Exogenous ligand binding to the [Fe4S4] cluster in Pyrococcus furiosus ferredoxin

Conover, Richard C.; Park, Jae Bum; Adams, Michael W. W.; Johnson, Michael K.

doi:10.1021/ja00007a091

Cited by 34 publications

(38 citation statements)

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“…Incubation of reduced NsrR with CN - results in a disappearance of the g = 5.2 signal and produces a new S = 1/2 signal (Figure 7B) which now accounts for 0.64 spin per cluster. These observations are consistent with coordination of exogenous CN - to convert the [4Fe-4S] + to a pure S = 1/2 species as observed for Pyrococcus furiosus ferredoxin (22). The remaining spin count missing in the EPR spectra may be due to chelation by the high concentration of CN - (20 mM), incomplete reduction of the 4Fe-4S cluster, and/or an overestimation of the cluster concentration caused by adventitious iron in the preparation.…”

Section: Resultssupporting

confidence: 88%

Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe−4S Cluster

et al. 2008

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In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S] 2+ cluster which reacts with NO to form dinitrosyl iron complexes. This method of NO sensing is analogous to that of the FNR protein of Escherichia coli. The Fe-S cluster of NsrR is also reactive toward other exogenous ligands such as cyanide, dithiothreitol, and O 2 . These results, together with the presence of only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a non-cysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and crosslinking experiments show that NsrR adopts a dimeric structure in its [4Fe-4S] 2+ holo form as well as in the apo form. These findings provide a first steppingstone to investigate the mechanism of NO sensing in NsrR.The ability to adapt to anaerobic conditions is vital for a great diversity of microorganisms. It is particularly true of pathogenic organisms which may encounter oxygen limitation within their hosts. Such bacteria also commonly face other stresses such as nitric oxide (NO) produced by phagocytic cells as part of the immune response of the host (1). Thus, an understanding of how bacteria sense and respond to these conditions is of considerable importance.An example of the bacterial response to oxygen limitation comes from Bacillus subtilis which, in the absence of oxygen, can grow by nitrate respiration (2). The ResDE two-component regulatory system is required for the induction of genes involved in nitrate respiration (3). These genes include fnr, (the gene encoding anaerobic transcription factor) (4), nasDEF (nitrite reductase genes) (5), and hmp (flavohemoglobin gene) (6). However, anaerobic conditions alone are not sufficient for the full induction of the ResDE-controlled genes, in particular hmp and nasDEF, and the presence of NO is required to attain the full induction of these genes (7). The effect of NO is abrogated in an nsrR mutant strain, indicating a role for NsrR in NOmediated control of the ResDE regulon (8).NsrR belongs to the Rrf2 family of transcription regulators and is widely found in various bacteria (9). Sequence analyses predict that NsrR contains a helix-turn-helix which likely binds to the promoter region of its target genes (9). In vivo studies of nsrR mutants and the effect of * To whom correspondence should be addressed. Tel: 503-748-167, Fax: 503-7481464, Email: ploccoz@ebs.ogi Materials & Methods Purification of N-terminal and C-terminal His 6 -tagged NsrR proteins (NH-NsrR & CH-NsrR)The various strains and plasmids used in this study are listed in Table S1 (see Supporting Information). NH-NsrR expression plasmid, pMMN648, was previously cons...

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Section: Resultssupporting

confidence: 88%

Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe−4S Cluster

et al. 2008

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“…P. furiosus Fd in the oxidized 3Fe form was provided by Prof. M.W.W. Adams, University of Georgia, Athens, Ga., and prepared as previously described [69,70]. A. vinelandii Fd I was provided by Prof. B.K.…”

Section: Protein Samplesmentioning

confidence: 99%

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

Telser

Lee²,

Hoffman³

2000

J Biol Inorg Chem

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We have studied four proteins containing oxidized 3Fe clusters ([Fe3S4]+, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T1, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2-4.2 K. The T1, values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g3 values and in 57 Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T1 is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at approximately 20 cm(-1) above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported 57Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J13=J, J12=J(1+epsilon'), J23=J(1+epsilon), where Jij is the isotropic exchange coupling between ferric ions i and j, and epsilon' and epsilon' are measures of coupling inequivalence. We have extended their theory to include the effects of epsilon' not equal to 0 and thus derived an exact expression for the energy of the doublet excited state for any epsilon, epsilon'. This excited state energy corresponds roughly to epsilonJ and is in the range 5-10 cm(-1) for each of these four 3Fe proteins. This magnitude of the product epsilonJ, determined by our time-domain relaxation studies in the temperature range 2-4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5-50 K, NMR studies in the range 293-303 K, and static susceptibility measurements in the range 1.8-200 K. We suggest that an apparent disagreement as to the individual values of J and epsilon be resolved in favor of the values obtained by susceptibility and NMR (J > or approximately 200 cm(-1) and epsilon> or =0.02 cm(-1)). as opposed to a smaller J and larger r as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters.

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“…This small redox protein (M r 7500) contains a single [4Fe-4S] cluster and is extremely thermostable, being unaffected by 12 h at 95°C [34]. It has several unusual properties compared to mesophilic ferredoxins [35][36][37][38][39]. The ferredoxin is the physiological electron donor for the hydrogenase of P. furiosus, the enzyme responsible for catalyzing H 2 production [28].…”

Section: The Sulfur-dependent Hyperthermophilesmentioning

confidence: 99%

Biochemical diversity among sulfur-dependent, hyperthermophilic microorganisms

Adams

1994

FEMS Microbiology Reviews

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Hyperthermophiles are a recently discovered group of microorganisms that grow at and above 90°C. They currently comprise over 20 different genera, and except for two novel bacleria, all are classified as Archaea. The majority of these organisms are obligately anaerobic heterotrophs that reduce elemental sulfur (S °) to H2S. The best studied from a biochemical perspective are the archaeon, ~'rococcus furiosus, and the bacterium, Thermotoga rnaritirna, both of which are saccharolytic. P. furiosus is thought to contain a new type of Entner-Doudoroff pathway for the conversion of carbohydrates ultimately to acetate, H 2 and CO~. The pathway is independent of nicotinamide nucleotides and involves novel types of ferredoxin-linked oxidoreductases, one of which has tungsten, a rarely used element, as a prosthetic group. The only site of energy conservation is at the level of acetyl CoA, which in the presence of ADP and phosphate is converted to acetate and ATP in a single step. In contrast, 72 maritima utilizes a conventional Embden-Meyerhof pathway for sugar oxidation. P. furiosus also utilizes peptides as a sole carbon and energy source. Amino acid oxidation is thought to involve glutamate dehydrogenase together with at least three types of novel ferredoxin-linked oxidoreductases which catalyze the oxidation of 2-ketoglutarate, aryl pyruvates and formaldehyde. One of these enzymes also utilizes tungsten. In P. furiosus, virtually all of the reductant that is generated during the catabolism of both carbohydrates and peptides is channeled to a cytoplasmic hydrogenase. This enzyme is now termed sulhydrogenase, as it reduces both protons to H 2 and S ° (or polysulfide) to H2S. S ° reduction appears to lead to the conservation of energy in P. furiosus but not in 72 rnaritima, although the mechanism by which this occurs is not known.

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Exogenous ligand binding to the [Fe4S4] cluster in Pyrococcus furiosus ferredoxin

Cited by 34 publications

References 0 publications

Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe−4S Cluster

Transcription Factor NsrR from Bacillus subtilis Senses Nitric Oxide with a 4Fe−4S Cluster

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

Biochemical diversity among sulfur-dependent, hyperthermophilic microorganisms

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