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The iron-sulfur cluster containing protein Fumarate and Nitrate Reduction (FNR) is the master regulator for the switch between anaerobic and aerobic respiration in Escherichia coli and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O 2 that leads to conversion to a [2Fe-2S] form with loss of high-affinity DNA binding. Here, we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a [3Fe-3S] cluster and persulfide-coordinated [2Fe-2S] clusters [[2Fe-2S](S) n , where n = 1 or 2]. Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion and not as a subsequent, secondary reaction to generate [2Fe-2S](S) n species. This methodology shows great potential for broad application to studies of protein cofactor-small molecule interactions., Competing Interests: The authors declare no conflict of interest.

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2.

The Fumarate nitrate reduction (FNR) regulator from Escherichia coli controls expression of >300 genes in response to O2 through reaction with its [4Fe-4S] cluster cofactor. FNR is the master switch for the transition between anaerobic and aerobic respiration. In response to physiological concentrations of nitric oxide (NO), FNR also regulates genes, including the nitrate reductase (nar) operon, a major source of endogenous cellular NO, and hmp, which encodes an NO-detoxifying enzyme. Here we show that the [4Fe-4S] cluster of FNR reacts rapidly in a multiphasic reaction with eight NO molecules. Oxidation of cluster sulfide ions (S(2-)) to sulfane (S(0)) occurs, some of which remains associated with the protein as Cys persulfide. The nitrosylation products are similar to a pair of dinuclear dinitrosyl iron complexes, [Fe(I)2(NO)4(Cys)2](0), known as Roussin's red ester. A similar reactivity with NO was reported for the Wbl family of [4Fe-4S]-containing proteins found only in actinomycetes, such as Streptomyces and Mycobacteria. These results show that NO reacts via a common mechanism with [4Fe-4S] clusters in phylogenetically unrelated regulatory proteins that, although coordinated by four Cys residues, have different cluster environments. The reactivity of E. coli FNR toward NO, in addition to its sensitivity toward O2, is part of a hierarchal network that monitors, and responds to, NO, both endogenously generated and exogenously derived.

The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile(12) and Val(14) are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile(12) on one subunit and Val(14) on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport.

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster. The reaction of the [4Fe-4S](2+) cluster with O(2) has been shown to proceed via a 2-step process, an O(2)-dependent 1-electron oxidation to yield a [3Fe-4S](+) intermediate with release of 1 Fe(2+) ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe(3+) and 2 S(2-) ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S](2+) cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S](2+) cluster in the presence of O(2). Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with O(2) is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O(2) interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O(2) concentrations.

The metabolic flexibility of bacteria is key to their ability to survive and thrive in a wide range of environments. Optimal switching from one metabolic pathway to another is a key requirement for this flexibility. Respiration is a good example: many bacteria utilize O(2) as the terminal electron acceptor, but can switch to a range of other acceptors, such as nitrate, when O(2) becomes limiting. Sensing environmental levels of O(2) is the key step in switching from aerobic to anaerobic respiration. In Escherichia coli, the fumarate and nitrate reduction transcriptional regulator (FNR) controls this switch. Under O(2)-limiting conditions, FNR binds a [4Fe-4S](2+) cluster, generating a transcriptionally active dimeric form. Exposure to O(2) results in conversion of the cluster into a [2Fe-2S](2+) form, leading to dissociation of the protein into inactive monomers. The mechanism of cluster conversion, together with the nature of the reaction products, is of considerable current interest, and a near-complete description of the process has now emerged. The [4Fe-4S](2+) into [2Fe-2S](2+) cluster conversion proceeds via a two-step mechanism. In step 1, a one-electron oxidation of the cluster takes place, resulting in the release of a Fe(2+) ion, the formation of an intermediate [3Fe-4S](1+) cluster, together with the generation of a superoxide anion. In step 2, the intermediate [3Fe-4S](1+) cluster rearranges spontaneously to form the [2Fe-2S](2+) cluster, releasing two sulfide ions and an Fe(3+) ion in the process. The one-electron activation of the cluster, coupled to catalytic recycling of the superoxide anion back to oxygen via superoxide dismutase and catalase, provides a novel means of amplifying the sensitivity of [4Fe-4S](2+) FNR to its signal molecule.

The Escherichia coli fumarate and nitrate reductase (FNR) regulator protein is an important transcriptional regulator that controls the expression of a large regulon of more than 100 genes in response to changes in oxygen availability. FNR is active when it acquires a [4Fe-4S](2+) cluster under anaerobic conditions. The presence of the [4Fe-4S](2+) cluster promotes protein dimerization and site-specific DNA binding, facilitating activation or repression of target promoters. Oxygen is sensed by the controlled disassembly of the [4Fe-4S](2+) cluster, ultimately resulting in inactive, monomeric, apo-FNR. The FNR [4Fe-4S](2+) cluster is also sensitive to nitric oxide, such that under anaerobic conditions the protein is inactivated by nitrosylation of the iron-sulfur cluster, yielding a mixture of monomeric and dimeric dinitrosyl-iron cysteine species. This chapter describes some of the methods used to produce active [4Fe-4S] FNR protein and investigates the reaction of the [4Fe-4S](2+) cluster with nitric oxide and oxygen in vitro.

A search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome bo3, E. coli, has been carried out using site directed nitroxide spin labeling (SDSL) of cysteine residues. These were positioned at R134 and R309, on loops that link helices II and III and VI and VII at the entrances to the D and K proton channels, respectively. The motional characteristics of both labels have been determined using X- and W-band EPR spectroscopy at room temperature in selected redox levels in the reaction sequence of QO with oxygen, namely, the mixed valence carbon monoxide form (COMV), the oxidized (O) and super-oxidized (PM) states. The O to PM step is accompanied by the uptake of protons through the K pathway. We find no evidence for changes in the motional characteristics of either label that are expected to be associated with helical motions at the entrances to the channels. Because kinetic studies of mutants show that the redox gating of protons occurs deep within the D channel close to the heme-copper site, the present study implies that no motion is transmitted to the ends of the helices.

Shewanella oneidensis MR-1 has the metabolic capacity to grow anaerobically using Fe(III) as a terminal electron acceptor. Growth under these conditions results in the de novo synthesis of a number of periplasmic c-type cytochromes, many of which are multiheme in nature and are thought to be involved in the Fe(III) respiratory process. To begin a biochemical study of these complex cytochromes, the mtrA gene that encodes an approximate 32-kDa periplasmic decaheme cytochrome has been heterologously expressed in Escherichia coli. Co-expression of mtrA with a plasmid that contains cytochrome c maturation genes leads to the assembly of a correctly targeted holoprotein, which covalently binds ten hemes. The recombinant MtrA protein has been characterized by magnetic circular dichroism, which shows that all ten hemes have bis-histidine axial ligation. EPR spectroscopy detected only eight of these hemes, all of which are low spin and provides evidence for a spin-coupled pair of hemes in the oxidized state. Redox titrations of MtrA have been carried out with optical- and EPR-monitored methods, and the hemes are shown to reduce over the potential range -100 to -400 mV. In intact cells of E. coli, MtrA is shown to obtain electrons from the host electron transport chain and pass these onto host oxidoreductases or a range of soluble Fe(III) species. This demonstrates the promiscuous nature of this decaheme cytochrome and its potential to serve as a soluble Fe(III) reductase in intact cells.

The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe- 4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2- mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanentmonomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2. [ABSTRACT FROM AUTHOR]

nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme-copper dinuclear site in Escherichia coli cytochrome bo a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T, of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T, of the dinuclear coupled pair Fe(III)-Cu(II) in the oxidized form of QO that is too short to measure directly. In the case of the F′ state, the relaxation properties of the heme-copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu(II) coupled by approximately 1.5-3 cm to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine-histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements. [ABSTRACT FROM AUTHOR]

In Escherichia coli, the switch between aerobic and anaerobic metabolism is controlled primarily by FNR (regulator of fumarate and nitrate reduction), the protein that regulates the transcription of >100 genes in response to oxygen. Under oxygen-limiting conditions, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Upon exposure to oxygen the cluster converts to a [2Fe-2S]2+ form, leading to dissociation of the protein into monomers, which are incapable of binding DNA with high affinity. The mechanism of cluster conversion together with the nature of the products of conversion is of considerable current interest. Here, we demonstrate that [4Fe-4s]2+ to [2Fe-2S)2+ cluster conversion, in both native and reconstituted [4Fe-4S] FNR, proceeds via a one electron oxidation of the cluster, to give a [3Fe-4S]1+ cluster intermediate, with the release of one Fe2+ ion and a superoxide ion. The cluster intermediate subsequently rear-ranges spontaneously to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and, as previously shown, two sulfide ions. Superoxide ion undergoes dismutation to hydrogen peroxide and oxygen. This mechanism, a one electron activation of the cluster, coupled to catalytic recycling of the resulting superoxide ion back to oxygen, provides a means of amplifying the sensitivity of [4Fe-4S] FNR to its signal molecule. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a signalling and defence molecule of major importance in biology. The flavohaemoglobin Hmp of Escherichia coli is involved in protective responses to NO. Because hmp gene transcription is repressed by the O2-responsive [ABSTRACT FROM AUTHOR]

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53% identity with mammalian cytosolic aconitase (c-aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe-4S]+-containing, inactive state, AcaA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aconitase) but is even more similar to the signal form c-aconitase. This is perhaps related to the greater similarity of AcnA amino acid sequence with c-aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe-4S] cluster of AcnA must be similar to, but ot identical to that of m-aconitase. Whilst the[3Fe-4S] clusters form both of these enzymes display some features in their MCD spectra common to [3Fe-4S] clusters in general, their spectra overall are unique and indicate that the Fea atom of the [4Fe-4S] form is not the only unusual feature of the [Fe-S] clusters of aconites. Active [4Fe-4S]-containing AcnA can be reduced to yield an EPR signal due to a [4Fe-4S]+ cluster which is indistinguishable from the signals from the [4Fe-4S]+ cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore,the catalytic activity of [4Fe-4S]2+-containing AcnA is fivefold higher than that of m-aconitase. The mechanistic implications of these data are discussed. A novel S =1/2 EPR signal with g ≈2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction. [ABSTRACT FROM AUTHOR]

Room-temperature (295 K) magnetic-circular­dichroism spectra at 280–2500 nm have been recorded for Escherichia coli cytochrome bo in its fast form (which has a g = 3.7 EPR signal and reacts rapidly with cyanide) and for its formate, fluoride, cyanide and hydrogen-peroxide derivatives. The spectra of all forms are dominated by signals from low-spin ferric heme b. These include a porphyrin-to-ferric ion charge-transfer transition in the near-infrared region (the near-infrared charge-transfer band) at 1610 nm. High-spin ferric heme o gives rise to a negative magnetic-circular-dichroism feature at 635, 642 and 625 nm (corresponding to a shoulder observed in the electronic absorption spectra) and a derivative charge-transfer feature at 1100, 1180 and 940 nm for the fast, formate and fluoride forms, respectively. The energies of these bands confirm that fluoride and formate are ligands to heme o. The energies of the analogous bands in the spectrum of fast cytochrome bo are typical tot high-spin ferric hemes with histidine and water axial ligands. Addition of cyanide ion to fast cytochrome bo causes a red shift in the position of the Soret absorption peak, from 406.5 nm to 413 nm, and results in the loss of the 635-nm feature form the magnetic-circular-dichroism spectrum and of the corresponding shoulder in the electronic absorption spectrum. In the magnetic-circular-dichroism spectrum, the intensities of the Soret and α.β bands are significantly increased. New near-infrared charge-transfer intensity is observed at 1000–2300 nm with a peak 2050 nm. These changes are interpreted as resulting from a high-spin to low-spin transition at ferric heme o brought about by the binding of cyanide ion. The energy of the near-infrared charge-transfer band suggests that the cyanide ion is bridged to the CuB of the binuclear site. Treatment of fast cytochrome bo with hydrogen peroxide also causes a red shift in the position of the Soret absorbance, to 412 nm. and a loss of the 625-nm absorption shoulder: Changes in the magnetic-circular-dichroism spectrum at 450–600 nm are observed, but there is no significant increase in the intensity of the magnetic-circular-dichroism Soret band and no new near-infrared charge-transfer bands are detected, ruling out a similar high-spin to low-spin transition at heme o. Subtraction of the contribution of low-spin ferric heme b from the magnetic-circular-dichroism spectrum reveals a spectrum characteristic of ferryl [Fe(IV)] heme o. [ABSTRACT FROM AUTHOR]

For the study of the dinuclear center of heme-copper oxidases cytochrome bo3 from Escherichia coli offers several advantages over the extensively charactererized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo3 is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in C cO. The absence of CuA and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o3. Careful analysis allows us to distinguish between small molecules that bind to heme o3, those which are ligands of CuB, and those which react to yield higher oxidation states of heme o3. Here we review results from our studies of the reactions of fast cytochrome bo3 with formate, fluoride, chloride, azide, cyanide, NO, and H2O2. [ABSTRACT FROM AUTHOR]

The facultative anaerobe Escherichia coli adopts different metabolic modes in response to the availability of oxygen. The global transcriptional regulator FNR (fumarate-nitrate reduction) monitors the availability of oxygen in the environment. Binding as a homodimer to palindromic sequences of DNA, FNR carries a sensory domain, remote from the DNA binding helix-turn-helix motif, which responds to oxygen. The sensing mechanism involves the transformation of a [4Fe-4S]2+ cluster into a [2Fe-2S] form in vitro on reaction with oxygen. Evidence is presented to show that this process proceeds by at least two steps, the first, an oxidative one, being the formation, on reaction with O2, of a [3Fe-4S]1+ cluster as an intermediate accompanied by the production of hydrogen peroxide. This is followed by a slower, non-redox, pseudo-first order step in which the [3Fe4S]1+ form converts to a [2Fe-2S]2+ cluster. This must be accompanied by a substantial protein conformational change since the four cysteine ligands that bind the two forms of the FeS clusters have different spatial disposition. Hydrogen peroxide is also an oxidant of the [4Fe4S]2+, causing a similar cluster transformation to a [2Fe2S] form. Either the hydrogen peroxide formed on reaction with oxygen can be recycled by intracellular catalase or it can be used to oxidize further Fe-S clusters. In both cases, the efficacy of oxygen sensing by FNR will be increased. [ABSTRACT FROM AUTHOR]

The Escherichia coli Fumarate-Nitrate Reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233 and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly-responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2. [ABSTRACT FROM AUTHOR]

The Fumarate nitrate reduction (FNR) regulator from Escherichia coli controls expression of >300 genes in response to O2 through reaction with its [4Fe-4S] cluster cofactor. FNR is the master switch for the transition between anaerobic and aerobic respiration. In response to physiological concentrations of nitric oxide (NO), FNR also regulates genes, including the nitrate reductase (nar) operon, a major source of endogenous cellular NO, and hmp, which encodes an NO-detoxifying enzyme. Here we show that the [4Fe-4S] cluster of FNR reacts rapidly in a multiphasic reaction with eight NO molecules. Oxidation of cluster sulfide ions (S2-) to sulfane (S0) occurs, some of which remains associated with the protein as Cys persulfide. The nitrosylation products are similar to a pair of dinuclear dinitrosyl iron complexes, [Fe(I)2(NO)4(Cys)2]0, known as Roussin's red ester. A similar reactivity with NO was reported for the Wbl family of [4Fe-4S]-containing proteins found only in actinomycetes, such as Streptomyces and Mycobacteria. These results show that NO reacts via a common mechanism with [4Fe-4S] clusters in phylogenetically unrelated regulatory proteins that, although coordinated by four Cys residues, have different cluster environments. The reactivity of E. coli FNR toward NO, in addition to its sensitivity toward O2, is part of a hierarchal network that monitors, and responds to, NO, both endogenously generated and exogenously derived. [ABSTRACT FROM AUTHOR]

In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlled by the fumarate and nitrate reduction transcriptional regulator FNR. In the absence of O2, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Exposure to O2 results in the conversion of the cluster to a [2Fe-2S]2+ form, leading to dissociation of the protein into transcriptionally inactive monomers. The [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion proceeds in two steps. Step 1 involves the one-electron oxidation of the cluster, resulting in the release of Fe2+ generating a [3Fe-4S]1+ cluster intermediate, and a superoxide ion. In step 2, the cluster intermediate spontaneously rearranges to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and two sulfide ions. Here, we demonstrate that, in both native and reconstituted [4Fe-4S] FNR, the reaction environment and, in particular, the presence of Fe2+ and/or Fe3+ chelators can influence significantly the cluster conversion reaction. We demonstrate that while the rate of step 1 is largely insensitive to chelators, that of step 2 is significantly enhanced by both Fe2+ and Fe3+ chelators. We show that, for reactions in Fe3+-coordinating phosphate buffer, step 2 is enhanced to the extent that step 1 becomes the rate determining step and the [3Fe-4S]1+ intermediate is no longer detectable. Furthermore, Fe3+ released during this step is susceptible to reduction in the presence of Fe2+ chelators. This work, which may have significance for the in vivo FNR cluster conversion reaction in the cell cytoplasm, provides an explanation for apparently contradictory results reported from different laboratories. [ABSTRACT FROM AUTHOR]

The Escherichia coli FNR protein regulates the transcription of >100 genes in response to environmental O2, thereby coordinating the response to anoxia. Under O2-limiting conditions, FNR binds a [4Fe-4S]2+ cluster through four cysteine residues (Cys20, Cys23, Cys29, Cys122). The acquisition of the [4Fe-4S]2+ cluster converts FNR into the transcriptionally active dimeric form. Upon exposure to O2, the cluster converts to a [2Fe-2S]2+ form, generating FNR monomers that no longer bind DNA with high affinity. The mechanism of the cluster conversion reaction and the nature of the released iron and sulfur are of considerable current interest. Here, we report the application of a novel in vitro method, involving 5,5′-dithiobis-(2-nitrobenzoic acid), for determining the oxidation state of the sulfur atoms released during FNR cluster conversion following the addition of O2. Conversion of [4Fe-4S]2+ to [2Fe-2SI2+ clusters by O2 for both native and reconstituted FNR results in the release of ~2 sulfide ions per [4Fe-4S]2+ cluster. This demonstrates that the reaction between O2 and the [4Fe-4S]2+ cluster does not require sulfide oxidation and hence must entail iron oxidation. [ABSTRACT FROM AUTHOR]

WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained ∼4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g ∼2.06, 1.94) consistent with a one-electron reduction of a [4Fe-4S]2+ cluster to a [4Fe-4S]1+ state with electron spin of S = ½. The anaerobically reconstituted [4Fe-4S] cluster was oxygen sensitive. Upon exposure to air, absorption at 410 and 505 nm first increased and then showed a steady decrease with time until the protein was colorless in the near UV/visible region. These changes are consistent with an oxygen-induced change from a [4Fe-4S] to a [2Fe-2S] cluster, followed by complete loss of cluster from the protein. Each of the four conserved cysteine residues, Cys-23, -53, -56, and -62, was essential for WhiD function in vivo. [ABSTRACT FROM AUTHOR]