Cluster Ligands Research Articles

One-Pot Synthesis of Antigen-Bearing, Lysine-Based Cluster Mannosides Using Two Orthogonal Chemoselective Ligation Reactions

Successive hydrazone/thioether orthogonal ligations of a dendrimeric lysine core with antigenic peptides and mannoside derivatives are described (see scheme). This method provides a flexible one-pot synthesis for artificial conjugates of clustered carbohydrate receptor ligands bearing antigens.

Substitution of Leucine 28 with Histidine in theEscherichia 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.

Slow formation of [3Fe–4S] 1+ clusters in mutant forms of Desulfovibrio africanus ferredoxin III

Desulfovibrio africanus ferredoxin III ( Da FdIII) readily interconverts between a 7Fe and an 8Fe form with Asp-14 believed to provide a cluster ligand in the latter form. To investigate the factors important for cluster interconversion in Fe/S cluster-containing proteins we have studied two variants of Da FdIII produced by site-directed mutagenesis, Asp14Glu and Asp14His, with cluster incorporation performed in vitro. Characterisation of these proteins by UV/visible, EPR and 1H NMR spectroscopies revealed that the formation of the stable 7Fe form of these proteins takes some time to occur. Evidence is presented which indicates the [4Fe–4S] 2+ cluster is incorporated prior to the [3Fe–4S] 1+ cluster.

Synthesis and spectroscopic characterisation of compounds with formula [{Fe 2S 2(CO) 5}Ph 2P(CH 2) nPPh 2{Fe(CO) 4}] and [{Fe 2S 2(CO) 5} 2Ph 2P(CH 2) nPPh 2] ( n=3, 4 and 6)

The work described herein concerns the synthesis and characterisation of compounds with formula [{Fe 2S 2(CO) 5}Ph 2P(CH 2) n PPh 2{Fe(CO) 4}] and [{Fe 2S 2(CO) 5} 2Ph 2P(CH 2) n PPh 2] ( n=3, 4, 6). The iron bis-phosphine cluster compounds with formula [{Fe 3(CO) 11} 2Ph 2P(CH 2) n PPh 2] and [{Fe 3(CO) 11}Ph 2P(CH 2) n PPh 2{Fe(CO) 4}] ( n=3, 4, 6)] were reacted with stoichiometric amounts of cyclohexene episulfide [SC 6H 10] in toluene at 70°C for 30–60 min. Optimum yields of 42.1–78.2%, were obtained for {Fe 2S 2Fe} type species when a 1:4 cluster–ligand mole ratio was used, while the {Fe 2S 2} 2 type species were prepared in yields of 30–38% when a 1:8 cluster–ligand mole ratio was used. The volume of reaction solvent was kept to a minimum. For the latter cluster compounds, three isomeric products were isolated from each reaction; a justification for their presence is offered. All of these reactions afford clusters with fewer iron atoms but with S atoms incorporated. In these reactions the Fe 3-unit is ‘degraded’ but at the same time the cluster is expanded from three atoms {Fe 3} to four {Fe 2S 2}.

Generation of metal–ligand cluster ion beams through pulsed discharge ionization and ablation

Pulsed capacitor discharge ionization in supersonic expansions was investigated for the production of intense beams of molecular cluster ions from seeded and ablated compounds. A pulsed discharge based on a triggered spark gap switch was designed and used as a method for ionization and ablation. Several combinations of nozzle geometry and electrode arrangement in front of a pulsed valve, were made to optimize the intensity of the ion beam as well as its composition. The cationic metal–ligand complexes Cu +–(methanol) n , Cu +–(acetone) n , Cu +–(toluene) n , Cu +–(water) n , and Al +–(water) n were synthesized by ablation of the metal from metallic discharge electrodes in a discharge gas mixture of helium seeded with the ligand of choice. The cluster mass spectra of the expanded plasmas show little background ion signal besides the metal–ligand species. Charge exchange processes in the expansion guarantee high ionization yields of the desired species and account for low backgrounds. Changes in the successive binding energy of Cu +–(water) n clusters n = 1–4 are clearly observed in the cluster mass spectra as step formation. A similar pattern found in Cu +–(acetone) n suggests the same trend in the successive binding energy as known for water.

Mixed-Valent Heptairon Complex with a Ground-State Spin Value ofS=12/2 Constructed from a Triiron Cluster Ligand

The anionic triiron(III) cluster ligand [Fe(III)(3)(µ(3)-O)(bpca)(2)Cl(4)(EtO)(2)](-) (1; Hbpca=bis(2-pyridylcarbonyl)amine) was prepared as a building block for constructing larger metal assemblies. This "metal cluster complex ligand" was used in the synthesis of the mixed-valent heptairon complex [Fe(II)(1)(2)(EtOH)(2)], which has a ground-state spin value of S=12/2.

Mixed-Valent Heptairon Complex with a Ground-State Spin Value ofS=12/2 Constructed from a Triiron Cluster Ligand

Der anionische Trieisen(III)-Clusterligand [Fe(μ3-O)(bpca)2Cl4(EtO)2]− 1, (Hbpca=Bis(2-pyridylcarbonyl)amin) wurde als Baustein für die Konstruktion größerer Metallaggregate hergestellt. Dieser „Metallcluster-Komplexligand”︁ wurde in der Synthese des gemischtvalenten Heptaeisenkomplexes [FeII(1)2(EtOH)2] verwendet, dessen Grundzustandsspin S=12/2 beträgt.

Mixed-Valent Heptairon Complex with a Ground-State Spin Value of S=12/2 Constructed from a Triiron Cluster Ligand

Mixed-Valent Heptairon Complex with a Ground-State Spin Value of S=12/2 Constructed from a Triiron Cluster Ligand

Electron-Nuclear Interactions in Two Prototypical [2Fe–2S] Proteins: Selective (Chiral) Deuteration and Analysis of 1H and 2H NMR Signals from the Alpha and Beta Hydrogens of Cysteinyl Residues That Ligate the Iron in the Active Sites of Human Ferredoxin and Anabaena 7120 Vegetative Ferredoxin

A vertebrate ferredoxin (human ferredoxin) and a plant-type ferredoxin (the ferredoxin from the vegetative form of Anabaena 7120) were labeled selectively with deuterium at their active site cysteines. The recombinant proteins were produced in Escherichia coli and labeled by replacing natural abundance cysteine in the defined culture medium with [2Hα]-cysteine, [2Hβ2, 2Hβ3]-cysteine, or [2Hβ2]-cystine. The chiral labeled cystine ([2Hβ2]-cystine) was prepared by selective hydrogen exchange catalyzed by cystathionine γ-synthase. NMR spectra of these samples in their oxidized and reduced states support unambiguous identifications by atom type of 1H and 2H NMR signals from the cysteine alpha and beta hydrogens. These signals lie outside the normal diamagnetic spectral region as a result of interaction of the hydrogens with unpaired electron density from the iron–sulfur cluster, and their chemical shifts are highly dependent on local conformation at the active site. The very different chemical properties of the iron centers of plant-type and vertebrate ferredoxins reflect relatively small differences in the conformation of the iron–sulfur cluster ligands.

P2O, the Phosphorus Analogue of N2O, as a Ligand in a Tetranuclear Cluster

P2O, the Phosphorus Analogue of N2O, as a Ligand in a Tetranuclear Cluster

Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP.

The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with S gamma of Cys77, one of the cluster ligands. The reduction potential of the S79P variant was 104 +/- 3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins. These results suggest that the influence of amide group 79 on the reduction potential of C. vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins.

Self-Assembly of a Three-Dimensional [Ga6(L2)6] Metal-Ligand “Cylinder”

A near trigonal antiprism with metal–metal distances in the nanometer regime is formed by the six metal ions in the crystalline, homochiral [Ga6(L2)6] (see structure). This metal–ligand “cylinder” is based on a threefold symmetric, β-diketone ligand, and represents a new geometry for metal–ligand clusters.

Protonation and substitution reactions of Fe–S ‘basket’ clusters including extracted FeMo-cofactor of nitrogenase

In an extension to our earlier work on the mechanisms of reactions of Fe–S-based clusters, we now report studies on the substitution kinetics of structurally more ‘open’ Fe–S clusters. In particular, we concentrate on protonation of the ‘basket’ cluster [Fe 6S 6Cl 2(PEt 3) 4]. Protonation at a μ n -S can either accelerate or inhibit the rate of substitution of the cluster depending on: the number of protons added and whether substitution is associative or dissociative. Analysis of the kinetics allows us to calculate the p K a values of the protonated clusters and comparison with a variety of other Fe–S-based clusters shows that, irrespective of the structure and charge, the first (p K a 1=17.9–18.9) is rather insensitive to the cluster ligands whilst the second (p K a 2=13.6–16.9) is markedly more sensitive. Analogous studies on extracted FeMo-cofactor [MoFe 7S 9(R-homocitrate)(NMF) 2] (NMF= N-methylformamide) from the nitrogenase of Klebsiella pneumoniae shows the same general protonation/substitution characteristics as synthetic Fe–S-based clusters.

The syntheses and X-ray crystal structures of novel transition metal cluster arrays containing 2–5 coordinated [(CO) 9Co 3(μ 3-CCOO)] − ligands in a variety of geometries

Reaction of (CO) 9Co 3(μ 3-CCOOH) with metal trifluoroacetates leads to the isolation of Cr 2{μ-OOC-CCo 3(CO) 9} 2{μ-OOCCF 3} 2(THF) 2, 1, and Sm 2{μ-OOCCCo 3(CO) 9} 2{μ-OOCCF 3} 4{(CO) 9Co 3CCOOH} 2(THF) 2·2THF, 2, which are cluster substituted analogs of known organic carboxylates. Reaction of Pt(II) acetate, prepared in situ, with (CO) 9Co 3(μ 3-CCOOH) leads to PtCo{μ-OOCCCo 3(CO) 9} 3(μ-OOCCH 3){(CO) 9Co 3CCOOH}, 3, which contains an unusual Pt(II)Co(II) dimeric core. Reaction of Zn(OH) 2 with (CO) 9Co 3(μ 3-CCOOH) in THF leads to Zn 4(μ 4-O){μ-OOC-CCo 3(CO) 9} 6 but in 2-MeTHF the reaction leads to ZnCo{μ-OOCCCo 3(CO) 9} 3{Co(CO) 4}{2-(CH 3)C 4H 7O}, 4. This compound features the presence of a [Co(CO) 4] − moiety coordinated to a Co(II) center. Reaction of Cr(II) acetate with (CO) 9Co 3(μ 3-CCOOH) in noncoordinating solvents results in CoCr 2(μ 3-O){μ-OOCCCo 3(CO) 9} 4(μ-OOCCH 3) 2{(CO) 9Co 3CCOOH} 2(H 2O)·C 7H 8, 5a, and Cr 3(μ 3-O){μ-OOCCCo 3(CO) 9} 4(μ-OOCCH 3) 2{(CO) 9Co 3CCOOH}(CH 3OOH) 2·C 7H 8, 5b, which are cluster ligand analogs of known organic carboxylate oxo metal trimers. Finally, deprotonation of (CO) 9Co 3(μ 3-CCOOH) with a bulky base inhibits the base degradation of the tricobalt cluster sufficiently such that [C 10H 6(N(CH 3) 2) 2H][Co 2{μ-OOCCCo 3(CO) 9} 3{OOCCCo 3(CO) 9} 2], 6, can be isolated. All compounds are structurally characterized by X-ray crystallography and selected spectroscopic techniques.

ChemInform Abstract: Binding and Reactivity of Thiophene‐Type Ligands in Transition Metal Complexes and Clusters

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas.

Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe2S2 prosthetic group. Tdx was first identified in a strain of Pseudomonas sp. capable of using alpha-terpineol as sole carbon source. The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E. coli. Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdxo) has been determined. High-resolution homo- and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein. The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra. Essentially complete 1H and 15N NMR resonance assignments have been made for the diamagnetic region of Tdxo (ca. 80% of the protein). A large five-stranded beta-sheet and a smaller two-stranded beta-sheet were identified, along with three alpha-helices. A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein. 1H/2H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure. In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state. Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed. Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component.

Role of Urokinase Receptor and Caveolin in Regulation of Integrin Signaling

Emerging evidence indicates a prominent role for non-integrin membrane adaptors in the dynamic regulation of integrin signaling. Two such integrin-associated proteins are the glycosylphosphatidyl-inositol (GPI)-linked urokinase receptor (u-PAR) and the cholesterol-binding protein, caveolin-1. Recent studies indicate that caveolin is required for the association of Src-family kinases with beta 1 integrins. Loss of caveolin/beta 1 integrin association results in loss of ligand-induced focal adhesion kinase (FAK) phosphorylation and impaired development of focal adhesion sites. Similarly, fibronectin-dependent fyn signaling through alpha 5/beta 1 leading to mitogen-activated protein (MAP) kinase activation requires the presence of caveolin-1. Caveolin binds Src-family kinases and such binding maintains these kinases in an inactive state. Current evidence favors a model in which ligand-induced integrin clustering, a central event in integrin activation, promotes caveolin oligomerization leading to release and/or activation of Src-family kinases and initiation of integrin signaling. The presence of u-PAR promotes these events because the extracellular domain(s) of u-PAR binds to beta 1 and beta 2 integrins and the GPI anchor of u-PAR, like that of other GPI-anchored proteins, interacts with cholesterol-rich membrane domains enriched in caveolin and tyrosine kinases. Integrins, caveolin, and u-PAR form interdependent functional complexes, promoting the association of integrins with caveolin-rich signaling domains. During states of accelerated cellular migration, such as during inflammation and tumorigenesis, expression of u-PAR may be a key facilitator of integrin signaling. Interruption of u-PAR/integrin interactions may be a strategy to regulate cellular migration in these settings.

Detection and classification of hyperfine-shifted 1H, 2H, and 15N resonances of the Rieske ferredoxin component of toluene 4-monooxygenase.

T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preliminary 1H NMR studies of oxidized and reduced T4MOC [Markley, J. L., Xia, B., Chae, Y. K., Cheng, H., Westler, W. M., Pikus, J. D., and Fox, B. G. (1996) in Protein Structure Function Relationships (Zaidi, Z., and Smith, D., Eds.) pp 135-146, Plenum Press, London] revealed the presence of hyperfine-shifted 1H resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperfine-shifted 1H, 2H, and 15N signals from T4MOC. Selective deuteration led to identification of signals from the four Hbeta atoms of cluster ligands C45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state, the Curie temperature dependence of the Hbeta protons corresponded to that predicted from the simple vector spin-coupling model for nuclei associated with the localized ferric site. The signal at 25.5 ppm in the 1H spectrum of reduced T4MOC was assigned on the basis of selective 2H labeling to the His Hepsilon1 atom of one of the cluster ligands (H47 or H67). This assignment was corroborated by a one bond 1H-13C correlation (at 25.39 ppm 1H and 136.11 ppm 13C) observed in spectra of [U-13C]T4MOC with a 1H-13C coupling constant of approximately 192 Hz. The carbon chemical shift and one bond coupling constant are those expected for 1Hepsilon1-13Cepsilon1 in the imidazolium ring of histidine and are inconsistent with values expected for cysteine 1Halpha-13Calpha. The His Hepsilon1 proton exhibited weak Curie temperature dependence from 283 to 303 K, contrary to the anti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A 1H peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspond to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results with T4MOC call into question certain 1H assignments recently reported on the basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Py2 [Holz, R. C., Small, F. J., and Ensign, S. A, (1997) Biochemistry 36, 14690-14696]. Selective 15N labeling was used to identify hyperfine-shifted 15N NMR signals from the backbone nitrogens of all four cluster ligands (C45, H47, C64, and H67), from the Nepsilon2 atoms of the two histidine ligands (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) present in the cluster-binding motif of T4MOC in the oxidized and reduced states. The results indicate that the Ndelta1 of each of the two ligand histidines of T4MOC are ligated to an iron atom and reveal a pattern of H-bonding to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T4MOC) of the five backbone amide H-bonds expected on the basis of comparison with the crystal structures of other related Rieske proteins; the fifth backbone amide (I50 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske proteins that may be related to functional diversity in this protein family.

Substitution Site Specificity on the Ru10C2 Cluster Framework. Multiple Convergent Pathways to the Mixed Hydrocarbon Ligand Derivative Ru10C2(CO)21(NBD)(C2R2)

The substitution of carbonyl ligands in the edge-shared bioctahedral cluster [PPN]2[Ru10C2(CO)24] by two types of 4e donor π-bonding ligands, viz., a diene (norbornadiene) and an alkyne (diphenylacetylene), has been investigated under various conditions. The reaction of [PPN]2[Ru10C2(CO)24] with norbornadiene (NBD) in diglyme at 140 °C has provided the anionic derivative [PPN]2[Ru10C2(CO)22(NBD)] (1). Oxidation of this compound with [Cp2Fe][BF4] affords the neutral derivative Ru10C2(CO)23(NBD) (2), which can also be prepared by direct oxidative substitution of [Ru10C2(CO)24]2- with 2[Cp2Fe][BF4] in the presence of NBD. Spectroscopic and crystallographic studies of 1 and 2 show that the NBD ligand occupies a chelating position on one of the “outer” ruthenium atoms in the bifurcated Ru10C2 framework. This location contrasts with that adopted by the alkyne ligand in both [PPN]2[Ru10C2(CO)22(C2Ph2)] (3) and Ru10C2(CO)23(C2Ph2) (4). In these derivatives the alkyne moiety is located in an “inner” site bridging two apical ruthenium atoms. The generality of these substitution sites was probed by preparing the mixed ligand derivative Ru10C2(CO)21(NBD)(C2Ph2) (5). This compound can be synthesized in four distinct ways: (1) by oxidation of 1 with 2[Cp2Fe][BF4] in the presence of C2R2; (2) by oxidation of 3 with 2[Cp2Fe][BF4] in the presence of NBD; (3) by substitution of two carbonyl ligands in 2 by C2R2 in refluxing toluene; and (4) by substitution of two carbonyl ligands in 4 by NBD in refluxing toluene. The substitution sites observed for the individual ligands are maintained in the mixed ligand derivative. The new compounds were characterized by analytical and spectroscopic methods including negative ion FAB mass spectroscopy and 1H NMR spectroscopy as well as by X-ray crystallographic studies of compounds 2 and 5.

Evidence That the Fourth Ligand to the [2Fe-2S] Cluster in Animal Ferrochelatase Is a Cysteine: CHARACTERIZATION OF THE ENZYME FROM DROSOPHILA MELANOGASTER

In a previous study, site-directed mutagenesis experiments identified three of the four ligands to the [2Fe-2S] cluster in animal ferrochelatase as conserved cysteines in the COOH-terminal extension, Cys-403, Cys-406, and Cys-411 in human ferrochelatase (Crouse, B. R., Sellers, V. M., Finnegan, M. G., Dailey, H. A. & Johnson, M. K. (1996) Biochemistry 35, 16222-16229). The nature of the fourth ligand was left unresolved, and spectroscopic studies raised the possibility of one noncysteinyl, oxygenic ligand. In this work, we report two lines of evidence that strongly suggest the fourth ligand is a cysteine residue. Cysteine at position 196 in human recombinant ferrochelatase when changed to a serine results in an inactive enzyme that is lacking the [2Fe-2S] cluster. Furthermore, whole cell EPR studies demonstrate that in the C196S mutant the cluster fails to assemble. Additionally, the cloning and expression of Drosophila melanogaster ferrochelatase has allowed the identification, by EPR and UV-visible spectroscopy, of a [2Fe-2S]2+ cluster with properties analogous to those of animal ferrochelatases. The observation that Drosophila ferrochelatase contains only four conserved cysteines at positions 196, 403, 406, and 411, is in accord with the proposal that these residues function as cluster ligands. In the case of the ferrochelatase iron-sulfur cluster ligands, NH2-Cys-X206-Cys-X2-Cys-X4-Cys-COOH, the position distant from other ligands may lead to a spatial positioning of the cluster near the enzyme active site or at the interface of two domains, thereby explaining the loss of enzyme activity that accompanies cluster degradation and reinforcing the idea that the cluster functions as a regulatory switch.