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1.
Redox Biol ; 72: 103152, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38593630

RESUMO

Cellular magnetic field effects are assumed to base on coherent singlet-triplet interconversion of radical pairs that are sensitive to applied radiofrequency (RF) and weak magnetic fields (WEMFs), known as radical pair mechanism (RPM). As a leading model, the RPM explains how quantum effects can influence biochemical and cellular signalling. Consequently, radical pairs generate reactive oxygen species (ROS) that link the RPM to redox processes, such as the response to hypoxia and the circadian clock. Therapeutic nuclear magnetic resonance (tNMR) occupies a unique position in the RPM paradigm because of the used frequencies, which are far below the range of 0.1-100 MHz postulated for the RPM to occur. Nonetheless, tNMR was shown to induce RPM like effects, such as increased extracellular H2O2 levels and altered cellular bioenergetics. In this study we compared the impact of tNMR and intermittent hypoxia on the circadian clock, as well as the role of superoxide in tNMR induced ROS partitioning. We show that both, tNMR and intermittent hypoxia, exert on/off effects on cellular clocks that are dependent on the time of application (day versus night). In addition, our data provide further evidence that superoxide plays a central role in magnetic signal transduction. tNMR used in combination with scavengers, such as Vitamin C, led to strong ROS product redistributions. This discovery might represent the first indication of radical triads in biological systems.


Assuntos
Relógios Circadianos , Campos Magnéticos , Superóxidos , Superóxidos/metabolismo , Animais , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Humanos , Hipóxia Celular , Oxirredução , Camundongos
2.
Front Physiol ; 15: 1348395, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38370016

RESUMO

Biological magnetic field sensing that gives rise to physiological responses is of considerable importance in quantum biology. The radical pair mechanism (RPM) is a fundamental quantum process that can explain some of the observed biological magnetic effects. In magnetically sensitive radical pair (RP) reactions, coherent spin dynamics between singlet and triplet pairs are modulated by weak magnetic fields. The resulting singlet and triplet reaction products lead to distinct biological signaling channels and cellular outcomes. A prevalent RP in biology is between flavin semiquinone and superoxide (O2 •-) in the biological activation of molecular oxygen. This RP can result in a partitioning of reactive oxygen species (ROS) products to form either O2 •- or hydrogen peroxide (H2O2). Here, we examine magnetic sensing of recombinant human electron transfer flavoenzyme (ETF) reoxidation by selectively measuring O2 •- and H2O2 product distributions. ROS partitioning was observed between two static magnetic fields at 20 nT and 50 µT, with a 13% decrease in H2O2 singlet products and a 10% increase in O2 •- triplet products relative to 50 µT. RPM product yields were calculated for a realistic flavin/superoxide RP across the range of static magnetic fields, in agreement with experimental results. For a triplet born RP, the RPM also predicts about three times more O2 •- than H2O2, with experimental results exhibiting about four time more O2 •- produced by ETF. The method presented here illustrates the potential of a novel magnetic flavoprotein biological sensor that is directly linked to mitochondria bioenergetics and can be used as a target to study cell physiology.

3.
Bioengineering (Basel) ; 11(1)2023 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-38247887

RESUMO

The impact of magnetic fields on cellular function is diverse but can be described at least in part by the radical pair mechanism (RPM), where magnetic field intervention alters reactive oxygen species (ROS) populations and downstream cellular signaling. Here, cellular migration within three-dimensional scaffolds was monitored in an applied oscillating 1.4 MHz radiofrequency (RF) magnetic field with an amplitude of 10 µT and a static 50 µT magnetic field. Given that cellular bioenergetics can be altered based on applied RF magnetic fields, this study focused on a magnetic field configuration that increased cellular respiration. Results suggest that RF accelerated cell clustering and elongation after 1 day, with increased levels of clustering and cellular linkage after 7 days. Cell distribution analysis within the scaffolds revealed that the clustering rate during the first day was increased nearly five times in the RF environment. Electron microscopy provided additional topological information and verified the development of fibrous networks, with a cell-derived matrix (CDM) visualized after 7 days in samples maintained in RF. This work demonstrates time-dependent cellular migration that may be influenced by quantum biology (QB) processes and downstream oxidative signaling, enhancing cellular migration behavior.

4.
Biochemistry ; 56(25): 3234-3247, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28525271

RESUMO

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514-3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.


Assuntos
Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/química , S-Adenosilmetionina/química , Enxofre/química , Catálise , Domínio Catalítico , Clostridium/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/química , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Oxirredução , Conformação Proteica , S-Adenosilmetionina/metabolismo , Enxofre/metabolismo
5.
PLoS One ; 12(3): e0171836, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28296892

RESUMO

Cryptochromes are evolutionarily conserved blue-light absorbing flavoproteins which participate in many important cellular processes including in entrainment of the circadian clock in plants, Drosophila and humans. Drosophila melanogaster cryptochrome (DmCry) absorbs light through a flavin (FAD) cofactor that undergoes photoreduction to the anionic radical (FAD•-) redox state both in vitro and in vivo. However, recent efforts to link this photoconversion to the initiation of a biological response have remained controversial. Here, we show by kinetic modeling of the DmCry photocycle that the fluence dependence, quantum yield, and half-life of flavin redox state interconversion are consistent with the anionic radical (FAD•-) as the signaling state in vivo. We show by fluorescence detection techniques that illumination of purified DmCry results in enzymatic conversion of molecular oxygen (O2) to reactive oxygen species (ROS). We extend these observations in living cells to demonstrate transient formation of superoxide (O2•-), and accumulation of hydrogen peroxide (H2O2) in the nucleus of insect cell cultures upon DmCry illumination. These results define the kinetic parameters of the Drosophila cryptochrome photocycle and support light-driven electron transfer to the flavin in DmCry signaling. They furthermore raise the intriguing possibility that light-dependent formation of ROS as a byproduct of the cryptochrome photocycle may contribute to its signaling role.


Assuntos
Criptocromos/metabolismo , Drosophila melanogaster/metabolismo , Luz , Fotoperíodo , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Humanos , Cinética , Teoria Quântica , Spodoptera
6.
Sci Rep ; 6: 38543, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27995996

RESUMO

Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2•-) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning to cellular bioenergetics.


Assuntos
Metabolismo Energético , Células Endoteliais da Veia Umbilical Humana/metabolismo , Teoria Quântica , Espécies Reativas de Oxigênio/metabolismo , Simulação por Computador , Humanos , Campos Magnéticos , Análise Numérica Assistida por Computador , Quinonas/química , Quinonas/metabolismo , Superóxidos/metabolismo
7.
J Biol Chem ; 291(34): 17816-28, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27307041

RESUMO

Members of the antibiotic biosynthesis monooxygenase family catalyze O2-dependent oxidations and oxygenations in the absence of any metallo- or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pKa The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O2-dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O2 reactivity via markedly different means.


Assuntos
Proteínas de Bactérias/química , Dinitrocresóis/química , Oxigenases de Função Mista/química , Streptomyces/enzimologia , Superóxidos/química , Catálise
8.
Appl Magn Reson ; 46(3): 349-355, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29051687

RESUMO

Polymeric nanohybrid P22 virus capsids were used as templates for high density Gd3+ loading to explore magnetic field-dependent (0.5-7.0 T) proton relaxivity. The field-dependence of relaxivity by the spatially constrained Gd3+ in the capsids was similar when either the loading of the capsids or the concentration of capsids was varied. The ionic longitudinal relaxivity, r1, decreased from 25-32 mM-1 s-1 at 0.5 T to 6-10 mM-1 s-1 at 7 T. The ionic transverse relaxivity, r2, increased from 28-37 mM-1 s-1 at 0.5 T to 39-50 mM-1 s-1 at 7 T. The r2/r1 ratio increased linearly with increasing magnetic field from about 1 at 0.5 T, which is typical of T1 contrast agents, to 5-8 at 7 T, which is approaching the ratios for T2 contrast agents. Increases in electron paramagnetic resonance line widths at 80 and 150 K and higher microwave powers required for signal saturation indicate enhanced Gd3+ electron spin relaxation rates for the Gd3+-loaded capsids than for low concentration Gd3+. The largest r2/r1 at 7 T was for the highest cage loading, which suggests that Gd3+-Gd3+ interactions within the capsid enhance r2 more than r1.

9.
PLoS One ; 9(3): e93065, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24681944

RESUMO

The effects of weak magnetic fields on the biological production of reactive oxygen species (ROS) from intracellular superoxide (O2•-) and extracellular hydrogen peroxide (H2O2) were investigated in vitro with rat pulmonary arterial smooth muscle cells (rPASMC). A decrease in O2•- and an increase in H2O2 concentrations were observed in the presence of a 7 MHz radio frequency (RF) at 10 µTRMS and static 45 µT magnetic fields. We propose that O2•- and H2O2 production in some metabolic processes occur through singlet-triplet modulation of semiquinone flavin (FADH•) enzymes and O2•- spin-correlated radical pairs. Spin-radical pair products are modulated by the 7 MHz RF magnetic fields that presumably decouple flavin hyperfine interactions during spin coherence. RF flavin hyperfine decoupling results in an increase of H2O2 singlet state products, which creates cellular oxidative stress and acts as a secondary messenger that affects cellular proliferation. This study demonstrates the interplay between O2•- and H2O2 production when influenced by RF magnetic fields and underscores the subtle effects of low-frequency magnetic fields on oxidative metabolism, ROS signaling, and cellular growth.


Assuntos
Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Animais , Células Cultivadas , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Campos Magnéticos , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo/fisiologia , Artéria Pulmonar/metabolismo , Ratos , Transdução de Sinais/fisiologia
10.
J Appl Phys ; 112(8): 84701, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23152643

RESUMO

Electron magnetic resonance (EMR) spectroscopy was used to determine the magnetic properties of maghemite (γ-Fe(2)O(3)) nanoparticles formed within size-constraining Listeria innocua (LDps)-(DNA-binding protein from starved cells) protein cages that have an inner diameter of 5 nm. Variable-temperature X-band EMR spectra exhibited broad asymmetric resonances with a superimposed narrow peak at a gyromagnetic factor of g ≈ 2. The resonance structure, which depends on both superparamagnetic fluctuations and inhomogeneous broadening, changes dramatically as a function of temperature, and the overall linewidth becomes narrower with increasing temperature. Here, we compare two different models to simulate temperature-dependent lineshape trends. The temperature dependence for both models is derived from a Langevin behavior of the linewidth resulting from "anisotropy melting." The first uses either a truncated log-normal distribution of particle sizes or a bi-modal distribution and then a Landau-Liftshitz lineshape to describe the nanoparticle resonances. The essential feature of this model is that small particles have narrow linewidths and account for the g ≈ 2 feature with a constant resonance field, whereas larger particles have broad linewidths and undergo a shift in resonance field. The second model assumes uniform particles with a diameter around 4 nm and a random distribution of uniaxial anisotropy axes. This model uses a more precise calculation of the linewidth due to superparamagnetic fluctuations and a random distribution of anisotropies. Sharp features in the spectrum near g ≈ 2 are qualitatively predicted at high temperatures. Both models can account for many features of the observed spectra, although each has deficiencies. The first model leads to a nonphysical increase in magnetic moment as the temperature is increased if a log normal distribution of particles sizes is used. Introducing a bi-modal distribution of particle sizes resolves the unphysical increase in moment with temperature. The second model predicts low-temperature spectra that differ significantly from the observed spectra. The anisotropy energy density K(1), determined by fitting the temperature-dependent linewidths, was ∼50 kJ/m(3), which is considerably larger than that of bulk maghemite. The work presented here indicates that the magnetic properties of these size-constrained nanoparticles and more generally metal oxide nanoparticles with diameters d < 5 nm are complex and that currently existing models are not sufficient for determining their magnetic resonance signatures.

11.
J Phys Chem B ; 115(16): 4613-20, 2011 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-21469686

RESUMO

Dendrimers are attractive templates to display functional molecular components. Since the behavior of dendrimer systems can depend greatly on the accessibility of these molecular components to the external environment, and on the spatial arrangement of functional groups attached to the dendrimer terminal branches (end-groups), techniques to determine the locations of end-groups are highly desirable. In this report, we describe a method to analyze the EPR spectra of multiple generations of poly(amidoamine) (PAMAM) dendrimers which have spin-labels attached to end-groups in variable percentages of the total number of available sites. The spectra are treated as a convolution of a narrow spin-label spectrum and a variable line broadening function. Trends in the parameters that describe the best-fit line broadening function with spin-label loading reveal the spatial arrangements and homogeneity of spin environments of the labels. We observe a shift in the end-group distribution from generation 3 (G(3)) to G(4) dendrimers that indicates a change in morphology from an open, extended structure to a more dense, compact arrangement.

12.
J Am Chem Soc ; 133(12): 4156-9, 2011 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-21388197

RESUMO

This work describes an approach for calculating and measuring dipolar interactions in multispin systems to monitor conformational changes in icosahedral protein cages using site-directed spin labeling. Cowpea chlorotic mottle virus (CCMV) is used as a template that undergoes a pH-dependent reversible capsid expansion wherein the protein cage swells by 10%. The sequence-position-dependent geometric presentation of attached spin-label groups provides a strategy for targeting amino acid residues most probative of structural change. The labeled protein cage residues and structural transition were found to affect the local mobility and dipolar interactions of the spin label, respectively. Line-shape changes provided a spectral signature that could be used to follow the conformational change in CCMV coat dynamics. The results provide evidence for a concerted swelling process in which the cages exist in only two structural forms, with essentially no intermediates. This methodology can be generalized for all symmetry types of icosahedral protein architectures to monitor protein cage dynamics.


Assuntos
Bromovirus/química , Bromovirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Marcadores de Spin , Capsídeo/química , Proteínas do Capsídeo/química , Concentração de Íons de Hidrogênio , Conformação Proteica
13.
Biochemistry ; 47(34): 8894-901, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18672901

RESUMO

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.


Assuntos
Flavoproteínas Transferidoras de Elétrons/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Enxofre/metabolismo , Animais , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/genética , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Ligação de Hidrogênio , Ferro/química , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Estrutura Secundária de Proteína , Enxofre/química , Suínos , Temperatura , Treonina/química , Treonina/genética , Treonina/metabolismo
14.
Biochemistry ; 47(1): 92-100, 2008 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-18069858

RESUMO

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.


Assuntos
Proteínas de Bactérias/metabolismo , Flavoproteínas Transferidoras de Elétrons/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Estrutura Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Potenciometria , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo
15.
J Magn Reson ; 190(2): 222-32, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18037314

RESUMO

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a membrane-bound electron transfer protein that links primary flavoprotein dehydrogenases with the main respiratory chain. Human, porcine, and Rhodobacter sphaeroides ETF-QO each contain a single [4Fe-4S](2+,1+) cluster and one equivalent of FAD, which are diamagnetic in the isolated enzyme and become paramagnetic on reduction with the enzymatic electron donor or with dithionite. The anionic flavin semiquinone can be reduced further to diamagnetic hydroquinone. The redox potentials for the three redox couples are so similar that it is not possible to poise the proteins in a state where both the [4Fe-4S](+) cluster and the flavoquinone are fully in the paramagnetic form. Inversion recovery was used to measure the electron spin-lattice relaxation rates for the [4Fe-4S](+) between 8 and 18K and for semiquinone between 25 and 65K. At higher temperatures the spin-lattice relaxation rates for the [4Fe-4S](+) were calculated from the temperature-dependent contributions to the continuous wave linewidths. Although mixtures of the redox states are present, it was possible to analyze the enhancement of the electron spin relaxation of the FAD semiquinone signal due to dipolar interaction with the more rapidly relaxing [4Fe-4S](+) and obtain point-dipole interspin distances of 18.6+/-1A for the three proteins. The point-dipole distances are within experimental uncertainty of the value calculated based on the crystal structure of porcine ETF-QO when spin delocalization is taken into account. The results demonstrate that electron spin relaxation enhancement can be used to measure distances in redox poised proteins even when several redox states are present.


Assuntos
Proteínas de Bactérias/química , Espectroscopia de Ressonância de Spin Eletrônica , Flavoproteínas Transferidoras de Elétrons/química , Proteínas Ferro-Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Ubiquinona/química , Animais , Transporte de Elétrons , Humanos , Fígado/enzimologia , Oxirredução , Rhodobacter sphaeroides/enzimologia , Suínos
16.
J Phys Chem B ; 109(46): 21532-8, 2005 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-16853796

RESUMO

Starburst dendrimers are receiving considerable attention as templates for the assembly of structured arrays of molecular components. This research motivates the development of improved methods for dendrimer characterization-specifically, for determining the numbers, distributions of numbers, and spatial distribution of molecular species synthetically attached to macromolecular templates. Such information provides the basis for advancing strategies aimed at controlling dendrimer functionalization, and thus represents enabling technology for tailoring the composition and structure of molecular arrays fashioned on dendrimer templates. Moreover, this information is vital to the proper interpretation of ongoing experiments in which dendrimers sparsely functionalized with reporter groups are used as probes. In this article, we report MALDI-TOF mass spectrometry and EPR spectroscopy of heterogeneously functionalized G(4)-PAMAM dendrimers bearing nitroxide spin-labels.


Assuntos
Poliaminas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Dendrímeros , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Estrutura Molecular , Poliaminas/síntese química , Sensibilidade e Especificidade
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