Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation

Mitochondrial aconitase (m-aconitase) contains a [4Fe-4S](2+) cluster in its active site that catalyzes the stereospecific dehydration-rehydration of citrate to isocitrate in the Krebs cycle. It has been proposed that the [4Fe-4S](2+) aconitase is oxidized by superoxide, generating the inactive [3Fe-4S](1+) aconitase. In this reaction, the likely products are iron(II) and hydrogen peroxide. Consequently, the inactivation of m-aconitase by superoxide may increase the formation of hydroxyl radical ((*)OH) through the Fenton reaction in mitochondria. In this work, evidence for the generation of (*)OH from the reaction of m-aconitase with superoxide is provided using ESR spin trapping experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide and alpha-phenyl-N-tert-butylnitrone. Formation of free ( small middle dot)OH was verified with the (*)OH scavenger Me(2)SO, which forms methyl radical upon reacting with (*)OH. The addition of Me(2)SO to incubation mixtures containing m-aconitase and xanthine/xanthine oxidase yielded methyl radical, which was detected by ESR spin trapping. Methyl radical formation was further confirmed using [(13)C]Me(2)SO. Parallel low temperature ESR experiments demonstrated that the generation of the [3Fe-4S](1+) cluster increased with increasing additions of superoxide to m-aconitase. This reaction was reversible, as >90% of the initial aconitase activity was recovered upon treatment with glutathione and iron(II). This mechanism presents a scenario in which (*)OH may be continuously generated in the mitochondria.     

Escherichia coli biotin synthase produces selenobiotin. Further evidence of the involvement of the [2Fe-2S]2+ cluster in the sulfur insertion step

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin. The as-isolated enzyme contains a [2Fe-2S](2+) cluster, but the active enzyme requires an additional [4Fe-4S](2+) cluster, which is formed in the presence of Fe(NH(4))(2)(SO(4))(2) and Na(2)S in the in vitro assay. The role of the [4Fe-4S](2+) cluster is to mediate the electron transfer to SAM, while the [2Fe-2S](2+) cluster is involved in the sulfur insertion step. To investigate the selenium version of the reaction, we have depleted the enzyme of its iron and sulfur and reconstituted the resulting apoprotein with FeCl(3) and Na(2)Se to yield a [2Fe-2Se](2+) cluster. This enzyme was assayed in vitro with Na(2)Se in place of Na(2)S to enable the formation of a [4Fe-4Se](2+) cluster. Selenobiotin was produced, but the activity was lower than that of the as-isolated [2Fe-2S](2+) enzyme in the presence of Na(2)S. The [2Fe-2Se](2+) enzyme was additionally assayed with Na(2)S, to reconstitute a [4Fe-4S](2+) cluster, in case the latter was more efficient than a [4Fe-4Se](2+) cluster for the electron transfer. Indeed, the activity was improved, but in that case, a mixture of biotin and selenobiotin was produced. This was unexpected if one considers the [2Fe-2S](2+) center as the sulfur source (either as the ultimate donor or via another intermediate), unless some exchange of the chalcogenide has taken place in the cluster. This latter point was seen in the resonance Raman spectrum of the reacted enzyme which clearly indicated the presence of both the [2Fe-2Se](2+) and [2Fe-2S](2+) clusters. No exchange was observed in the absence of reaction. These observations bring supplementary proof that the [2Fe-2S](2+) cluster is implicated in the sulfur insertion step.     

Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.     

Characterization of the cofactor composition of Escherichia coli biotin synthase

The cofactor content of in vivo, as-isolated, and reconstituted forms of recombinant Escherichia coli biotin synthase (BioB) has been investigated using the combination of UV-visible absorption, resonance Raman, and M?ssbauer spectroscopies along with parallel analytical and activity assays. In contrast to the recent report that E. coli BioB is a pyridoxal phosphate (PLP)-dependent enzyme with intrinsic cysteine desulfurase activity (Ollagnier-deChoudens, S., Mulliez, E., Hewitson, K. S., and Fontecave, M. (2002) Biochemistry 41, 9145-9152), no evidence for PLP binding or for PLP-induced cysteine desulfurase or biotin synthase activity was observed with any of the forms of BioB investigated in this work. The results confirm that BioB contains two distinct Fe-S cluster binding sites. One site accommodates a [2Fe-2S](2+) cluster with partial noncysteinyl ligation that can only be reconstituted in vitro in the presence of O(2). The other site accommodates a [4Fe-4S](2+,+) cluster that binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster and undergoes O(2)-induced degradation via a distinct type of [2Fe-2S](2+) cluster intermediate. In vivo M?ssbauer studies show that recombinant BioB in anaerobically grown cells is expressed exclusively in an inactive form containing only the as-isolated [2Fe-2S](2+) cluster and demonstrate that the [2Fe-2S](2+) cluster is not a consequence of overexpressing the recombinant enzyme under aerobic growth conditions. Overall the results clarify the confusion in the literature concerning the Fe-S cluster composition and the in vitro reconstitution and O(2)-induced cluster transformations that are possible for recombinant BioB. In addition, they provide a firm foundation for assessing cluster transformations that occur during turnover and the catalytic competence of the [2Fe-2S](2+) cluster as the immediate S-donor for biotin biosynthesis.     

The novel structure and chemistry of iron-sulfur clusters in the adenosylmethionine-dependent radical enzyme biotin synthase

Biotin synthase is an adenosylmethionine-dependent radical enzyme that catalyzes the substitution of sulfur for hydrogen at the saturated C6 and C9 positions in dethiobiotin. The structure of the biotin synthase monomer is an (alpha/beta)(8) barrel that contains one [4Fe-4S](2+) cluster and one [2Fe-2S](2+) cluster that encapsulate the substrates AdoMet and dethiobiotin. The air-sensitive [4Fe-4S](2+) cluster and the reductant-sensitive [2Fe-2S](2+) cluster have unique coordination environments that include close proximity to AdoMet and DTB, respectively. The relative positioning of these components, as well as several conserved protein residues, suggests at least two potential catalytic mechanisms that incorporate sulfur from either the [2Fe-2S](2+) cluster or a cysteine persulfide into the biotin thiophane ring. This review summarizes an accumulating consensus regarding the physical and spectroscopic properties of each FeS cluster, and discusses possible roles for the [4Fe-4S](2+) cluster in radical generation and the [2Fe-2S](2+) cluster in sulfur incorporation.     

Optical and EPR characterization of different species of active and inactive aconitase

It has been shown by spectroscopic (Kent, T. A., Dreyer, J-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1096-1100) and chemical (Kennedy, M.C., Emptage, M.H., Dryer, J-L., and Beinert, H. (1983) J. Biol. Chem. 258, 11098-11105) methods that interconversion of [3Fe-4S] and [4Fe-4S] clusters underlies activation and inactivation of aconitase. Since Fe-S clusters can assume different oxidation states, a number of different species of the enzyme can be expected to exist. Observations on activation-inactivation, as well as light absorption and EPR spectra, can be interpreted in terms of four species: [3Fe-4S]1+, the oxidized inactive enzyme as obtained on aerobic preparation from mitochondria; [3Fe-4S]0, the reduced inactive form as obtained on reduction in the presence of EDTA; [4Fe-4S]2+, the oxidized active form as obtained on reductive activation; and [4Fe-4S]1+, the reduced active form prepared by photoreduction of active aconitase. The light absorption spectra of each species are presented. Oxidized inactive aconitase shows EPR spectra typical of oxidized 3Fe clusters (g = 2.01), and reduced active enzyme shows spectra typical of reduced ferredoxins (g1,2,3 = 2.06, 1.93, 1.86). The EPR spectrum of the latter is drastically changed (g1,2,3 = 2.04, 1.85, 1.78) on addition of substrate. The active enzyme can be quantitatively converted to inactive enzyme by titration with ferricyanide in the presence of substrate. The correlation of EPR and optical spectra with enzymatic activity observed during titration demonstrates further that active aconitase requires an intact [4Fe-4S] cluster. A model of aconitase incorporating the four cluster species is presented, and explanations for some previous conflicting data concerning aconitase are offered.     

Biotin synthase contains two distinct iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions.

Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV. BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.     

Iron-sulfur clusters of biotin synthase in vivo: a Mössbauer study

Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe M?ssbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.     

Reactivity of nitric oxide with the [4Fe-4S] cluster of dihydroxyacid dehydratase from Escherichia coli

Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.     

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.     

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.     

Spectroscopic changes during a single turnover of biotin synthase: destruction of a [2Fe-2S] cluster accompanies sulfur insertion.

Biotin synthase catalyzes the insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Catalysis requires AdoMet and flavodoxin and generates 5'-deoxyadenosine and methionine, suggesting that biotin synthase is an AdoMet-dependent radical enzyme. Biotin synthase (BioB) is aerobically purified as a dimer of 38.4 kDa monomers that contains 1-1.5 [2Fe-2S](2+) clusters per monomer and can be reconstituted with exogenous iron, sulfide, and reductants to contain up to two [4Fe-4S] clusters per monomer. The iron-sulfur clusters may play a dual role in biotin synthase: a reduced iron-sulfur cluster is probably involved in radical generation by mediating the reductive cleavage of AdoMet, while recent in vitro labeling studies suggest that an iron-sulfur cluster also serves as the immediate source of sulfur for the biotin thioether ring. Consistent with this dual role for iron-sulfur clusters in biotin synthase, we have found that the protein is stable, containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. In the present study, we demonstrate that this mixed cluster state is essential for optimal activity. We follow changes in the Fe and S content and UV/visible and EPR spectra of the enzyme during a single turnover and conclude that during catalysis the [4Fe-4S](2+) cluster is preserved while the [2Fe-2S](2+) cluster is destroyed. We propose a mechanism for incorporation of sulfur into dethiobiotin in which a sulfur atom is oxidatively extracted from the [2Fe-2S](2+) cluster.     

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, M?ssbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel biotin formation. The initial decay rate of the [2Fe-2S](2+) cluster is about 1 order of magnitude faster than the initial formation rate of biotin, indicating that if the [2Fe-2S] cluster is the immediate S donor for biotin synthesis, insertion of S into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe-2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe-2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.     

Detection of a [3Fe-4S] cluster intermediate of cytosolic aconitase in yeast expressing iron regulatory protein 1. Insights into the mechanism of Fe-S cluster cycling.

Interconversion of iron regulatory protein 1 (IRP1) with cytosolic aconitase (c-aconitase) occurs via assembly/disassembly of a [4Fe-4S] cluster. Recent evidence implicates oxidants in cluster disassembly. We investigated H(2)O(2)-initiated Fe-S cluster disassembly in c-aconitase expressed in Saccharomyces cerevisiae. A signal for [3Fe-4S] c-aconitase was detected by whole-cell EPR of aerobically grown, aco1 yeast expressing wild-type IRP1 or a S138A-IRP1 mutant (IRP1(S138A)), providing the first direct evidence of a 3Fe intermediate in vivo. Exposure of yeast to H(2)O(2) increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity. Untreated yeast expressing IRP1(S138D) or IRP1(S138E), which mimic phosphorylated IRP1, failed to give a 3Fe signal. H(2)O(2) produced a weak 3Fe signal in yeast expressing IRP1(S138D). Yeast expressing IRP1(S138D) or IRP1(S138E) were the most sensitive to inhibition of aconitase-dependent growth by H(2)O(2) and were more responsive to changes in media iron status. Ferricyanide oxidation of anaerobically reconstituted c-aconitase yielded a strong 3Fe EPR signal with wild-type and S138A c-aconitases. Only a weak 3Fe signal was obtained with S138D c-aconitase, and no signal was obtained with S138E c-aconitase. This, paired with loss of c-aconitase activity, strongly argues that the Fe-S clusters of these phosphomimetic c-aconitase mutants undergo more complete disassembly upon oxidation. Our results demonstrate that 3Fe c-aconitase is an intermediate in H(2)O(2)-initiated Fe-S cluster disassembly in vivo and suggest that cluster assembly/disassembly in IRP1 is a dynamic process in aerobically growing yeast. Further, our results support the view that phosphorylation of IRP1 can modulate its response to iron through effects on Fe-S cluster stability and turnover.     

M?ssbauer studies of Escherichia coli biotin synthase: evidence for reversible interconversion between [2Fe-2S](2+) and [4Fe-4S](2+) clusters.

The nature and properties of the iron-sulphur (Fe-S) cluster in as-prepared and reduced biotin synthase of Escherichia coli have been investigated by M?ssbauer spectroscopy. Our data clearly demonstrate that in the as-prepared sample, the cluster is present as [2Fe-2S](2+) with isomer shift, delta = 0.29 mm/s and quadrupole splitting, DeltaE(Q) = 0.53 mm/s, indicating incomplete cysteinyl-S coordination. Anaerobic reduction by dithionite in the presence of 55% (v/v) glycerol converts this form to [4Fe-4S](2+) (delta = 0.45 mm/s and DeltaE(Q) = 1.11 mm/s) and is accompanied by some destruction to Fe(2+). This cluster conversion is reversible and when exposed to air, the [4Fe-4S](2+) cluster is quantitatively reconverted to the [2Fe-2S](2+) cluster without any further cluster degradation.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.