IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins.

IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA/SoxS/Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S](1+) cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells.

Jac1, a mitochondrial J-type chaperone, is involved in the biogenesis of Fe/S clusters in Saccharomyces cerevisiae.

A minor Hsp70 chaperone of the mitochondrial matrix of Saccharomyces cerevisiae, Ssq1, is involved in the formation or repair of Fe/S clusters and/or mitochondrial iron metabolism. Here, we report evidence that Jac1, a J-type chaperone of the mitochondrial matrix, is the partner of Ssq1 in this process. Reduced activity of Jac1 results in a decrease in activity of Fe/S containing mitochondrial proteins and an accumulation of iron in mitochondria. Fe/S enzyme activities remain low in both jac1 and ssq1 mutant mitochondria even if normal mitochondrial iron levels are maintained. Therefore, the low activities observed are not solely due to oxidative damage caused by excess iron. Rather, these molecular chaperones likely play a direct role in the normal assembly process of Fe/S clusters.

A tribute to sulfur.

Recent progress in a number of areas of biochemistry and biology has drawn attention to the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved. This brief review is intended as a broad overview of this currently rapidly moving field of sulfur biochemistry, for those who are interested or are involved in one or the other aspect of it, a synopsis by one who has stumbled into this field from several directions in the course of time. Only for iron are metal-sulfur relationships discussed in detail, as the iron-sulfur subfield is one of the most active areas.

Iron-sulfur proteins: ancient structures, still full of surprises.

This article is a survey of the properties and functions of Fe-S proteins under the following headings: sulfur and iron; iron-sulfur clusters; evolution of cofactor use; early observations; complex and extended clusters; sulfur exchange and core interconversions; synthesis and biosynthesis of Fe-S clusters; functions of Fe-S clusters: electron transfer, electron delocalization, spin states and magnetism, covalency of sulfur bonds; non-electron transfer functions of Fe-S clusters: substrate binding and catalysis, regulatory and sensing functions.

Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1.

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.

Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the [4Fe-4S](2+) cluster to oxygen.

To understand the role of the [4Fe-4S](2+) cluster in controlling the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) during changes in O(2) availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions. The His-28 substitution appears to stabilize the [4Fe-4S](2+) cluster of FNR-L28H in the presence of O(2) because air-exposed FNR-L28H did not undergo the rapid [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions. This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S](2+) cluster-deficient preparations of FNR-L28H. The Mössbauer spectra of FNR-L28H indicated that the coordination environment of the [4Fe-4S](2+) cluster did not differ from that of WT-FNR. Whole cell Mössbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the [4Fe-4S](2+) cluster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [4Fe-4S](2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.

Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae.

nifU of nitrogen-fixing bacteria is involved in the synthesis of the Fe-S cluster of nitrogenase. In a synthetic lethal screen with the mitochondrial heat shock protein (HSP)70, SSQ1, we identified a gene of Saccharomyces cerevisiae, NFU1, which encodes a protein with sequence identity to the C-terminal domain of NifU. Two other yeast genes were found to encode proteins related to the N-terminal domain of bacterial NifU. They have been designated ISU1 and ISU2. Isu1, Isu2, and Nfu1 are located in the mitochondrial matrix. ISU genes of yeast carry out an essential function, because a Deltaisu1Deltaisu2 strain is inviable. Growth of Deltanfu1Delta isu1 cells is significantly compromised, allowing assessment of the physiological roles of Nfu and Isu proteins. Mitochondria from Deltanfu1Deltaisu1 cells have decreased activity of several respiratory enzymes that contain Fe-S clusters. As a result, Deltanfu1Deltaisu1 cells grow poorly on carbon sources requiring respiration. Deltanfu1Deltaisu1 cells also accumulate abnormally high levels of iron in their mitochondria, similar to Deltassq1 cells, indicating a role for these proteins in iron metabolism. We suggest that NFU1 and ISU1 gene products play a role in iron homeostasis, perhaps in assembly, insertion, and/or repair of mitochondrial Fe-S clusters. The conservation of these protein domains in many organisms suggests that this role has been conserved throughout evolution.

Fe-S proteins in sensing and regulatory functions.

In the past five to ten years, it has become increasingly apparent that the function of Fe-S clusters is not limited to electron transfer, a function implicit in their discovery. We now know that the vulnerability of these structures to oxidative destruction is used by nature in sensing O2, iron, and possibly also nitric oxide. Changes in the oxidation state of Fe-S clusters can also serve as a reversible switch.

Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli.

The global regulator FNR (for fumarate nitrate reduction) controls the transcription of >100 genes whose products facilitate adaptation of Escherichia coli to growth under O2-limiting conditions. Previous Mössbauer studies have shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster that, on exposure to oxygen, is converted into a [2Fe-2S]2+ cluster, a process that decreases DNA binding by FNR. Using 57Fe Mössbauer spectroscopy of E. coli cells containing overexpressed FNR, we show here that the same cluster conversion also occurs in vivo on exposure to O2. Furthermore, the data show that a significant amount of the [4Fe-4S]2+ cluster is regenerated when the cells are shifted back to an anaerobic environment. The present study also demonstrates that 57Fe Mössbauer spectroscopy can be employed to study the in vivo behavior of (overexpressed) proteins. The use of this technique to study other iron-containing cell components is discussed.

S-Adenosylmethionine-dependent reduction of lysine 2,3-aminomutase and observation of the catalytically functional iron-sulfur centers by electron paramagnetic resonance.

Lysine 2,3-aminomutase catalyzes the interconversion of l-alpha-lysine and l-beta-lysine. The enzyme contains an iron-sulfur cluster with unusual properties, and it requires pyridoxal-5'-phosphate (PLP) and S-adenosylmethionine (AdoMet) for activity. The reaction proceeds by a substrate radical rearrangement mechanism, in which the external aldimine formed between PLP and lysine is initially converted into a lysyl-radical intermediate by hydrogen abstraction from C3. The present research concerns the mechanism by which a hydrogen-abstracting species is generated at the active site of lysine 2,3-aminomutase. Earlier tritium tracer experiments have implicated the 5'-deoxyadenosyl moiety of AdoMet in this process. AdoMet is here shown to interact with the iron-sulfur cluster at the active site of Clostridial lysine 2,3-aminomutase. Reduction of the iron-sulfur cluster from its EPR-silent form [4Fe-4S]2+ to the fully reduced form [4Fe-4S]1+ requires the presence of either AdoMet or S-adenosylhomocysteine (SAH) and a strong reducing agent such as dithionite or deazariboflavin and light. The reduced forms are provisionally designated E-[4Fe-4S]1+/AdoMet and E-[4Fe-4S]1+/SAH, and they display similar low-temperature EPR spectra centered at gav = 1.91. The reduced form E-[4Fe-4S]1+/AdoMet is fully active in the absence of any added reducing agent, whereas the form E-[4Fe-4S]1+/SAH is not active. It is postulated that the active form E-[4Fe-4S]1+/AdoMet is in equilibrium with a low concentration of a radical-initiating form that contains the 5'-deoxyadenosyl radical. Initiation of the radical rearrangement mechanism is postulated to take place by action of the 5'-deoxyadenosyl radical in abstracting a hydrogen atom from carbon-3 of lysine, which is bound as its external aldiminine with PLP. This process accounts for the results of tritium tracer experiments, it explains the radical rearrangement mechanism, and it rationalizes the roles of AdoMet and the [4Fe-4S] cluster in the reaction.

Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster.

FNR is a global regulator that controls transcription of genes whose functions facilitate adaptation to growth under O2 limiting conditions. It has long been appreciated that the activity of FNR must be regulated by O2 availability, since FNR dependent gene expression is observed in vivo only under anaerobic conditions, while similar levels of this protein are present in both aerobic and anaerobic grown cells. Recent progress in this field has shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster and that this [4Fe-4S]2+ cluster is sufficiently unstable toward O2 to make it suitable as an O2 sensor. The presence of the [4Fe-4S] cluster increases dimerization of FNR which is correlated with an increase in site-specific DNA binding of FNR, a property expected of transcription factors of the FNR/CRP family. According to Mössbauer spectroscopy on purified FNR and cells containing overexpressed FNR, the [4Fe-4S]2+ cluster of FNR is converted by O2 to a [2Fe-2S]2+ in high yield. The [2Fe-2S]2+ cluster can be reconverted to the [4Fe-4S]2+ cluster on reduction with dithionite in vitro raising the possibility that the [2Fe-2S]2+ cluster is a biologically inactive intermediate which may be more readily available for reconstitution into the [4Fe-4S]2+ form than the Fe-free apoform. The ability to observe, by Mössbauer spectroscopy, the Fe-S clusters of FNR in cells containing high levels of FNR should be of value in further unraveling how FNR functions in vivo. Attempts to reduce the [4Fe-4S]2+ cluster of FNR with dithionite indicated that the redox potential of the +1/+2 couple is < or = -650 mV and that the [4Fe-4S]+ cluster form is, therefore, not likely to occur in vivo.

Iron-sulfur clusters: nature's modular, multipurpose structures.

Iron-sulfur proteins are found in all life forms. Most frequently, they contain Fe2S2, Fe3S4, and Fe4S4 clusters. These modular clusters undergo oxidation-reduction reactions, may be inserted or removed from proteins, can influence protein structure by preferential side chain ligation, and can be interconverted. In addition to their electron transfer function, iron-sulfur clusters act as catalytic centers and sensors of iron and oxygen. Their most common oxidation states are paramagnetic and present significant challenges for understanding the magnetic properties of mixed valence systems. Iron-sulfur clusters now rank with such biological prosthetic groups as hemes and flavins in pervasive occurrence and multiplicity of function.

An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide.

Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide. Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein. We have undertaken the in vitro study of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants. Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms. This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clusters and the g approximately 2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d7 state. In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, g parallel = 2.11 and g perpendicular = 2.03, is observed. Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g approximately 2.04 species and in the case of mitochondrial aconitase, a transient signal at g approximately 2. 032 appeared. This signal is tentatively assigned to the d9 form of an iron-nitrosyl-histidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g = 2.02 [3Fe-4S] proteins.

Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O2: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity.

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]2+ cluster (delta = 0.45 mm/s; DeltaEQ = 1.22 mm/s) and that the [4Fe-4S]2+ cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow-green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]2+ cluster was converted in about 60% yield to a [2Fe-2S]2+ cluster (delta = 0.28 mm/s; DeltaEQ = 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]2+ cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]2+ cluster could be largely restored from the [2Fe-2S]2+ form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]2+ cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein.

Copper A of cytochrome c oxidase, a novel, long-embattled, biological electron-transfer site.

This review traces the history of understanding of the CuA site in cytochrome c oxidase (COX) from the beginnings, when few believed that there was any significant Cu in COX, to the verification of three atoms Cu/monomer and to the final identification of the site as a dinuclear, Cys-bridged average valence Cu1.5+ ... Cu1.5+ structure through spectroscopy, recombinant DNA techniques, and crystallography. The critical steps forward in understanding the nature of the CuA site are recounted and the present state (as of the end of 1996) of our knowledge of the molecular and electronic structure is discussed in some detail. The contributions made through the years by the development of methodology and concepts for solving the enigma of CuA are emphasized and impediments, often rooted in contemporary preconceptions and attitudes rather than solid data, are mentioned, which discouraged the exploitation of early valuable clues. Finally, analogies in construction principles of polynuclear Cu-S and Fe-S proteins are pointed out.

Copper in biological systems. A report from the 7th Manziana Conference, held at Santa Severa, September 11-15, 1995.

In this fifty-seven page report, the author attempts to give the essence of the twenty-four lectures and of an about equal number of posters, including subjects of discussion, that were presented at an international conference on copper proteins held in Italy. The report deals with research carried out up to mid-1995 and contains 140 literature references and thirty-three figures or schemes.

The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex.

It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of fluorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (-)-erythro diastereomer of 2-fluorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to fluoro-cis-aconitate, followed by addition of hydroxide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of fluorocitrate inhibition is supported by the crystal structure of the enzyme-inhibitor complex at 2.05-A resolution, release of fluoride stoichiometric with total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10(6)-fold excess of isocitrate, and previously published Mössbauer experiments. When (+)-erythro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative alpha-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds < 2.7 A in length involving HTn, H2O bound to the [4Fe-4S] cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.

Redox control of gene expression involving iron-sulfur proteins. Change of oxidation-state or assembly/disassembly of Fe-S clusters?

Attention is drawn to a mechanism of redox control of gene expression involving Fe-S proteins which depends on the disassembly and reassembly of Fe-S clusters rather than a change in oxidation state. Iron Regulatory Protein (IRP)/aconitase and FNR are discussed as examples for such a mechanism.

DNA binding and dimerization of the Fe-S-containing FNR protein from Escherichia coli are regulated by oxygen.

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 oxygen-stable 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 S2-/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.

Crystals and structures of cytochrome c oxidases--the end of an arduous road.

Crystal structures of cytochrome c oxidases, one of which is the largest membrane-bound protein complex crystallized to date in a form suitable for X-ray diffraction, have recently been solved. The information from these accomplishments confirms many of the structural properties known from earlier spectroscopic and analytical studies, and provides a basis for understanding the complex mechanisms of electron transfer and proton pumping.