Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein–ubiquinone oxidoreductase (ETF–QO)

Fielding, Alistair J.; Usselman, Robert J.; Watmough, Nicholas J.; Šimkovič, Martin; Frerman, Frank E.; Eaton, Gareth R.

doi:10.1016/j.jmr.2007.11.002

Cited by 16 publications

(23 citation statements)

References 55 publications

(97 reference statements)

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“…74−76 Because the two FeS clusters in HydE each have net S = 1 / 2 , this program also can be applied to mapping the cluster–cluster interaction. Figure S5 shows the inversion recovery curve at g ⊥ for the [2Fe-2S] + cluster signal in HydE at 40 K. Simulations based on an interspin distance of 45 Å (which is defined as providing no relaxation enhancement) do not adequately fit the experimental data.…”

Section: Resultsmentioning

confidence: 99%

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 99%

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

et al. 2017

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“…In general, the CSDs of only one partner are used to certify the interaction (61). Here we investigated CSDs of both K156 and LTR34 to map the interface of the peptide–DNA complex in the presence of Mg 2+ .…”

Section: Resultsmentioning

confidence: 99%

Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase α4 helix

Hobaika¹,

Zargarian

Boulard

et al. 2009

Nucleic Acids Research

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HIV-1 integrase integrates retroviral DNA through 3′-processing and strand transfer reactions in the presence of a divalent cation (Mg2+ or Mn2+). The α4 helix exposed at the catalytic core surface is essential to the specific recognition of viral DNA. To define group determinants of recognition, we used a model composed of a peptide analogue of the α4 helix, oligonucleotides mimicking processed and unprocessed U5 LTR end and 5 mM Mg2+. Circular dichroism, fluorescence and NMR experiments confirmed the implication of the α4 helix polar/charged face in specific and non-specific bindings to LTR ends. The specific binding requires unprocessed LTR ends—i.e. an unaltered 3′-processing site CA↓GT3′—and is reinforced by Mg2+ (Kd decreases from 2 to 0.8 nM). The latter likely interacts with the ApG and GpT3′ steps of the 3′-processing site. With deletion of GT3′, only persists non-specific binding (Kd of 100 μM). Proton chemical shift deviations showed that specific binding need conserved amino acids in the α4 helix and conserved nucleotide bases and backbone groups at LTR ends. We suggest a conserved recognition mechanism based on both direct and indirect readout and which is subject to evolutionary pressure.

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“…EPR (electron paramagnetic resonance) spectroscopy, signal quantitation and analysis of the data for [4Fe-4S] + at 15 K and for FAD semiquinone (SQ −• ) at 100 K were done by the methods of Fielding et al (18) for wild-type ETF-QO and Usselman et al (8) for ETF-QO mutants. EPR spectra of FAD SQ −• at 293 K were recorded in 1.0 mm ID capillaries, on a Varian E9 spectrometer using a microwave power of 0.5 mW, a 2.0 G modulation amplitude, a time constant of 0.25 seconds, and averaging 5, 120 second scans.…”

Section: Methodsmentioning

confidence: 99%

“…EPR spectra of FAD SQ −• at 293 K were recorded in 1.0 mm ID capillaries, on a Varian E9 spectrometer using a microwave power of 0.5 mW, a 2.0 G modulation amplitude, a time constant of 0.25 seconds, and averaging 5, 120 second scans. Spectra were quantitated by simulation (18) and compared with a tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinooxy) standard.…”

Section: Methodsmentioning

confidence: 99%

The Iron−Sulfur Cluster of Electron Transfer Flavoprotein−Ubiquinone Oxidoreductase Is the Electron Acceptor for Electron Transfer Flavoprotein

et al. 2008

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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 mV and −30 mV for wild type to −11 mV and −19 mV, respectively. The N338A mutation decreased the potentials to −37 mV and −49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e− 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.

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Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein–ubiquinone oxidoreductase (ETF–QO)

Cited by 16 publications

References 55 publications

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase α4 helix

The Iron−Sulfur Cluster of Electron Transfer Flavoprotein−Ubiquinone Oxidoreductase Is the Electron Acceptor for Electron Transfer Flavoprotein

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