Amino acid residues associated with cluster N3 in the NuoF subunit of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The NuoF subunit, which harbors NADH-binding site, of Escherichia coli NADH-quinone oxidoreductase (NDH-1) contains five conserved cysteine residues, four of which are predicted to ligate cluster N3. To determine this coordination, we overexpressed and purified the NuoF subunit and NuoF+E subcomplex in E. coli. We detected two distinct EPR spectra, arising from a [4Fe-4S] cluster (g(x,y,z)=1.90, 1.95, and 2.05) in NuoF, and a [2Fe-2S] cluster (g(x,y,z)=1.92, 1.95, and 2.01) in NuoE subunit. These clusters were assigned to clusters N3 and N1a, respectively. Based on the site-directed mutagenesis experiments, we identified that cluster N3 is ligated to the 351Cx2Cx2Cx40C398 motif.     

Electron transfer from heme bL to the [3Fe-4S] cluster of Escherichia coli nitrate reductase A (NarGHI)

We have investigated the functional relationship between three of the prosthetic groups of Escherichia coli nitrate reductase A (NarGHI): the two hemes of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster of the electron-transfer subunit (NarH). In two site-directed mutants (NarGHI(H56R) and NarGHI(H205Y)) that lack the highest potential heme of NarI (heme b(H)), a large negative DeltaE(m,7) is elicited on the NarH [3Fe-4S] cluster, suggesting a close juxtaposition of these two centers in the holoenzyme. In a mutant retaining heme b(H), but lacking heme b(L) (NarGHI(H66Y)), there is no effect on the NarH [3Fe-4S] cluster redox properties. These results suggest a role for heme b(H) in electron transfer to the [3Fe-4S] cluster. Studies of the pH dependence of the [3Fe-4S] cluster, heme b(H), and heme b(L) E(m) values suggest that significant deprotonation is only observed during oxidation of the latter heme (a pH dependence of -36 mV pH(-1)). In NarI expressed in the absence of NarGH [NarI(DeltaGH)], apparent exposure of heme b(H) to the aqueous milieu results in both it and heme b(L) having E(m) values with pH dependencies of approximately -30 mV pH(-1). These results are consistent with heme b(H) being isolated from the aqueous milieu and pH effects in the holoenzyme. Optical spectroscopy indicates that inhibitors such as HOQNO and stigmatellin bind and inhibit oxidation of heme b(L) but do not inhibit oxidation of heme b(H). Fluorescence quench titrations indicate that HOQNO binds with higher affinity to the reduced form of NarGHI than to the oxidized form. Overall, the data support the following model for electron transfer through the NarI region of NarGHI: Q(P) site --> heme b(L) --> heme b(H) --> [3Fe-4S] cluster.     

Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

The NADP-reducing hydrogenase from Desulfovibrio fructosovorans: functional interaction between the C-terminal region of HndA and the N-terminal region of HndD subunits

The hndABCD operon from Desulfovibrio fructosovorans encodes an uncommon heterotetrameric NADP-reducing iron hydrogenase. The presence of a [2Fe-2S] cluster likely located in the C-terminal region of the HndA subunit has already been revealed. We have cloned and expressed the truncated hndA gene in Escherichia coli to isolate the structural [2Fe-2S] module. Optical and EPR spectra are found identical to that of the native HndA subunit and the midpoint redox potential (-385 mV) is similar to that of the native protein (-395 mV). These results clearly demonstrate that the C-terminal region of HndA is a structurally independent [2Fe2S] ferredoxin-like domain. In the same way, the N-terminal domain of the HndD subunit was overproduced in E. coli and characterized. The presence of a [2Fe-2S] cluster was evidenced by optical spectroscopy. The midpoint redox potential (-380 mV) of this domain was found very close to that of the truncated HndA subunit but the EPR properties were significantly different. The various EPR properties allowed us to observe an electron exchange between the two [2Fe-2S] ferredoxin-like domains of the HndA and HndD subunits. Moreover, domain-domain interactions, observed by far-western experiments, indicate that these subunits are direct partners in the native complex.     

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.     

Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis.

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.     

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

The [2Fe-2S] cluster E(m) as an indicator of the iron-sulfur subunit position in the ubihydroquinone oxidation site of the cytochrome bc1 complex.

Recent crystallographic and kinetic data have revealed the crucial role of the large scale domain movement of the iron-sulfur subunit [2Fe-2S] cluster domain during the ubihydroquinone oxidation reaction catalyzed by the cytochrome bc(1) complex. Previously, the electron paramagnetic resonance signature of the [2Fe-2S] cluster and its redox midpoint potential (E(m)) value have been used extensively to characterize the interactions of the [2Fe-2S] cluster with the occupants of the ubihydroquinone oxidation (Q(o)) catalytic site. In this work we analyze these interactions in various iron-sulfur subunit mutants that carry mutations in its flexible hinge region. We show that the E(m) increases of the iron-sulfur subunit [2Fe-2S] cluster induced either by these mutations or by the addition of stigmatellin do not act synergistically. Moreover, the E(m) increases disappear in the presence of class I inhibitors like myxothiazol. Because various inhibitors are known to affect the location of the iron-sulfur subunit cluster domain, the measured E(m) value of the [2Fe-2S] cluster therefore reflects its equilibrium position in the Q(o) site. We also demonstrate the existence in this site of a location where the E(m) of the cluster is increased by about 150 mV and discuss its possible implications in term of Q(o) site catalysis and energetics.     

Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.     

Identification of an unusual [2Fe-2S]-binding motif in the CDP-6-deoxy-D-glycero-l-threo-4-hexulose-3-dehydrase from Yersinia pseudotuberculosis: implication for C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses

CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E(1)) catalyzes the C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. E(1) is a pyridoxamine 5'-phosphate (PMP)-dependent enzyme that also contains a [2Fe-2S] center. This iron-sulfur cluster is catalytically essential, since removal of the [2Fe-2S] center leads to inactive enzyme. To identify the [2Fe-2S] core in E(1) and to study the effect of impairing the iron-sulfur cluster on the activity of E(1), a series of E(1) cysteine mutants were constructed and their catalytic properties were characterized. Our results show that E(1) displays a cluster-binding motif (C-X(57)-C-X(1)-C-X(7)-C) that has not been observed previously for [2Fe-2S] proteins. The presence of such an unusual iron-sulfur cluster in E(1), along with the replacement of the active site lysine by a histidine residue (H220), reflects a distinct evolutionary path for this enzyme. The cysteine residues (C193, C251, C253, C261) implicated in the binding of the iron-sulfur cluster in E(1) are conserved in the sequences of its homologues. It is likely that E(1) and its homologues constitute a new subclass in the family of iron-sulfur proteins, which are distinguished not only by their cluster ligation patterns but also by the chemistry used in catalyzing a simple, albeit mechanistically challenging, reaction.     

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.     

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.     

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.     

Optical and EPR characterization of different species of active and inactive aconitase

It has been shown by spectroscopic (Kent, T. A., Dreyer, J-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1096-1100) and chemical (Kennedy, M.C., Emptage, M.H., Dryer, J-L., and Beinert, H. (1983) J. Biol. Chem. 258, 11098-11105) methods that interconversion of [3Fe-4S] and [4Fe-4S] clusters underlies activation and inactivation of aconitase. Since Fe-S clusters can assume different oxidation states, a number of different species of the enzyme can be expected to exist. Observations on activation-inactivation, as well as light absorption and EPR spectra, can be interpreted in terms of four species: [3Fe-4S]1+, the oxidized inactive enzyme as obtained on aerobic preparation from mitochondria; [3Fe-4S]0, the reduced inactive form as obtained on reduction in the presence of EDTA; [4Fe-4S]2+, the oxidized active form as obtained on reductive activation; and [4Fe-4S]1+, the reduced active form prepared by photoreduction of active aconitase. The light absorption spectra of each species are presented. Oxidized inactive aconitase shows EPR spectra typical of oxidized 3Fe clusters (g = 2.01), and reduced active enzyme shows spectra typical of reduced ferredoxins (g1,2,3 = 2.06, 1.93, 1.86). The EPR spectrum of the latter is drastically changed (g1,2,3 = 2.04, 1.85, 1.78) on addition of substrate. The active enzyme can be quantitatively converted to inactive enzyme by titration with ferricyanide in the presence of substrate. The correlation of EPR and optical spectra with enzymatic activity observed during titration demonstrates further that active aconitase requires an intact [4Fe-4S] cluster. A model of aconitase incorporating the four cluster species is presented, and explanations for some previous conflicting data concerning aconitase are offered.     

A [2Fe-2S] protein from the hyperthermophilic bacterium Aquifex aeolicus.

Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic [2Fe-2S](Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one [2Fe-2S] cluster per subunit. This protein is undamaged by heating to 100 degrees C for at least three hours. The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A. aeolicus protein relate it to well characterized [2Fe-2S] proteins from Clostridium pasteurianum and Azotobacter vinelandii. These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. The A. aeolicus [2Fe-2S] protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds.     

Characterization of quinolinate synthases from Escherichia coli, Mycobacterium tuberculosis, and Pyrococcus horikoshii indicates that [4Fe-4S] clusters are common cofactors throughout this class of enzymes

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate, affording quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Iminoaspartate is generated via the action of l-aspartate oxidase (NadB), which catalyzes the first step in the biosynthesis of NAD in most prokaryotes. NadA from Escherichia coli was hypothesized to contain an iron-sulfur cluster as early as 1991, because of its observed labile activity, especially in the presence of hyperbaric oxygen, and because its primary structure contained a CXXCXXC motif, which is commonly found in the [4Fe-4S] ferredoxin class of iron-sulfur (Fe/S) proteins. Indeed, using analytical methods in concert with Mossbauer and electron paramagnetic resonance spectroscopies, the protein was later shown to harbor a [4Fe-4S] cluster. Recently, the X-ray structure of NadA from Pyrococcus horikoshii was solved to 2.0 A resolution [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 26645-26648]. This protein does not contain a CXXCXXC motif, and no Fe/S cluster was observed in the structure or even mentioned in the report. Moreover, rates of quinolinic acid production were reported to be 2.2 micromol min (-1) mg (-1), significantly greater than that of E. coli NadA containing an Fe/S cluster (0.10 micromol min (-1) mg (-1)), suggesting that the [4Fe-4S] cluster of E. coli NadA may not be necessary for catalysis. In the study described herein, nadA genes from both Mycobacterium tuberculosis and Pyrococcus horikoshii were cloned, and their protein products shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the "activity" reported does not correspond to quinolinate formation.     

The prosthetic groups in succinate dehydrogenase. Number and stoichiometry

I. Succinate:Q oxidoreductase (EC 1.3.99.1) as present in beef-heart submitochondrial particles contains equal amounts of FAD, a [2Fe-2S] cluster and a [4Fe-4S] cluster. Both Fe-S clusters are reducible by succinate. 2. A second type of [2Fe-2S] cluster, called center S-2, that has been proposed to be present in purified preparations of succinate dehydrogenase and isolated Complex II (Ohnishi, T., Winter, D.B., Lim, J. and King, T.E. (1973) Biochem. Biophys. Res. Commun. 53, 231--237) is an artifact introduced by the purification procedure. 3. It is suggested that the 70 000 dalton subunit which is known to bind the flavin, accomodates also the [4Fe-4S] cluster whereas the 28 000 dalton subunit contains the [2Fe-2S] cluster.     

Subunit D of RNA polymerase from Methanosarcina acetivorans contains two oxygen-labile [4Fe-4S] clusters: implications for oxidant-dependent regulation of transcription

Subunit D of multisubunit RNA polymerase from many species of archaea is predicted to bind one to two iron-sulfur (Fe-S) clusters, the function of which is unknown. A survey of encoded subunit D in the genomes of sequenced archaea revealed six distinct groups based on the number of complete or partial [4Fe-4S] cluster motifs within domain 3. Only subunit D from strictly anaerobic archaea, including all members of the Methanosarcinales, are predicted to bind two [4Fe-4S] clusters. We report herein the purification and characterization of Methanosarcina acetivorans subunit D in complex with subunit L. Expression of subunit D and subunit L in Escherichia coli resulted in the purification of a D-L heterodimer with only partial [4Fe-4S] cluster content. Reconstitution in vitro with iron and sulfide revealed that the M. acetivorans D-L heterodimer is capable of binding two redox-active [4Fe-4S] clusters. M. acetivorans subunit D deleted of domain 3 (DΔD3) was still capable of co-purifying with subunit L but was devoid of [4Fe-4S] clusters. Affinity purification of subunit D or subunit DΔD3 from M. acetivorans resulted in the co-purification of endogenous subunit L with each tagged subunit D. Overall, these results suggest that domain 3 of subunit D is required for [4Fe-4S] cluster binding, but the [4Fe-4S] clusters and domain 3 are not required for the formation of the D-L heterodimer. However, exposure of two [4Fe-4S] cluster-containing D-L heterodimer to oxygen resulted in loss of the [4Fe-4S] clusters and subsequent protein aggregation, indicating that the [4Fe-4S] clusters influence the stability of the D-L heterodimer and therefore have the potential to regulate the assembly and/or activity of RNA polymerase in an oxidant-dependent manner.     

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.     

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.