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Biochemical characterization of a novel iron-sulfur flavoprotein from Methanosarcina thermophila strain TM-1Leartsakulpanich, Ubolsree 30 June 1999 (has links)
The iron-sulfur flavoprotein (Isf) from the acetate utilizing methanoarchaeon Methanosarcina thermophila was heterologously produced in Escherichia coli, purified to homogeneity, and characterized to determine the properties of the iron-sulfur cluster and FMN. Chemical and spectroscopic analyses indicated that Isf contained one 4Fe-4S cluster and one FMN per monomer. The midpoint potentials of the [4Fe-4S]2+/1+ center and FMN/FMNH2 redox couple were -394 and -277 mV respectively.
The deduced amino acid sequence of Isf revealed high identity with Isf homologues from the CO2 reducing methanoarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum. Extracts of H2-CO2-grown M. thermoautotrophicum cells were able to reduce Isf from M. thermophila using either H2 or CO as the reductant. Addition of ferredoxin A to the reaction further stimulated the rate of Isf reduction. These results suggest that Isf homologues are coupled to ferredoxin in electron transfer chains in methanoarchaea with diverse metabolic pathways.
Reconstituted systems containing carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS), ferredoxin A, Isf, and the designated electron carriers (NAD, NADP, F420, and 2-hydroxyphenazine) were used in an attempt to determine the electron acceptor for Isf. Isf was unable to reduce any of these compounds. Furthermore, 2-hydroxyphenazine competed with Isf to accept electrons from ferredoxin A indicating that ferredoxin A is a more favorable electron partner for 2-hydroxyphenazine. Thus, the physiological electron acceptor for Isf is unknown.
Amino acid sequence alignment of Isf sequences revealed a conserved atypical cysteine motif with the potential to ligate the 4Fe-4S cluster. Site-directed mutagenesis of the cysteine residues in this motif, and the two additional cysteines in the sequence, was used to investigate these cysteine residue as ligands for coordinating the 4Fe-4S center of Isf. Spectroscopic and biochemical analyses were consistent with the conserved cysteine motif functioning as ligating the 4Fe-4S center. Redox properties of the 4Fe-4S and FMN centers revealed a role for the 4Fe-4S center in the transfer of electrons from ferredoxin A to FMN. / Ph. D.
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Probing the dynamics and conformational landscape of neuronal nitric oxide synthaseSobolewska-Stawiarz, Anna January 2014 (has links)
Rat neuronal nitric oxide synthase (nNOS) is a flavo-hemoprotein that catalyses the NADPH and O2-dependent conversion of L-arginine (L-arg) to L-citrulline and nitric oxide (NO) via the intermediate N-hydroxyarginine. nNOS is a homodimer, where the subunits are modular and are comprised of an N-terminal oxygenase domain (nNOSoxy) that binds iron protoporphyrin IX (heme), (6R)-5,6,7,8-tetrahydro-biopterin (H4B) and L-arg, and a C-terminal flavoprotein or reductase domain (nNOSred) that binds NADPH, FAD and FMN. Regulation of NO biosynthesis by nNOS is primarily through control of interdomain electron transfer processes in NOS catalysis. The interdomain electrons transferred from the FMN to the heme domain are essential in the delivery of electrons required for O2 activation (which occurs in the heme domain) and the subsequent NO synthesis by NOS. Both spectroscopic and kinetic approaches have been used in studying the nature and control of interdomain electron transfer, reaction mechanism and structural changes during catalysis in WT and R1400E nNOS in both full length (FL) and nNOSred. Cytochrome c reduction activity of nNOS was used to determine kinetic parameters for NADPH for FL and nNOSred, WT and R1400E nNOS in the presence and absence of calmodulin (CaM). FL nNOS, where both domains (nNOSred and nNOSoxy) were present, was proven to be more stable and more catalytically efficient than nNOSred by itself. Additionally it was observed that R1400E is still promoting electron transfer despite being thought to lower the affinity of the enzyme to the substrate (NADPH); R1400E also showed lower catalytic efficiency and lower dependence on CaM/Ca2+ compared to the WT. The structure of the functional output state has not yet been determined. In the absence of crystallographic structural data for the NOS holoenzyme, it was important to experimentally determine conformational changes and distances between domains in nNOS. A pulsed EPR spectroscopy (PELDOR) approach has been utilised to gain important and unique information about the conformational energy landscape changes in nNOS. In the presence of CaM, PELDOR results for FL WT nNOS shows a complex energy landscape with multiple conformational states, while in the absence of CaM the interflavin distance distribution matches that exhibited by nNOSred CaM- in the presence of NADP+, suggesting that CaM binding affects some major large-scale conformational changes which are involved in internal electron transfer control in nNOS. A high-pressure stopped-flow technique was also used to perturb an equilibrium distribution of conformational states, to observe the effect of the pressure on the internal electron transfer and to study the kinetics of NADPH oxidation, flavin reduction by NADPH and NO formation. It was shown that high pressure is forcing major changes in the conformational energy landscape of the protein, affecting internal electron transfer. NO formation studies under pressure show that the R1400E mutation in FL nNOS may be affecting protein/NADPH affinity and flavin reduction, but it has no effect on the heme reduction step.
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Oxidative Biocatalysis with Novel NADH OxidasesJiang, Rongrong 28 June 2006 (has links)
Many oxidoreductases need nicotinamide cofactors for their reactions. The big obstacle of using these syntheses in industry is the high cost of these nicotinamide cofactors. The work here is about finding novel NADH oxidases from Lactococcus lactis and applying in a cofactor regeneration system with carbonyl reductase or alcohol dehydrogenase. NADH oxidases are useful biocatalysts for regenerating nicotinamide cofactors of biological redox reactions. The annotated alkyl hydroperoxide reductase (AhpR) and the H2O-forming enzyme (nox-2) genes from Lactococcus lactis (L. lactis, L.lac-Nox2) were cloned and proteins were expressed and characterized. They were compared with the H2O-former from Lactobacillus sanfranciscensis (L. sanfranciscensis, L.san-Nox2). AhpR is composed of H2O2-forming NADH oxidase (nox-1) and peroxidase and the net reaction of AhpR is the same as nox-2 when oxygen is the substrate. Both nox-1 and nox-2 are flavoproteins and turnover-limited. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2. L.san-Nox2 was crystallized and was found to have ADP ligand, but according to the HPLC results, no ADP ligand was found in the L. lac-Nox-2. Enzyme membrane reactor was used for the application of oxidative reaction of cyclohexanol to cyclohexanone, with isolated enzymes horse liver alcohol dehydrogenase and L.lac-Nox2.
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Characterisation of the active site of kynurenine 3-monooxygenaseBell, Helen Barbara January 2016 (has links)
Kynurenine 3-monooxygenase (KMO) is a flavoprotein which has been implicated in Huntington’s disease, Alzheimer’s disease and acute pancreatitis. Recently there has been important research published about this enzyme including the structure of a truncated Saccharomyces cerevisiae KMO enzyme and KMO inhibition studies in animal models of disease. In previous work from this research group the complete Pseudomonas fluorescens KMO enzyme has been successfully crystallised both with and without the substrate, L-kynurenine, from which significant insights were gained into function and the potential role of domain movement. To examine substrate binding in KMO and to consolidate previous structural studies, key residues in the active site have been investigated using site directed mutagenesis, crystallography and kinetic analysis using steady-state techniques. This analysis has identified the interactions between the enzyme and the substrate and provides a basis for inhibitor design. The residues implicated in substrate binding are N369, Y404 and R84. For N369 and Y404, minor changes to the amino acid in the mutations N369S and Y404F were shown to cause a decrease in binding affinity of the substrate but the enzyme remained active. For the mutations Y404A and R84K enzyme activity was significantly affected. Crystal structures of N369S, Y404F and R84K were also obtained. Another residue in the active site studied was H320 which is the only amino acid to differ in the active sites of the human and Pseudomonas fluorescens enzymes. This residue was therefore of interest to determine whether the bacterial enzyme used in this work is likely to be a good model for the human enzyme, which has not yet been successfully isolated in significant quantities for in vitro research. Modifying this residue to obtain H320F KMO revealed that this residue does not have a significant role in substrate binding. Potent inhibitor molecules have been studied with this enzyme and shown in kinetic assays to have nanomolar Ki values. These inhibitors are the most potent inhibitors studied with Pseudomonas fluorescens to date and continue previous inhibitor studies carried out with this enzyme. This group of inhibitors contain different substituents in the part of the molecule shown to bind closest to the C-terminal domain of the protein. These novel inhibitors do not allow the flavin to be reduced by NADPH (which results in unwanted peroxide production) unlike a number of previously studied molecules and therefore have the potential to be clinically useful. This research therefore answers many questions about this enzyme, in particular about the role of particular residues in the active site, substrate recognition and inhibition of this important drug target.
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Systematic Analysis of Structure-Function Relationships of Conserved Sequence Motifs in the NADH-Binding Lobe of Cytochrome <em>b<sub>5</sub></em> ReductaseRoma, Glenn W 15 July 2008 (has links)
NADH:Cytochrome b5 Reductase (cb5r) catalyzes the reduction of the ferric iron (Fe3+) atom of the heme cofactor found within cytochrome b5 (cb5) by the reduction of the FAD cofactor of cb5r from reducing equivalents of the physiological electron donor, reduced nicotinamide adenine dinucleotide (NADH). Cb5r is characterized by the presence of two domains necessary for proper enzyme function: a flavin-binding domain and a pyridine nucleotide-binding domain. Within these domains are highly conserved "motifs" necessary for the correct binding and orientation of both the NADH coenzyme and the FAD cofactor.
To address the importance of these conserved motifs, site-directed mutagenesis was utilized to generate a series of variants of residues located within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants found in proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis. In addition, a canine variant of the cb5r soluble domain was cloned, generated and characterized and compared with the WT rat domain.
The canine construct showed a high degree of sequence homology to that of the corresponding human and rat sequences. Characterization of the canine variant indicated that it possessed comparable functional characteristics to the rat variant.
Investigation of the pyrophosphate-associating residues, Y112 and Q210, indicated that each played a role in the proper association and anchoring of NADH to the enzyme. The RCM type I mutants, T116S and E212K, caused a moderate decrease in efficiency of the enzyme. The presence of both mutations interact synergistically to generate a more substantially decreased function
Analysis of the "180GtGitP185" NADH-binding motif and the preceding residue G179 revealed that these residues are vital in enabling proper NADH association. The residues of this motif were shown to be important in determining nucleotide specificity and properly positioning the NADH and flavin cofactor for efficient electron transfer. RCM variants A178T and A178V were shown to decrease catalytic efficiency or protein stability respectively, leading to disease phenotype.
Analysis of the NADH-binding motif "273CGxxxM278" indicated that this motif facilitates electron transfer from substrate to cofactor and is important in release of NAD+ from the enzyme after electron transfer.
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Structure-Function Studies of Conserved Sequence Motifs of Cytochrome <em>b</em><sub>5</sub> Reductase:Crowley, Louis J 11 April 2007 (has links)
NADH:Cytochrome b5 Reductase (cb5r) catalyzes the two electron reduction of the iron center of the heme cofactor found within cytochrome b5 (cb5) utilizing reducing equivalents of the nicotinamide adenine dinucleotide (NADH) coenzyme. Cb5r is characterized by two domains necessary for proper enzyme function: a flavin-binding domain and a pyridine nucleotide-binding domain. Within these domains are highly conserved "motifs" necessary for the proper binding and orientation of both the NADH coenzyme and the FAD cofactor.
To address the importance of these conserved motifs site-directed mutagenesis was utilized to generate a series of variants upon residues found within the motifs to allow for the full characterizations. Second, naturally occurring recessive congenital methemoglobinemia (RCM) mutants that are found within or in close proximity to these highly conserved motifs were analyzed utilizing site-directed mutagenesis.
The flavin-binding motif "91RxYSTxxSN97" was characterized by the generation of variants T94H, T94G, T94P, P95I, V96S, and S97N. In addition to this, the naturally occurring double mutant P92H/E255- was fully characterized to establish a role of the P92 residue giving rise to RCM.
The role of the "124GRxxST127" was determined by the introduction of a positive charge, charge reversal, and conserved amino acid mutations through site-directed mutagenesis of the G124, K125, and M126 residues. Based on the data presented here, each of the residues of the GRxxST motif are directly involved in maintaining the proper binding and orientation of the cb5r flavin prosthetic group.
Analysis of the NADH-binding motif "273CGxxx-M278" was accomplished through the characterization of the type II RCM variant M272- and the type I RCM variant P275L. This demonstrates that the deletion of the M272 residue causes a frame shift leading to the inability of the NADH substrate to bind. The introduction of the P275L variant showed that substrate affinity was diminished, yet turnover was comparable to wild-type cytochrome b5 reductase, indicating that although P275 is required for proper substrate binding it is not essential for overall catalytic function.
Finally, analysis of the naturally occurring double mutant G75S/V252M provided the first insight into a methemoglobinemia variant that possessed mutations in both the FAD-binding and NADH-binding domains.
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<sup>15</sup>N SOLID-STATE NMR DETECTION OF FLAVIN PERTURBATION BY H-BONDING IN MODELS AND ENZYME ACTIVE SITESCui, Dongtao 01 January 2010 (has links)
Massey and Hemmerich proposed that the different reactivities displayed by different flavoenzymes could be achieved as a result of dominance of different flavin ring resonance structures in different binding sites. Thus, the FMN cofactor would engage in different reactions when it had different electronic structures. To test this proposal and understand how different protein sites could produce different flavin electronic structures, we are developing solid-state NMR as a means of characterizing the electronic state of the flavin ring, via the 15N chemical shift tensors of the ring N atoms. These provide information on the frontier orbitals. We propose that the 15N chemical shift tensors of flavins engaged in different hydrogen bonds will differ from one another. Tetraphenylacetyl riboflavin (TPARF) is soluble in benzene to over 250 mM, so, this flavin alone and in complexes with binding partners provides a system for studying the effects of formation of specific hydrogen bonds. For N5, the redoxactive N atom, one of the chemical shift principle values (CSPVs) changed 10 ppm upon formation of a hydrogen bonded complex, and the results could be replicated computationally. Thus our DFT-derived frontier orbitals are validated by spectroscopy and can be used to understand reactivity. Indeed, our calculations indicate that the electron density in the diazabutadiene system diminishes upon H-bond complex formation, consistent with the observed 100 mV increase in reduction midpoint potential. Thus, the current studies of TPARF and its complexes provide a useful baseline for further SSNMR studies aimed at understanding flavin reactivity in enzymes.
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Redox and functional characterization of a surface loop spanning residues 536 to 541 in the flavin mononucleotide-binding domain of flavocytochrome P450BM-3 from Bacillus megateriumChen, Huai-Chun 27 August 2009 (has links)
No description available.
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Structural and mechanistic studies on eukaryotic UDP-galactopyranose mutasesOppenheimer, Michelle Lynn 26 April 2012 (has links)
Galactofuranose (Galf) is the five membered ring form of galactose. It is found on the cell wall and surface of many pathogens including Mycobacterium tuberculosis, Aspergillus fumigatus, Leishmania major, and Trypanosoma cruzi. Galf has been implicated in pathogenesis in these organisms; thus the biosynthetic pathway of Galf is a target for drug design. Galf is synthesized by the enzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). Solving the mechanism and structure of UGMs will aid in the development of specific inhibitors against these enzymes. Herein we present the detailed functional analysis of UGMs from A. fumigatus, T. cruzi, and L. major. The mechamism and structure these eukaryotic UGMs were examined by steady-state kinetics, rapid-reaction kinetics, trapping of reaction intermediates, fluorescence anisotropy, and X-ray crystallography. The mechanism first involves reduction of the required flavin by NADPH, followed by UDP-Galp binding and subsequent SN2 attack by the flavin on galactose displacing UDP to form a flavin N5-C1 galactose adduct. Next, the galactose ring opens forming an iminium ion allowing isomerization to occur. Lastly, the product is released and UGM is available to bind another substrate or be reoxidized by molecular oxygen. The three-dimensional structure of A. fumigatus UGM was solved using X-ray crystallography in four conformations: oxidized in complex with sulfate ions, reduced, reduced in complex with UDP, and reduced in complex with UDP-Galp, giving valuable information on the unique features of eukaryotic UGMs including features important for oligomerization and for substrate binding. The novel mechanism and structure provide valuable information for the development of specific inhibitors of eukaryotic UGMs. / Ph. D.
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PROTEIN SUPPRESSION OF FLAVIN SEMIQUINONE AS A MECHANISTICALLY IMPORTANT CONTROL OF REACTIVITY: A STUDY COMPARING FLAVOENZYMES WHICH DIFFER IN REDOX PROPERTIES, SUBSTRATES, AND ABILITY TO BIFURCATE ELECTRONSHoben, John Patrick 01 January 2018 (has links)
A growing number of flavoprotein systems have been observed to bifurcate pairs of electrons. Flavin-based electron bifurcation (FBEB) results in products with greater reducing power than that of the reactants with less reducing power. Highly reducing electrons at low reduction midpoint potential are required for life processes of both aerobic and anaerobic metabolic processes. For electron bifurcation to function, the semiquinone (SQ) redox intermediate needs to be destabilized in the protein to suppress its ability to trap electrons. This dissertation examines SQ suppression across a number of flavin systems for the purpose of better understanding the nature of SQ suppression within FBEB and elucidates potential mechanistic roles of SQ.
The major achievement of this work is advancing the understanding of SQ suppression and its utility in flavoproteins with the capacity to bifurcate pairs of electrons. Much of these achievements are highlighted in Chapter 6. To contextualize these mechanistic studies, we examined the kinetic and thermodynamic properties of non-bifurcating flavoproteins (Chapters 2 and 3) as well as bifurcating flavoproteins (Chapters 4 and 5). Proteins were selected as models for SQ suppression with the aim of elucidating the role of an intermediate SQ in bifurcation.
The chemical reactions of flavins and those mediated by flavoproteins play critical roles in the bioenergetics of all lifeforms, both aerobic and anaerobic. We highlight our findings in the context of electron bifurcation, the recently discovered third form of biological energy conservation.
Bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I (Nfn) and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin were studied by transient absorption spectroscopy to compare electron transfer rates and mechanisms in the picosecond range. Different mechanisms were found to dominate SQ decay in the different proteins, producing lifetimes ranging over 3 orders of magnitude. The presence of a short-lived SQ alone was found to be insufficient to infer bifurcating activity. We established a model wherein the short SQ lifetime in Nfn results from efficient electron propagation. Such mechanisms of SQ decay may be a general feature of redox active site ensembles able to carry out bifurcation.
We also investigated the proposed bifurcating electron transfer flavoprotein (Etf) from Pyrobaculum aerophilum (Pae), a hyperthermophilic archaeon. Unlike other Etfs, we observed a stable and strong charge transfer band (λmax= 724 nm) for Pae’s Etf upon reduction by NADH. Using a series of reductive titrations to probe bounds for the reduction midpoint potential of the two flavins, we argue that the heterodimer alone could participate in a bifurcation mechanism.
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