No abstract

The transcription factor FNR from Escherichia coli regulates transcription of genes in response to oxygen deprivation. To determine how the activity of FNR is regulated by oxygen, a form of FNR had to be isolated that had properties similar to those observed in vivo. This was accomplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen. Iron and sulfide analyses of this FNR fraction indicated the presence of an Fe-S cluster. To determine the type of Fe-S cluster present, an oxygenstable mutant protein LH28-DA154 was also analyzed since FNR LH28-DA154 purified anoxically contained almost 3-fold more iron and sulfide than the wild-type protein. Based on the sulfide analysis, the stoichiometry (3.3 mol of S 2؊ /FNR monomer) was consistent with either one [4Fe-4S] or two [2Fe-2S] clusters per mutant FNR monomer. However, since FNR has only four Cys residues as potential cluster ligands and an EPR signal typical of a 3Fe-4S cluster was detected on oxidation, we conclude that there is one [4Fe-4S] cluster present per monomer of FNR LH28-DA154. We assume that the wild type also contains one [4Fe-4S] cluster per monomer and that the lower amounts of iron and sulfide observed per monomer were due to partial occupancy. Consistent with this, the Fe-S cluster in the wild-type protein was found to be extremely oxygen-labile. In addition, molecular-sieve chromatographic analysis showed that the majority of the anoxically purified protein was a dimer as compared to aerobically purified FNR which is a monomer. The loss of the Fe-S cluster by exposure to oxygen was associated with a conversion to the monomeric form and decreased DNA binding. Taken together, these observations suggest that oxygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen.

Numerous degenerative disorders are associated with elevated levels of prooxidants and declines in mitochondrial aconitase activity. Deficiency in the mitochondrial iron-binding protein frataxin results in diminished activity of various mitochondrial iron-sulfur proteins including aconitase. We found that aconitase can undergo reversible citrate-dependent modulation in activity in response to pro-oxidants. Frataxin interacted with aconitase in a citrate-dependent fashion, reduced the level of oxidant-induced inactivation, and converted inactive [3Fe-4S]1+ enzyme to the active [4Fe-4S]2+ form of the protein. Thus, frataxin is an iron chaperone protein that protects the aconitase [4Fe-4S]2+ cluster from disassembly and promotes enzyme reactivation.

The aconitases (EC 4.2.1.3) are a family of dehyratases that catalyze the reversible isomerization of citrate and isocitrate via cis-aconitate (1). These enzymes, of which bovine mitochondrial aconitase (m-acon), 1 is the most extensively studied, contain unique [4Fe-4S] clusters in that one of the irons, Fe a , is not ligated to a protein residue but rather to a hydroxide from solvent (2). This is the same iron to which substrate binds during turnover. Inactivation of these enzymes occurs when Fe a is lost by oxidation of the Fe-S cluster with the formation of a cubane [3Fe-4S] cluster that is detectable by electron paramagnetic resonance spectroscopy (EPR) at very low temperatures at g Ϸ 2.02. To date there is no EPR evidence that this reaction occurs in vivo.The recent discovery that the apo-form of mammalian cytosolic aconitase (c-acon), is identical to an RNA-binding protein, iron-regulatory protein (IRP1), has led to increased interest in the study of this isoform of the enzyme (3-5). There is a second IRP, IRP2, with similarities both in structure and function to IRP1 that will not be dealt with in this study (6, 7). These iron-regulatory proteins bind to specific stem-loop structures called iron-responsive elements which occur in the untranslated regions of the mRNA of a number of proteins involved in iron and energy metabolism (8). When bound to iron-responsive elements located in the 5Ј-untranslated regions of the mRNA as occurs with the iron storage protein, ferritin, translation is blocked (9). In contrast to this, binding to the iron-responsive elements in the 3Ј-untranslated regions of the mRNA of the transferrin receptor stabilizes the message, allowing translation to occur (10). For example, in iron-deficient cells the binding of IRP1 (apo-c-acon) to these iron-responsive elements would increase the production of transferrin receptors, which are key for increasing intracellular iron concentrations, while decreasing the biosynthesis of ferritin. Of particular interest in this process of cellular iron regulation is the mechanism of the interconversion between c-acon, containing a [4Fe-4S] cluster, and the cluster-free IRP1, since it has been demonstrated that the de novo biosynthesis of protein is not involved (11). Furthermore, in vitro experiments have shown that the Fe-S cluster of c-acon is quite stable, particularly in the presence of substrate (3) which can be assumed to be present in all cells. Therefore, any mechanism proposed for the interconversion must take these facts into consideration.Two lines of evidence have led investigators to examine whether or not NO plays a role in the cellular process where c-acon is converted to IRP. First, it had been known from the work of Hibbs and co-workers (12, 13) that, when tumor target cells are cocultivated with activated macrophages, there is a loss of iron from the target cells that is associated with the inhibition of mitochondrial respiration and DNA replication as well as with the inactivation of m-acon. Later it was shown that this process oc...

In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia cofi aconitases (EC 4.2. In the past 10 years evidence has been obtained that intracellular iron levels are controlled by a posttranscriptional mechanism which correlates translation of mRNA for the H subunit of ferritin and stabilization of transferrin receptor mRNA. This is accomplished by the interaction ofa cytosolic protein with iron-responsive elements (IREs),-which are stem-loop structures located in the untranslated regions of the respective mRNAs (1-3). Small quantities (nanograms to micrograms) of a cytosolic protein of -100 kDa that binds to IREs (IRE binding protein, IRE-BP) have been isolated (4-6). This research took an unexpected turn when the cDNA sequence for the protein from human liver was determined and the protein sequence deduced was found to have a striking homology to the amino acid sequence of pig heart mitochondrial aconitase (m-aconitase) (7). All active-site residues identified in the aconitase crystal structure are conserved (8 MATERIALS AND METHODS m-Aconitase was prepared and enzyme activation, assay, and analysis for S2-, SO, and Fe were carried out as described (18)(19)(20). Protein was determined by a biuret method, standardized for m-or c-aconitase, respectively, by amino acid analysis.Purification of c-Aconitase. m-Aconitase is the least desirable contaminant of c-aconitase. Hence, we chose as the initial part of the purification procedure the separation of cytosol and mitochondria by a method previously used for the large-scale preparation of mitochondria from slaughterhouse tissue (21); the following modifications were incorporated: (i) 2 mM Hepes (pH 7.2) containing 2 mM citrate was used as buffer, (ii) the tissue grinding step was omitted, (iii) blending time was only 30 sec, and (iv) the homogenate was centrifuged at 1300 x g for 60 min. All manipulations were done at 0-40C and the initial ratio of liver to buffer was 1:3.5 (wt/vol). The supernatant obtained after the sedimentation of the mitochondria was made 20%o (vol/vol) 11730The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Prostate epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase activity that minimizes their ability to oxidize citrate. These cells also possess uniquely high cellular and mitochondrial zinc levels. Correlations among zinc, citrate, and m-aconitase in prostate indicated that zinc might be an inhibitor of prostate m-aconitase activity and citrate oxidation. The present studies reveal that zinc at near physiological levels inhibited m-aconitase activity of mitochondrial sonicate preparations obtained from rat ventral prostate epithelial cells. Corresponding studies conducted with mitochondrial sonicates of rat kidney cells revealed that zinc also inhibited the kidney m-aconitase activity. However the inhibitory effect of zinc was more sensitive with the prostate m-aconitase activity. Zinc inhibition fit the competitive inhibitor model. The inhibitory effect of zinc occurred only with citrate as substrate and was specific for the citrate 3 cis-aconitate reaction. Other cations (Ca 2؉ , Mn 2؉ , Cd 2؉) did not result in the inhibitory effects obtained with zinc. The presence of endogenous zinc inhibited the m-aconitase activity of the prostate mitochondrial preparations. Kidney preparations that contain lower endogenous zinc levels exhibited no endogenous inhibition of m-aconitase activity. Studies with pig prostate and seminal vesicle mitochondrial preparations also revealed that zinc was a competitive inhibitor against citrate of m-aconitase activity. The effects of zinc on purified beef heart m-aconitase verified the competitive inhibitor action of zinc. In contrast, zinc had no inhibitory effect on purified cytosolic aconitase. These studies reveal for the first time that zinc is a specific inhibitor of m-aconitase of mammalian cells. In prostate epithelial cells, in situ mitochondrial zinc levels inhibit m-aconitase activity, which provides a mechanism by which citrate oxidation is limited.Prostate secretory epithelial cells have the specialized function and capability of accumulating and secreting extraordinarily high levels of citrate. This is achieved by the existence of a uniquely limiting m-aconitase 1 activity that minimizes the oxidation of citrate via the Krebs cycle. Consequently, citrate synthesized by these cells is accumulated and secreted (which we refer to as "net citrate production"), thereby accounting for the extremely high (20 -150 mM) citrate content of human prostatic fluid. In typical mammalian cell metabolism, m-aconitase is not a regulatory, rate-limiting enzyme. Consequently, the steady-state citrate/isocitrate ratio of most cells is generally maintained at about 11/1, which is established by the aconitase equilibrium reaction, 88 citrate 7 4 cis-aconitate 7 8 isocitrate. In contrast, the citrate/isocitrate ratio in prostate is generally about 30/1. Also, the intracellular citrate concentration of prostate cells is estimated to be about 1.2 mM as compared with about 0.1-0.4 mM for typical mammalian cells. These and other relationships of prostate citrate metabolism an...

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 ⅐ OH may be continuously generated in the mitochondria.There is much debate in the literature on the relative importance of hydroxyl radical ( ⅐ OH) and peroxynitrite in free radical pathology (1). Clarification of the mechanism centered on this subject is of considerable importance, especially in mitochondria, cellular organelles that are constantly exposed to low levels of superoxide anion (2, 3). Several neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, and Lou Gehrig's disease or amyotrophic lateral sclerosis) and aging have been linked to mitochondrial oxidative damage that results in decreased mitochondrial function (3, 4). However, in biological systems, it is nearly impossible to associate a specific damage to a single oxidant. For example, superoxide and nitric oxide ( ⅐ NO) co-generated at very low levels (Ϸ10 Ϫ8 M) in cells will form peroxynitrite (ONOO Ϫ ) via a nearly diffusion-controlled reaction (5, 6). The toxicological significance of these species is clearly dependent on cell type, the biological targets, and their relationship to one another. One of the sensitive biological targets in oxidative damage to mitochondria is aconitase, an iron-sulfur protein that catalyzes the stereospecific dehydration-hydration of citrate to isocitrate in the Krebs cycle (7). Aconitase activity in mitochondria has been reported to be a sensitive redox sensor of reactive oxygen and nitrogen species in cells (8 -11). Aconitase contains a cubane-type [4Fe-4S] 2ϩ cluster in its active site with three iron atoms bound to cysteinyl groups and inorganic sulfur atoms and a fourth labile iron atom (Fe-␣). This Fe-␣ is unique in that it is not bound to a protein cysteine, but rather to a hydroxyl group of substrate and water (7 (13, 14). However, the reaction between aconitase and peroxynitrite is strongly inhibited by the addition of substrate that binds to the enzyme with high affinity (14).It was recently proposed that the reaction between mitochondrial aconitase (m-aconitase) 1 and superoxide plays a major role in mitochondrial oxidative damage (15)(16)(17). During this reaction, it has been proposed that iron is released from maconitase as iron(II) with the concomitant generation of hydrogen peroxide. This facilitates the formation of "free" hydroxyl radical in mitochondria. In the presence of intracellular reducing agents (e.g. glutathione, ascorbate, and NADPH), iron(II) is reincorporated into the inactive form of m-aconitase to regenerate the active form. According to this proposal, hydroxyl radical should be continuously generated in mitochondria as a result of the reaction between superoxide and aconitase. However, the experimental verification of this intriguing mechanism has so far been lacking.The objective of this study is to provide e...

The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (ERE-BP), with RNA stem-oop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S duster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.