: periplasmic compartmentalization and surface display in Escherichia coli. A chemical optimization of an IrCp* cofactor was performed. This led to 90 turnovers in the cell, affording a 69-fold increase in periplasmic product formation over the previously reported, sulfonamide-bearing IrCp* cofactor. These findings highlight the versatility of carbonic anhydrase as a promising scaffold for whole-cell catalysis with artificial metalloenzymes.
RESUMO
Bioorthogonal chemistry largely relies on the use of abiotic metals to catalyze new-to-nature reactions in living systems. Over the past decade, metal complexes and metal-encapsulated systems such as nanoparticles have been developed to unravel the reactivity of transition metals, including ruthenium, palladium, iridium, copper, iron, and gold in biological systems. Thanks to these remarkable achievements, abiotic catalysts are able to fluorescently label cells, uncage or form cytotoxic drugs and activate enzymes in cellulo/vivo. Recently, strategies for the delivery of such catalysts to specific cell types, cell compartments or proteins were established. These studies reveal the enormous potential of this emerging field and its application in both medicinal chemistry and in synthetic biology.
Assuntos
Química Inorgânica/métodos , Animais , Catálise , Humanos , Nanopartículas Metálicas/química , Metais/químicaRESUMO
The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The "normalization" of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the "weighted" contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.
Assuntos
Azotobacter vinelandii/enzimologia , Coenzimas/metabolismo , Nitrogenase/metabolismo , Azotobacter vinelandii/genética , Monóxido de Carbono/metabolismo , Hidrocarbonetos/metabolismo , Nitrogenase/genética , OxirreduçãoRESUMO
The vanadium (V)-nitrogenase of Azotobacter vinelandii catalyses the in vitro conversion of carbon monoxide (CO) to hydrocarbons. Here we show that an A. vinelandii strain expressing the V-nitrogenase is capable of in vivo reduction of CO to ethylene (C2H4), ethane (C2H6) and propane (C3H8). Moreover, we demonstrate that CO is not used as a carbon source for cell growth, being instead reduced to hydrocarbons in a secondary metabolic pathway. These findings suggest a possible role of the ancient nitrogenase as an evolutionary link between the carbon and nitrogen cycles on Earth and establish a solid foundation for biotechnological adaptation of a whole-cell approach to recycling carbon wastes into hydrocarbon products. Thus, this study has several repercussions for evolution-, environment- and energy-related areas.