RESUMO
The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observed the canonical flavodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize partner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the 19F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3-19F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer.
Assuntos
Proteínas de Bactérias , Flavodoxina , Modelos Moleculares , Oxirredução , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Flavodoxina/química , Flavodoxina/metabolismo , Conformação Proteica , Tirosina/química , Tirosina/metabolismoRESUMO
Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.
Assuntos
Flavoproteínas Transferidoras de Elétrons , Flavoproteínas , Flavoproteínas Transferidoras de Elétrons/metabolismo , Flavoproteínas/química , Oxirredução , Flavinas/metabolismo , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/metabolismoRESUMO
Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.
RESUMO
Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.
Assuntos
Arginina , Flavina-Adenina Dinucleotídeo/análogos & derivados , Monofosfato de Adenosina/metabolismo , Arginina/metabolismo , Transporte de Elétrons , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/química , Flavinas/metabolismo , OxirreduçãoRESUMO
From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.
Assuntos
Aminas , Flavoproteínas Transferidoras de Elétrons , Flavoproteínas Transferidoras de Elétrons/metabolismo , Oxirredução , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/metabolismoRESUMO
A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.
Assuntos
Proteínas de Bactérias/química , Flavina-Adenina Dinucleotídeo/química , Flavoproteínas/química , Rodopseudomonas/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Flavina-Adenina Dinucleotídeo/genética , Flavoproteínas/genética , Rodopseudomonas/genéticaRESUMO
Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.
Assuntos
Proteínas de Bactérias/metabolismo , Flavinas/metabolismo , Oxigenases de Função Mista/metabolismo , 4-Hidroxibenzoato-3-Mono-Oxigenase/genética , 4-Hidroxibenzoato-3-Mono-Oxigenase/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Biocatálise , Domínio Catalítico , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fenilacetatos/química , Fenilacetatos/metabolismo , Rhodococcus/enzimologia , Especificidade por SubstratoRESUMO
Flavodoxin is a small protein that employs a non-covalently bound flavin to mediate single-electron transfer at low potentials. The long-chain flavodoxins possess a long surface loop that is proposed to interact with partner proteins. We have incorporated 19F-labeled tyrosine in long-chain flavodoxin from Rhodopseudomonas palustris to gain a probe of possible loop dynamics, exploiting the presence of a Tyr in the long loop in addition to Tyr residues near the flavin. We report 19F resonance assignments for all four Tyrs, and demonstration of a pair of resonances in slow exchange, both corresponding to a Tyr adjacent to the flavin. We also provide evidence for dynamics affecting the Tyr in the long loop. Thus, we show that 19F NMR of 19F-Tyr labeled flavodoxin holds promise for monitoring possible changes in conformation upon binding to partner proteins.
Assuntos
Flavodoxina/química , Flavodoxina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Modelos Moleculares , Conformação ProteicaRESUMO
Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.
Assuntos
Proteínas Arqueais/metabolismo , Biocatálise , Flavoproteínas Transferidoras de Elétrons/metabolismo , NAD/metabolismo , PyrobaculumRESUMO
A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° flavin displays sequential one-electron (1-e-) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° flavin displayed a single two-electron (2-e-) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.
Assuntos
Monofosfato de Adenosina/química , Proteínas de Bactérias/química , Flavoproteínas Transferidoras de Elétrons/química , Flavina-Adenina Dinucleotídeo/química , Rodopseudomonas/enzimologia , TermodinâmicaRESUMO
The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.
Assuntos
Elétrons , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavinas/metabolismo , Modelos Biológicos , Sítios de Ligação , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/químicaRESUMO
Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I and can be an indication of capacity for electron bifurcation.
Assuntos
Proteínas de Bactérias/metabolismo , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavodoxina/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Nitrorredutases/metabolismo , Oxirredutases/metabolismo , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácido Benzoico/química , Ácido Benzoico/metabolismo , Biocatálise , Desulfovibrio vulgaris/enzimologia , Transporte de Elétrons , Enterobacter cloacae/enzimologia , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavodoxina/química , Flavodoxina/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , Nitrorredutases/química , Nitrorredutases/genética , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Pyrococcus furiosus/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mutação Silenciosa , Thermus thermophilus/enzimologia , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismoRESUMO
Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance.
Assuntos
Aminas/metabolismo , Fermentação , Nitrorredutases/metabolismo , Aminas/química , Sítios de Ligação , Vias Biossintéticas , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , NAD/química , NAD/metabolismo , Nitrorredutases/química , Oxirredução , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.
Assuntos
Archaea/enzimologia , Bactérias/enzimologia , Flavoproteínas Transferidoras de Elétrons/classificação , Flavoproteínas Transferidoras de Elétrons/metabolismo , Motivos de Aminoácidos , Archaea/genética , Bactérias/genética , Biologia Computacional , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/genética , Modelos Moleculares , Oxirredução , Conformação ProteicaRESUMO
The catalytic active site of Mn-specific superoxide dismutase (MnSOD) is organized around a redox-active Mn ion. The most highly conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the active sites of these two types of SODs. Mutation of this Gln69 to Glu in Escherichia coli FeSOD increased the Fe3+/2+ reduction midpoint potential by >0.6 V without disrupting the structure or Fe binding [ Yikilmaz, E., Rodgers, D. W., and Miller, A.-F. ( 2006 ) Biochemistry 45 ( 4 ), 1151 - 1161 ]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, Q146E-apoMnSOD displays an apparent melting midpoint temperature (Tm) 35 °C higher that of wild-type (WT) apoMnSOD, whereas the Tm of WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, the Tm attributed to Q146E-holoMnSOD is 40 °C lower than that of Q146E-apoMnSOD. Thus, our data refute the notion that the WT residues optimize the structural stability of the protein and instead are consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy to favor metal ion binding.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Manganês/química , Mutação de Sentido Incorreto , Superóxido Dismutase/química , Substituição de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanidina/química , Humanos , Ferro , Superóxido Dismutase/genéticaRESUMO
The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.
Assuntos
Azotobacter vinelandii/enzimologia , Modelos Moleculares , Complexos Multienzimáticos/química , Nitrogenase/química , Catálise , Transporte de Elétrons/fisiologia , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nitrogenase/genética , Nitrogenase/metabolismo , Estrutura Quaternária de ProteínaRESUMO
Nuclear magnetic resonance is applied to investigate the relative positions and interactions between cationic and non-ionic carbohydrate-based surfactants in mixed micelles with D2 O as the solvent. This is accomplished by using relaxation measurements [spin-lattice (T1 ) and spin-spin (T2 ) analysis] and nuclear Overhauser effect spectroscopy (NOESY). This study focuses on the interactions of n-octyl ß-d-glucopyranoside (C8G1) and ß-d-xylopyranoside (C8X1) with the cationic surfactant hexadecyltrimethylammonium bromide (C16 TAB). Whereas the interactions between carbohydrate and cationic surfactants are thermodynamically favorable, the NOESY results suggest that both of the sugar head groups are located preferentially at the interior core of the mixed micelles, so that they are not directly exposed to the bulk solution. The more hydrophilic sugar headgroups of C8G1 have more mobility than sugar heads of C8X1 owing to increased hydration. Herein, an inverted carbohydrate configuration in mixed micelles is proposed for the first time and supported by fluorescence spectroscopy experiments. This inverted carbohydrate headgroup configuration would limit the use of these mixed surfactants when access to the carbohydrate headgroup is important, but may present new opportunities where the carbohydrate-rich core of the micelles can be exploited.
Assuntos
Compostos de Cetrimônio/química , Glicosídeos/química , Micelas , Tensoativos/química , Cátions/química , Cetrimônio , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s(-1), indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s(-1)). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s(-1) and a dissociation constant of 0.51 ± 0.04 mM. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines.
Assuntos
Enterobacter cloacae/enzimologia , Escherichia coli/enzimologia , Modelos Químicos , Nitrorredutases/química , Nitrorredutases/metabolismo , Aerobiose , Aminas/metabolismo , Anaerobiose , Biodegradação Ambiental , Dinitrocresóis/metabolismo , Ativação Enzimática/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , NAD/metabolismo , Nitrobenzoatos/metabolismo , Oxirredução , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
In order to properly evaluate the ecological and human health risks of ZnO manufactured nanomaterials (MNMs) released to the environment, it is critical to understand the likely transformation products in various environments, such as soils, surface and ground waters, and wastewater treatment processes. To address this knowledge gap, we examined the transformation of 30 nm ZnO MNMs in the presence of different concentrations of phosphate as a function of time and pH using a variety of orthogonal analytical techniques. The data reveal that ZnO MNMs react with phosphate at various concentrations and transform into two distinct morphological/structural phases: a micrometer scale crystalline zinc phosphate phase (hopeite-like) and a nanoscale phase that likely consists of a ZnO core with an amorphous Zn3(PO4)2 shell. The P species composition was also pH dependent, with 82% occurring as hopeite-like P at pH 6 while only 15% occurred as hopeite-like P at pH 8. These results highlight how reactions of ZnO MNMs with phosphate are influenced by environmental variables, including pH, and may ultimately result in structurally and morphologically heterogeneous end products.