Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5'-phosphosulfate reductase from Mycobacterium tuberculosis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.     

The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation

It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5'-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5'-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank(TM), revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. M?ssbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways.     

A dimer of the FeS cluster biosynthesis protein IscA from cyanobacteria binds a [2Fe2S] cluster between two protomers and transfers it to [2Fe2S] and [4Fe4S] apo proteins.

Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocystis PCC 6803. One of them, the product of slr1417 which accounts for 0.025% of the total soluble protein of Synechocystis was over-expressed in E. coli and purified. The purified protein was found to be mainly dimeric and did not contain any cofactor. Incubation with iron ions, cysteine and Synechocystis IscS led to the formation of one [2Fe2S] cluster at an IscA dimer as demonstrated (by the binding of about one iron and one sulfide ion per IscA monomer) by UV/Vis, EPR and M?ssbauer spectroscopy. M?ssbauer spectroscopy further indicated that the FeS cluster was bound by four cysteine residues. Site-directed mutagenesis revealed that of the five cysteine residues only C110 and C112 were involved in cluster binding. It was therefore concluded that the [2Fe2S] cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers. The cluster could be transferred to apo ferredoxin, a [2Fe2S] protein, with a half-time of 10 min. Surprisingly, incubation of cluster-containing IscA with apo adenosine 5'-phosphosulfate reductase led to a reactivation of the enzyme which requires the presence of a [4Fe4S] cluster. This demonstrates that it is possible to build [4Fe4S] clusters from [2Fe2S] units.     

M?ssbauer studies of Escherichia coli sulfite reductase complexes with carbon monoxide and cyanide. Exchange coupling and intrinsic properties of the [4Fe-4S] cluster

M?ssbauer studies of the hemoprotein subunit (SiR) of E. coli sulfite reductase have shown that the siroheme and the [4Fe-4S] cluster are exchange-coupled. Here we report M?ssbauer studies of SiR complexed with either CO or CN- and of SiR in the presence of the chaotropic agent dimethyl sulfoxide (Me2SO). The spectra of one-electron-reduced SiR X CN show that all five iron atoms reside in a diamagnetic environment; the ferroheme X CN complex is low spin and the [4Fe-4S] cluster is in the 2+ oxidation state. Titration with ferricyanide affords a CN- complex of oxidized SiR in which the siroheme iron is low spin ferric, with the cluster remaining in the 2+ state. At low temperatures, paramagnetic hyperfine interactions are observed for the iron sites of the cluster, suggesting that it is exchange-coupled to the heme iron. Reduction of one-electron-reduced SiR X CN and SiR X CO yields complexes with "g = 1.94"-type EPR signals showing that the second electron is accommodated by the iron-sulfur cluster. The fully reduced complexes yield well resolved M?ssbauer spectra which were analyzed in the spin Hamiltonian formalism. The analysis shows that the cluster subsites are equivalent in pairs, one pair having properties reminiscent of ferric sites whereas the other pair has features more typical of ferrous sites. The M?ssbauer spectra of oxidized SiR kept in 60% (v/v) Me2SO are virtually identical with those observed for SiR in standard buffer, implying that the coupling is maintained in the presence of the chaotrope. Fully reduced SiR displays an EPR signal with g values of g = 2.53, 2.29, and 2.07. In 60% Me2SO, this signal vanishes and a g = 1.94 signal develops; this transition is accompanied by a change in the spin state of the heme iron from S = 1 (or 2) to S = O.     

Characterization of a sulfite reductase from Desulfovibrio vulgaris. Evidence for the presence of a low-spin siroheme and an exchange-coupled siroheme-[4Fe-4S] unit

We have studied a low-molecular-weight (Mr = 27,200) sulfite reductase from Desulfovibrio vulgaris (Hildenborough, NCIB 8303) with M?ssbauer, EPR, and chemical techniques. This sulfite reductase was found to contain one siroheme and one [4Fe-4S] cluster. As purified, the siroheme is low-spin ferric (S = 1/2) which exhibits characteristic EPR resonances at g = 2.44, 2.36, and 1.77. At 150 K, the observed M?ssbauer parameters, delta EQ = 2.49 +/- 0.02 mm/s and delta = 0.31 +/- 0.02 mm/s, for the siroheme are typical for low-spin ferric complexes. The [4Fe-4S] cluster is in the 2+ state. The M?ssbauer parameters, delta EQ = 0.95 +/- 0.02 mm/s and delta = 0.38 +/- 0.02 mm/s, for the cluster are almost identical to those observed for the [4Fe-4S]2+ cluster in the hemoprotein subunit of the sulfite reductase from Escherichia coli. Similar to the hemoprotein subunit of E. coli sulfite reductase, low-temperature M?ssbauer spectra of D. vulgaris sulfite reductase recorded with weak and strong applied fields also show evidence for an exchange-coupled siroheme-[4Fe-4S] unit.     

M?ssbauer study of Clostridium pasteurianum hydrogenase II. Evidence for a novel three-iron cluster

Hydrogenase II contains two iron-sulfur clusters, one of the [4Fe-4S] type and one of unknown structure with unusual spectral properties (H-cluster). Using M?ssbauer spectroscopy we have studied the H-cluster under a variety of conditions. In the reduced state the cluster exhibits, in zero magnetic field, spectra with the typical 2:1 quadrupole pattern of reduced [3Fe-4S] clusters. However, whereas the latter are paramagnetic (S = 2) the H-cluster is diamagnetic (S = 0). Upon oxidation and exposure to CO the H-cluster exhibits an S = 1/2 EPR spectrum with g values at 2.03, 2.02, and 2.00. In this state, the M?ssbauer spectra reveal two cluster subsites with magnetic hyperfine coupling constants AI = +26.5 MHz and AII = -30 MHz. ENDOR data obtained by Hoffman and co-workers (Telser, J., Benecky, M. J., Adams, M. W. W., Mortenson, L. E., and Hoffman, B. M. (1986) J. Biol. Chem. 261, 13536-13541) show a 57Fe resonance at AIII approximately equal to 9.5 MHz. Analysis of the M?ssbauer spectra shows that this resonance represents one iron site. Our studies of the reduced and CO-bound oxidized states of hydrogenase II suggest that the H-cluster contains three iron atoms. The data obtained for the oxidized H-cluster suggest a novel type of 3-Fe cluster and bear little resemblance to those reported for oxidized [3Fe-4S] clusters with g = 2.01 EPR signals. In the reduced sample the [4Fe-4S]1+ cluster appears to occur in a mixture of two distinct electronic states.     

M?ssbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5

The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides. We have studied oxidized samples of the enzyme with M?ssbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01. The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances. One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz. These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. The M?ssbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms. Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated [4Fe-4S] cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a [4Fe-4S] core. The M?ssbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type [4Fe-4S] clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme.     

M?ssbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters

Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our M?ssbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared M?ssbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state.     

Unusual features in EPR and M?ssbauer spectra of the 2[4Fe-4Se]+ ferredoxin from Clostridium pasteurianum

The electronic and magnetic properties of the selenium-substituted 2[4Fe-4Se]2+/+ ferredoxin (Fd) from Clostridium pasteurianum have been investigated by EPR and M?ssbauer spectroscopy. The [4Fe-4Se]2+ clusters of oxidized Fd are diamagnetic and the M?ssbauer spectra are nearly identical to those of oxidized 2[4Fe-4S]2+ Fd. The addition of 2e- per molecule of Se-substituted Fd causes the simultaneous appearance of three EPR signals: one (g1,2,3 = 2.103, 1.940, 1.888) is reminiscent of [4Fe-4S]+ EPR spectra and accounts for 0.7 to 0.8 spin/molecule. The two others consist of a broad signal with g = 4.5, 3.5, and approximately 2 (0.7 to 0.8 spin/molecule) and of a narrow peak at g = 5.172 which is observed up to 60 K. Peculiar features are also present in the M?ssbauer spectra of 2[4Fe-4Se]+ Fd below 20 K: a subcomponent with lines near to +/- 4 mm/s and accounting for 20% of the total iron corresponds to two antiferromagnetically coupled sites in approximately a 3:1 ratio and displays fully developed paramagnetic hyperfine interactions at 4.2 K without any applied field. At 77 K, however, the reduced Se-substituted Fd yields a M?ssbauer spectrum similar to that of 2[4Fe-4S]+ Fd. The new EPR and M?ssbauer spectroscopic features of the 2[4Fe-4Se]+ Fd are attributed to S = 3/2 and S = 7/2 spin states which accompany the classical S = 1/2 state of [4Fe-4X]+ (X = S, Se) structures.     

The putative moss 3'-phosphoadenosine-5'-phosphosulfate reductase is a novel form of adenosine-5'-phosphosulfate reductase without an iron-sulfur cluster

Sulfate assimilation provides reduced sulfur for synthesis of the amino acids cysteine and methionine and for a range of other metabolites. Sulfate has to be activated prior to reduction by adenylation to adenosine 5'-phosphosulfate (APS). In plants, algae, and many bacteria, this compound is reduced to sulfite by APS reductase (APR); in fungi and some cyanobacteria and gamma-proteobacteria, a second activation step, phosphorylation to 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is necessary before reduction to sulfite by PAPS reductase (PAPR). We found previously that the moss Physcomitrella patens is unique among these organisms in possessing orthologs of both APR and PAPR genes (Koprivova, A., Meyer, A. J., Schween, G., Herschbach, C., Reski, R., and Kopriva, S. (2002) J. Biol. Chem. 277, 32195-32201). To assess the function of the two enzymes, we compared their biochemical properties by analysis of purified recombinant proteins. APR from Physcomitrella is very similar to the well characterized APRs from seed plants. On the other hand, we found that the putative PAPR preferentially reduces APS. Sequence analysis, analysis of UV-visible spectra, and determination of iron revealed that this new APR, named PpAPR-B, does not contain the FeS cluster, which was previously believed to determine the substrate specificity of the otherwise relatively similar enzymes. The lack of the FeS cluster in PpAPR-B catalysis is connected with a lower turnover rate but higher stability of the protein. These findings show that APS reduction without the FeS cluster is possible and that plant sulfate assimilation is predominantly dependent on reduction of APS.     

M?ssbauer studies on the active Fe ... [2Fe-2S] site of putidamonooxin, its electron transport and dioxygen activation mechanism

Putidamonooxin, the oxygenase of a 4-methoxybenzoate monooxygenase enzyme system, catalyzes the oxidative O-demethylation of the substrate 4-methoxybenzoate in conjunction with the NADH:putidamonooxin oxidoreductase. Putidamonooxin is a conjugated iron-sulfur protein which needs iron ions as cofactors for its enzymatic activity. Putiamonooxin was isolated from Pseudomonas putida, which was grown on a 57Fe-enriched culture medium. Thus putidamonooxin was enriched in vivo with 57Fe up to about 80%. During our M?ssbauer study of putidamonooxin a number of parameters have been varied: (a) the oxidation state of putidamonooxin (oxidized, reduced and aerobically reoxidized); (b) the substrate bound to putidamonooxin (4-methoxybenzoate, benzoate, 4-tert-butylbenzoate); (c) the temperature between 2.7 K and 245 K; (d) the applied magnetic field between 0 and 0.1 T and (e) the amount of iron cofactor. From our M?ssbauer results it is obvious that the iron-sulfur centers of putidamonooxin are [2 Fe-2S] clusters similar to those of the plant-type ferredoxins. Further, we have evidence for the existence of iron ions (one per [2 Fe-2S] cluster), which serve as cofactors for the dioxygen activation, functioning as the dioxygen binding site and mediating the electron flow from the [2 Fe-2S] cluster to dioxygen.     

M?ssbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase.

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. M?ssbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. M?ssbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.     

The iron-sulfur clusters in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. Biochemical and spectroscopic investigations.

The reversible dehydration of (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA is catalysed by the combined action of two oxygen-sensitive enzymes from Acidaminococcus fermentans, the homodimeric component A (2 x 27 kDa) and the heterodimeric component D (45 and 50 kDa). Component A was purified to homogeneity (specific activity 25-30 s-1) using streptavidin-tag affinity chromatography. In the presence of 5 mM MgCl2 and 1 mM ADP or ATP, component A could be stabilized and stored for 4-5 days at 4 degrees C without loss of activity. The purification of component D from A. fermentans was also improved as indicated by the 1.5-fold higher specific activity (15 s-1). The content of 1.0 riboflavin 5'-phosphate (FMN) per heterodimer could be confirmed, whereas in contrast to an earlier report only trace amounts of riboflavin (< 0.1) could be detected. Each active component contains an oxygen sensitive diamagnetic [4Fe-4S]2+ cluster as revealed by UV-visible, EPR and M?ssbauer spectroscopy. Reduction of the [4Fe-4S]2+ cluster in component A with dithionite yields a paramagnetic [4Fe-4S]1+ cluster with the unusual electron spin ground state S = 3/2 as indicated by strong absorption type EPR signals at high g values, g = 4-6. Spin-Hamiltonian simulations of the EPR spectra and of magnetic M?ssbauer spectra were performed to determine the zero field splitting (ZFS) parameters of the cluster and the 57Fe hyperfine interaction parameters. The electronic properties of the [4Fe-4S]2+, 1+ clusters of component A are similar to those of the nitrogenase iron protein in which a [4Fe-4S]2+ cluster bridges the two subunits of the homodimeric protein. Under air component A looses its activity within seconds due to irreversible degradation of its [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster. The [4Fe-4S]2+ cluster of component D could not be reduced to a [4Fe-4S]1+ cluster, even with excess of Ti(III)citrate or dithionite. Exposure to oxic conditions slowly converts the diamagnetic [4Fe-4S]2+ cluster of component D to a paramagnetic [3Fe-4S]+ cluster concomitant with loss of activity (30% within 24 h at 4 degrees C).     

Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis

L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5'-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5'-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 +/- 0.46 eq of iron and 0.670 +/- 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g axially = 2.03 and g radially = 1.93. M?ssbauer spectra of the (57)Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S](2+) clusters, as evidenced by the typical M?ssbauer parameters-isomer shift, delta = 0.47 mm/s, quadrupole splitting of Delta E(Q) = 1.14 mm/s, and a diamagnetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.     

M?ssbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2

We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with M?ssbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits M?ssbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the M?ssbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and M?ssbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.     

Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide

Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)). In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. M?ssbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S](2+). When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. M?ssbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of [4Fe-4S](2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 [4Fe-4S](2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the M?ssbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.     

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.     

Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis

The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.     

The function of the [4Fe-4S] clusters and FAD in bacterial and archaeal adenylylsulfate reductases. Evidence for flavin-catalyzed reduction of adenosine 5'-phosphosulfate

The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase catalyzes reversibly the 2-electron reduction of APS to sulfite and AMP, a key step in the biological sulfur cycle. APS reductase from one archaea and three different bacteria has been purified, and the molecular and catalytic properties have been characterized. The EPR parameters and redox potentials (-60 and -520 mV versus NHE) have been assigned to the two [4Fe-4S] clusters I and II observed in the three-dimensional structure of the enzyme from Archaeoglobus fulgidus (Fritz, G., Roth, A., Schiffer, A., Büchert, T., Bourenkov, G., Bartunik, H. D., Huber, H., Stetter, K. O., Kroneck, P. M. H., and Ermler, U. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 1836-1841). Sulfite binds to FAD to form a covalent FAD N(5)-sulfite adduct with characteristic UV/visible spectra, in accordance with the three-dimensional structure of crystalline enzyme soaked with APS. UV/visible monitored titrations reveal that the substrates AMP and APS dock closely to the FAD cofactor. These results clearly document that FAD is the site of the 2-electron reduction of APS to sulfite and AMP. Reaction of APS reductase enzyme with sulfite and AMP leads to partial reduction of the [4Fe-4S] centers and formation of the anionic FAD semiquinone. Thus, both [4Fe-4S] clusters function in electron transfer and guide two single electrons from the protein surface to the FAD catalytic site.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.