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Jeffrey A Gralnick - One of the best experts on this subject based on the ideXlab platform.
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flavin electron shuttles dominate extracellular electron transfer by shewanella oneidensis
Mbio, 2013Co-Authors: Nicholas J Kotloski, Jeffrey A GralnickAbstract:ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete Flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe ( b acterial f lavin adenine dinucleotide [FAD] e xporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular Flavins, while overexpression of bfe increased the concentration of extracellular Flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous Flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δ bfe mutant. With the same concentration of supplemented Flavins, the two strains produced similar amounts of current. However, when exogenous Flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete Flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.
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flavin electron shuttles dominate extracellular electron transfer by shewanella oneidensis
Mbio, 2013Co-Authors: Nicholas J Kotloski, Jeffrey A GralnickAbstract:ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete Flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe ( b acterial f lavin adenine dinucleotide [FAD] e xporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular Flavins, while overexpression of bfe increased the concentration of extracellular Flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous Flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δ bfe mutant. With the same concentration of supplemented Flavins, the two strains produced similar amounts of current. However, when exogenous Flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete Flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.
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the mtr respiratory pathway is essential for reducing Flavins and electrodes in shewanella oneidensis
Journal of Bacteriology, 2010Co-Authors: Dan Coursolle, Daniel B Baron, Daniel R Bond, Jeffrey A GralnickAbstract:The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated Flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External Flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external Flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of Flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external Flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce Flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.
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the mtr respiratory pathway is essential for reducing Flavins and electrodes in shewanella oneidensis
Journal of Bacteriology, 2010Co-Authors: Dan Coursolle, Daniel B Baron, Daniel R Bond, Jeffrey A GralnickAbstract:The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated Flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External Flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external Flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of Flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external Flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce Flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.
Dan Coursolle - One of the best experts on this subject based on the ideXlab platform.
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the mtr respiratory pathway is essential for reducing Flavins and electrodes in shewanella oneidensis
Journal of Bacteriology, 2010Co-Authors: Dan Coursolle, Daniel B Baron, Daniel R Bond, Jeffrey A GralnickAbstract:The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated Flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External Flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external Flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of Flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external Flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce Flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.
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the mtr respiratory pathway is essential for reducing Flavins and electrodes in shewanella oneidensis
Journal of Bacteriology, 2010Co-Authors: Dan Coursolle, Daniel B Baron, Daniel R Bond, Jeffrey A GralnickAbstract:The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated Flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External Flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external Flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of Flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external Flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce Flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.
Vincent Massey - One of the best experts on this subject based on the ideXlab platform.
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reaction of reduced Flavins and flavoproteins with diphenyliodonium chloride
Journal of Biological Chemistry, 2002Co-Authors: Sumita Chakraborty, Vincent MasseyAbstract:Abstract The reaction of diphenyliodonium chloride with free reduced Flavins has been studied by stopped flow spectrophotometry under anaerobic conditions, and second order rate constants were determined as a function of pH. The reactive flavin species was identified as the reduced anion, based on an observed reaction pK of 6.7. The product mixture was independent of the initial concentration of reactant and contained ∼20% oxidized flavin. The results can be modeled quantitatively on a modification of the mechanism proposed by Tew (Tew, D. G. (1993)Biochemistry 32, 10209–10215). The composition of the complex reaction mixture has been analyzed, and four flavin-phenyl adducts with distinctive absorbance and fluorescence characteristics have been identified, involving substitution at the flavin C4a, N5, and C8 positions. Inactivation of flavoprotein enzymes by diphenyliodonium has also been studied, and several examples were found where inactivation occurs readily, despite noninvolvement of radical intermediates in their reaction mechanisms. It can be concluded that inactivation by phenyliodonium species is not a valid indicator of catalytic mechanism involving radical intermediates. One of the several factors determining inactivation is maintenance of the enzyme flavin in the reduced form in the steady state of catalysis, the other factors being redox potential and accessibility of the inhibitor to the flavin active site.
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old yellow enzyme reduction of nitrate esters glycerin trinitrate and propylene 1 2 dinitrate
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Younus Meah, Sumita Chakraborty, B J Brown, Vincent MasseyAbstract:The reaction of the old yellow enzyme and reduced Flavins with organic nitrate esters has been studied. Reduced Flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M−1s−1 and 5.8 M−1s−1, respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme–substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme–substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzyme-catalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the free-flavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced Flavins with GTN, reduced 5-deazaFlavins have been found to react some 4–5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.
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the chemical and biological versatility of riboflavin
Biochemical Society Transactions, 2000Co-Authors: Vincent MasseyAbstract:Since their discovery and chemical characterization in the 1930s, Flavins have been recognized as being capable of both one- and two-electron transfer processes, and as playing a pivotal role in coupling the two-electron oxidation of most organic substrates to the one-electron transfers of the respiratory chain. In addition, they are now known as versatile compounds that can function as electrophiles and nucleophiles, with covalent intermediates of flavin and substrate frequently being involved in catalysis. Flavins are thought to contribute to oxidative stress through their ability to produce superoxide, but at the same time Flavins are frequently involved in the reduction of hydroperoxides, products of oxygen-derived radical reactions. Flavoproteins play an important role in soil detoxification processes via the hydroxylation of many aromatic compounds, and a simple flavoprotein in liver microsomes catalyses many reactions similar to those carried out by cytochrome P450 enzymes. Flavins are involved in the production of light in bioluminescent bacteria, and are intimately connected with light-initiated reactions such as plant phototropism and nucleic acid repair processes. Recent reports also link them to programmed cell death. The chemical versatility of flavoproteins is clearly controlled by specific interactions with the proteins with which they are bound. One of the main thrusts of current research is to try to define the nature of these interactions, and to understand in chemical terms the various steps involved in catalysis by flavoprotein enzymes.
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structure function correlations of the reaction of reduced nicotinamide analogues with p hydroxybenzoate hydroxylase substituted with a series of 8 substituted Flavins
Biochemistry, 1999Co-Authors: Mariliz Ortizmaldonado, David P Ballou, Domenico L Gatti, Vincent MasseyAbstract:Structural and kinetic studies have revealed two flavin conformations in p-hydroxybenzoate hydroxylase (PHBH), the in-position and the out-position. Conversion between these two conformations is believed to be essential during catalysis. Although substrate hydroxylation occurs while the flavin in PHBH is in the in-conformation, the position of the flavin during reduction by NADPH is uncertain. To investigate the catalytic importance of the out-conformation of the flavin and to clarify the mechanism of flavin reduction in PHBH, we report quantitative structure-reactivity relationships (QSAR) using PHBH substituted separately with nine derivatives of FAD modified in the 8-position and four dihydronicotinamide analogues as reducing agents. The 8-position of the FAD isoalloxazine ring was chosen for modification because in PHBH it has minimal interactions with the protein and is accessible to solvent. The chemical sequence of events during catalysis by PHBH was not altered when using any of the modified Flavins, and normal products were obtained. Although the rate of reduction of PHBH reconstituted with flavin derivatives is expected to be dependent on the redox potential of the flavin, no strict correlation was observed. Instead, the rate of reduction correlated with the kappa-substituent constant, which is based on size and hydrophobicity of the 8-substituent on the FAD. Substituents that sterically hinder attainment of the out-conformation decreased the rate of flavin reduction much more than expected on the basis of the redox potential of the flavin. The results of this QSAR analysis are consistent with the hypothesis that the flavin in PHBH must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction.
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oxygen activation by Flavins and pterins
1995Co-Authors: Bruce A. Palfey, David P Ballou, Vincent MasseyAbstract:As discussed in other chapters in this volume, the high reduction potential of O2 makes it thermodynamically an excellent oxidizing agent for most biochemicals. However, the activation energy for these reactions is rather high, and O2 is kinetically unreactive without the intervention of a catalyst. Furthermore, the products generated by the uncontrolled reduction of O2 are often toxic. Such reductions usually produce highly reactive radicals, which react nonspecifically with the many reducing cellular compounds. Thus, the reactions of O2 must be strictly controlled. The task of oxygen-utilizing enzymes, then, is to accelerate the reaction of O2 at the enzyme active site and control the fate of the activated oxygen intermediate so that only a specific organic compound is oxidized. Enzymes that activate O2 generally require the participation of either metals or organic cofactors. Metals are discussed in other chapters. The most widely occurring and best-studied organic cofactors that activate O2 are the Flavins, derivatives of vitamin B2 (riboflavin; FMN without the phosphate). The structures of the two most common Flavins, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), are shown overleaf.
Javier Abadia - One of the best experts on this subject based on the ideXlab platform.
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iron deficient medicago scutellata grown in nutrient solution at high ph accumulates and secretes large amounts of Flavins
Plant Science, 2021Co-Authors: Zahra Gheshlaghi, Adrian Luisvillarroya, Ana Alvarezfernandez, Reza Khorassani, Javier AbadiaAbstract:Flavin synthesis and secretion is an integral part of the toolbox of root-borne Fe facilitators used by Strategy I species upon Fe deficiency. The Fe-deficiency responses of the wild legume Medicago scutellata grown in nutrient solution have been studied at two different pH values (5.5 and 7.5). Parameters studied include leaf chlorophyll, nutrient solution pH, concentrations and contents of micronutrients, flavin accumulation in roots, flavin export to the medium, and root ferric chelate reductase and acidification activities. Results show that M. scutellata behaves upon Fe deficiency as a Strategy I species, with a marked capacity for synthesizing Flavins (riboflavin and three hydroxylated riboflavin derivatives), which becomes more intense at high pH. Results also show that this species is capable of exporting a large amount of Flavins to the external medium, both at pH 5.5 and 7.5. This is the first report of a species having a major flavin secretion at pH 7.5, in contrast with the very low flavin secretion found in other flavin-producing species such as Beta vulgaris and M. truncatula. These results provide further support to the hypothesis that flavin secretion is relevant for Fe acquisition at high pH, and open the possibility to improve the Fe-efficiency responses in legumes of agronomic interest.
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Flavins secreted by roots of iron deficient beta vulgaris enable mining of ferric oxide via reductive mechanisms
New Phytologist, 2016Co-Authors: Patricia Sisoterraza, Javier Abadia, Juan J Rios, Anunciacion Abadia, Ana AlvarezfernandezAbstract:Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete Flavins upon Fe deficiency, to study whether or not Flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of Flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of Flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular Flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of Flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms.
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characterization of Flavins in roots of fe deficient strategy i plants with a focus on medicago truncatula
Plant and Cell Physiology, 2011Co-Authors: Jorge Rodriguezcelma, Ana Alvarezfernandez, Javier Abadia, Anunciacion Abadia, Saul Vazquezreina, Jesus Orduna, Anaflor LopezmillanAbstract:The root accumulation and excretion of riboflavin (Rbfl) and Rbfl derivatives have been studied in the model legume species Medicago truncatula, grown in hydroponics in two different Fe deficiency conditions, with and without CaCO(3). Using high resolution mass spectrometry techniques coupled to liquid chromatography, three different flavin derivatives not previously reported in plants, putatively identified as 7-hydroxy-Rbfl, 7α-hydroxy-Rbfl and 7-carboxy-Rbfl, were found along with Rbfl in Fe-deficient M. truncatula roots. In the presence of CaCO(3) most of the Flavins were accumulated in the roots, whereas in the absence of CaCO(3) there was partial export to the nutrient solution. The major Flavins in roots and nutrient solution were Rbfl and 7-hydroxy-Rbfl, respectively. Flavins were located in the root cortex and epidermal cells, preferentially in a root region near the apex that also exhibited increased ferric chelate reductase (FCR) activity. Six out of 15 different species of horticultural interest showed root increases in both Rbfl (four of them also having Rbfl derivatives) and FCR. No significant correlation was found between Rbfl and either phosphoenolpyruvate carboxylase or FCR activities, whereas the latter two showed a good correlation between them. The possible roles of Rbfl and Rbfl derivatives in roots and nutrient solutions are discussed. Medicago truncatula is proposed as a model system for flavin studies.
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riboflavin 3 and 5 sulfate two novel Flavins accumulating in the roots of iron deficient sugar beet beta vulgaris
Journal of Biological Chemistry, 1993Co-Authors: Santos A Susin, Anunciacion Abadia, Joaquin Abian, Francisco Sanchezbaeza, M L Peleato, E Gelpi, Javier AbadiaAbstract:Roots from iron-deficient sugar beet grown in the presence of calcium carbonate exhibit a yellow color and autofluorescence typical of flavin-like compounds, whereas roots of control, iron-sufficient plants exhibited no yellow color and extremely low autofluorescence. The two major Flavins whose accumulation is induced by iron deficiency have been shown to be different from riboflavin, FMN, and FAD by reversed-phase high performance liquid chromatography. These Flavins, accounting for 82 and 15% of the total flavin concentration in deficient roots, have been shown unequivocally to be riboflavin 3‘-sulfate and riboflavin 5‘-sulfate, respectively, by electrospray-mass spectrometry, inductively coupled plasma emission spectroscopy, infrared spectrometry, and 1H nuclear magnetic resonance. These flavin sulfates have not been found previously in biological systems. The localization of riboflavin sulfates in deficient roots is similar, but not identical, to that of high iron reductase activity. The concentration of riboflavin sulfates has been estimated from root extracts to be at least 1 mM. We hypothesize, based on the similar localization of flavin and that of iron reduction, that the accumulation of riboflavin sulfates induced by iron deficiency may be an integral part of the turbo iron-reducing system in sugar beet roots.
Annefrances Miller - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic evidence for direct flavin flavin contact in a bifurcating electron transfer flavoprotein
Journal of Biological Chemistry, 2020Co-Authors: Diessel H Duan, Nishya Mohamedraseek, Annefrances MillerAbstract: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.
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distinct properties underlie flavin based electron bifurcation in a novel electron transfer flavoprotein fixab from rhodopseudomonas palustris
Journal of Biological Chemistry, 2018Co-Authors: Diessel H Duan, Carolyn E Lubner, Monika Tokminalukaszewska, George H Gauss, Brian Bothner, Paul W King, John W Peters, Annefrances MillerAbstract: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.
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solid state nmr of Flavins and flavoproteins
Methods of Molecular Biology, 2014Co-Authors: Annefrances MillerAbstract:: Why apply solid-state NMR (SSNMR) to Flavins and flavoproteins? NMR provides information on an atom-specific basis about chemical functionality, structure, proximity to other groups, and dynamics of the system. Thus, it has become indispensable to the study of chemicals, materials, catalysts, and biomolecules. It is no surprise then that NMR has a great deal to offer in the study of Flavins and flavoenzymes. In general, their catalytic or electron-transfer activity resides essentially in the flavin, a molecule eminently accessible by NMR. However, the specific reactivity displayed depends on a host of subtle interactions whereby the protein biases and reshapes the flavin's propensities to activate it for one reaction while suppressing other aspects of this cofactor's prodigious repertoire (Massey et al., J Biol Chem 244:3999-4006, 1969; Muller, Z Naturforsch 27B:1023-1026, 1972; Joosten and van Berkel, Curr Opin Struct Biol 11:195-202, 2007). Thus, we are fascinated to learn about how the flavin cofactor of one enzyme is, and is not, like the flavin cofactor of another. In what follows, we describe how the capabilities of SSNMR can help and are beginning to bear fruit in this exciting endeavor.