The [4Fe-4S] cluster of quinolinate synthase from Escherichia coli: investigation of cluster ligands

Nicotinamide adenine dinucleotide (NAD) derives from quinolinic acid which is synthesized in Escherichia coli from l-aspartate and dihydroxyacetone phosphate through the concerted action of l-aspartate oxidase and the [4Fe-4S] quinolinate synthase (NadA). Here, we addressed the question of the identity of the cluster ligands. We performed in vivo complementation experiments as well as enzymatic, spectroscopic and structural in vitro studies using wild-type vs. Cys-to-Ala mutated NadA proteins. These studies reveal that only three cysteine residues, the conserved Cys113, Cys200 and Cys297, are ligands of the cluster. This result is in contrast to the previous proposal that pointed the three cysteines of the C(291)XXC(294)XXC(297) motif. Interestingly, we demonstrated that Cys291 and Cys294 form a disulfide bridge and are important for activity.     

Quinolinate synthetase, an iron-sulfur enzyme in NAD biosynthesis

Nicotinamide adenine dinucleotide (NAD) plays a crucial role as a cofactor in numerous essential redox biological reactions. NAD derives from quinolinic acid which is synthesized in Escherichia coli from L-aspartate and dihydroxyacetone phosphate (DHAP) as the result of the concerted action of two enzymes, L-aspartate oxidase (NadB) and quinolinate synthetase (NadA). We report here the characterization of NadA protein from E. coli. When anaerobically purified, the isolated soluble protein contains 3-3.5 iron and 3-3.5 sulfide/polypeptide chain. M?ssbauer spectra of the 57Fe-protein revealed that the majority of the iron is in the form of a (4Fe-4S)2+ cluster. An enzymatic assay for quinolinate synthetase activity was set up and allowed to demonstrate that the cluster is absolutely required for NadA activity. Exposure to air leads to degradation of the cluster and inactivate enzyme.     

Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis

NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low-GC Gram-positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe-4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe-4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species-specific between B. subtilis and Escherichia coli.     

Characterization of the iron-sulfur cluster N7 (N1c) in the subunit NuoG of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX(27)C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated Delta N1b, Delta N1c, Delta N4, and Delta N5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g(x,y,z) = 1.89, 1.94, and 2.06, and g(x,y,z) = 1.91, 1.94, and 2.05 at 6-20 K in wild type, Delta N1b, and Delta N5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in Delta N4 and Delta N1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with "cluster N7." In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knock-out strain, the implicated structural importance of the iron-sulfur binding domains is discussed.     

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

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

Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer.     

Regulation of the activity of Escherichia coli quinolinate synthase by reversible disulfide-bond formation

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between dihydroxyacetone phosphate and iminoaspartate, yielding inorganic phosphate, 2 mol of water, and quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide and its derivatives. The enzyme from Escherichia coli contains a C (291)XXC (294)XXC (297) motif in its primary structure. Bioinformatics analysis indicates that only Cys297 serves as a ligand to a [4Fe-4S] cluster that is required for turnover. In this report, we show that the two remaining cysteines, Cys291 and Cys294, undergo reversible disulfide-bond formation, which regulates the activity of the enzyme. This mode of redox regulation of NadA appears physiologically relevant, since disulfide-bond formation and reduction are effected by oxidized and reduced forms of E. coli thioredoxin. A midpoint potential of -264 +/- 1.77 mV is approximated for the redox couple.     

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>; evidence for a [2Fe-2S] cluster with Asp(Cys)<Subscript>3</Subscript> ligands

The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus. The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da. The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a [2Fe-2S]2+,+ prosthetic group. The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands. The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum. The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a [3Fe-4S]+,0 cluster, and a [4Fe-4S]2+,+ cluster. The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV. The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster. The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH. SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase. However, P. furiosus sulfide dehydrogenase does not have glutamate synthase activity. SudB is highly homologous to HydG, the gamma-subunit of P. furiosus NiFe hydrogenase. The latter enzyme also has sulfide dehydrogenase activity. The P. furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme. Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.     

Reversible inactivation of E. coli endonuclease III via modification of its [4Fe-4S] cluster by nitric oxide

Endonuclease III, a highly conserved enzyme initiating the base excision repair of oxidized DNA bases, hosts a [4Fe-4S] cluster. Unlike many other iron-sulfur clusters, the [4Fe-4S] cluster of endonuclease III is stable and resistant to both oxidation and reduction. Here we show that the [4Fe-4S] cluster of the E. coli endonuclease III can be readily modified by nitric oxide forming the protein-bound dinitrosyl iron complex in vitro and in vivo. Modification of the [4Fe-4S] cluster completely inhibits the DNA glycosylase activity of the endonuclease III. Remarkably, the enzymatic activity is restored when the [4Fe-4S] cluster is re-assembled in the endonuclease III dinitrosyl iron complex with L-cysteine, cysteine desulfurase (IscS) and ferrous iron in vitro. Furthermore, the nitric oxide-modified [4Fe-4S] cluster in endonuclease III is efficiently repaired in aerobically growing E. coli cells, and this repair does not require new protein synthesis. These results suggest that the E. coli endonuclease III can be reversibly inactivated by nitric oxide via modification of its [4Fe-4S] cluster.     

Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA: optical and EPR studies

An unprecedented [4Fe-4S] iron-sulfur cluster was found in RumA, the enzyme that methylates U1939 in Escherichia coli 23 S ribosomal RNA (Agarwalla, S., Kealey, J. T., Santi, D. V., and Stroud, R. M. (2002) J. Biol. Chem. 277, 8835-8840; Lee, T. T., Agarwalla, S., and Stroud, R. M. (2004) Structure 12, 397-407). Methyltransferase reactions do not involve a redox step. To understand the structural and functional roles of the cluster in RumA, we have characterized redox reactions of the iron-sulfur cluster. As isolated aerobically, RumA exhibits a visible absorbance maximum at 390 nm and is EPR silent. It cannot be reduced by anaerobic additions of dithionite. Photoreduction by deazariboflavin/EDTA gives EPR spectra, the quantity (56% of S = 1/2 species) and details (g(av) approximately 1.96-1.93) of which indicate a [4Fe-4S](1+) cluster in the reduced RumA. Oxidation of RumA by ferricyanide leads to loss of the 390-nm band and appearance of lower intensity bands at 444 and 520 nm. EPR spectra of ferricyanide-oxidized RumA show a fraction (<8%) of the FeS cluster trapped in the [3Fe-4S](1+) form (g(av) approximately 2.011) together with unusual radical-like spectrum (g' values 2.015, 2.00, and 1.95). RumA also reacts with nitric oxide to give EPR spectra characteristic of the protein-bound iron dinitrosyl species. Oxidation of the cluster leads to its decomposition and that could be a mechanism for regulating the activity of RumA under conditions of oxidative stress in the cell. Sequence data base searches revealed that RumA homologs are widespread in various kingdoms of life and contain a conserved and unique iron-sulfur cluster binding motif, CX(5)CGGC.     

Expression, purification and molecular modelling of the Iro protein from Acidithiobacillus ferrooxidans Fe-1

The Iro protein was proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, it is a member of HiPIP family with the iron-sulfur cluster for electron transfer. The gene of Iro protein from A. ferrooxidans Fe-1 was cloned and then successfully expressed in Escherichia coli, finally purified by one-step affinity chromatography to homogeneity. The recombinant protein was observed to be dimer. The molecular mass of a monomer containing the [Fe4S4] cluster was 6847.35 Da by MALDI-TOF-MS. The optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Molecular modelling for the protein revealed that Cys20, Cys23, Cys32 and Cys45 were in ligation with the iron-sulfur cluster, and Tyr10 was important for the stability of the [Fe4S4] cluster. As we know, this is the first report of expression in E. coli of the Iro protein from A. ferrooxidans Fe-1.     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.     

Determination of nonligand amino acids critical to [4Fe-4S]2+/+ assembly in ferredoxin maquettes.

The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single [4Fe-4S]2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the. CIACGAC. consensus ferredoxin core motif observed in natural protein sequences. Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little [4Fe-4S]2+/+ cluster, 6 and 7%, respectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC. or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7. Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino acids. Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of [4Fe-4S] cluster binding relative to that of the glycine variant. The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence analysis, suggesting they are applicable to both the de novo design and structure-based redesign of natural proteins.     

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.     

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.     

Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY.

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7, 8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mismatches in DNA [Michaels et al. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 7022-7025]. MutY prevents DNA mutations resulting from the misincorporation of A opposite OG by using N-glycosylase activity to remove the adenine base. An interesting feature of MutY is that it contains a [4Fe-4S]2+ cluster that has been shown to play an important role in substrate recognition [Porello, S. L., Cannon, M. J., David, S. S. (1998) Biochemistry 37, 6465-6475]. Herein, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, and alanine. The extent to which the various mutations reduce the levels of protein overexpression suggests that coordination of the [4Fe-4S]2+ cluster provides stability to MutY in vivo. The ability of the mutated enzymes to bind to a substrate analogue DNA duplex and their in vivo activity were evaluated. Remarkably, the effects are both substitution and position dependent. For example, replacement of cysteine 199 with histidine provides a mutated enzyme that is expressed at high levels and exhibits DNA binding and in vivo activity similar to the WT enzyme. These results suggest that histidine coordination to the iron-sulfur cluster may be accommodated at this position in MutY. In contrast, replacement of cysteine 192 with histidine results in less efficient DNA binding and in vivo activity compared to the WT enzyme without affecting levels of overexpression. The results from the site-directed mutagenesis suggest that the structural properties of the iron-sulfur cluster coordination domain are important for both substrate DNA recognition and the in vivo activity of MutY.     

Characterization of the iron-sulfur cluster coordinated by a cysteine cluster motif (CXXCXXXCX27C) in the Nqo3 subunit in the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is composed of 14 subunits (designated Nqo1-14). This NDH-1 houses nine putative iron-sulfur binding sites, eight of which are generally found in bacterial NDH-1 and its mitochondrial counterpart (complex I). The extra site contains a CXXCXXXCX(27)C motif and is located in the Nqo3 subunit. This motif was originally found in Escherichia coli NDH-1 and was assigned to a binuclear cluster (g(z, y, x) = 2.00, 1.95, 1.92) and named N1c. In this report, the Thermus Nqo3 fragment containing this motif was heterologously overexpressed, using a glutathione S-transferase fusion system. This fragment contained a small amount of iron-sulfur cluster, whose content was significantly increased by in vitro reconstitution. The UV-visible and EPR spectroscopic properties of this fragment indicate that the ligated iron-sulfur cluster is tetranuclear with nearly axial symmetry (g( parallel, perpendicular) = 2.045, approximately 1.94). Site-directed mutants show that all four cysteines participate in the ligation of a [4Fe-4S] cluster. Considering the fact that the same motif coordinates only tetranuclear clusters in other enzymes so far known, we propose that the CXXCXXXCX(27)C motif in the Nqo3 subunit most likely ligates the [4Fe-4S] cluster.     

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.     

Biogenesis of iron-sulfur clusters in photosystem I: holo-NfuA from the cyanobacterium Synechococcus sp. PCC 7002 rapidly and efficiently transfers [4Fe-4S] clusters to apo-PsaC in vitro

The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, M?ssbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro.