Radical-mediated dehydration reactions in anaerobic bacteria

Most dehydratases catalyse the elimination of water from beta-hydroxy ketones, beta-hydroxy carboxylic acids or beta-hydroxyacyl-CoA. The electron-withdrawing carbonyl functionalities acidify the alpha-hydrogens to enable their removal by basic amino acid side chains. Anaerobic bacteria, however, ferment amino acids via alpha- or gamma-hydroxyacyl-CoA, dehydrations of which involve the abstraction of a beta-hydrogen, which is ostensibly non-acidic (pK ca. 40). Evidence is accumulating that beta-hydrogens are acidified via transient conversion of the CoA derivatives to enoxy radicals by one-electron transfers, which decrease the pK to 14. The dehydrations of (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA are catalysed by heterodimeric [4Fe-4S]-containing dehydratases, which require reductive activation by an ATP-dependent one-electron transfer mediated by a homodimeric protein with a [4Fe-4S] cluster between the two subunits. The electron is further transferred to the substrate, yielding a ketyl radical anion, which expels the hydroxyl group and forms an enoxy radical. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA involves a similar mechanism, in which the ketyl radical anion is generated by one-electron oxidation. The structure of the FAD- and [4Fe-4S]-containing homotetrameric dehydratase is related to that of acyl-CoA dehydrogenases, suggesting a radical-based mechanism for both flavoproteins.     

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (Etf_Af) and butyryl-CoA dehydrogenase (Bcd_Af) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. Etf_Af contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The Etf_Af-NAD⁺ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH⁻ is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH⁻ by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD^⨪, which donates this electron further to Dh-FAD of Bcd_Af after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH^•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH⁻ that converts crotonyl-CoA to butyryl-CoA.     

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.     

Fermentation of 4-aminobutyrate by Clostridium aminobutyricum: cloning of two genes involved in the formation and dehydration of 4-hydroxybutyryl-CoA

Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate. The genes coding for the enzymes that catalyse the interconversion of these intermediates are arranged in the order abfD (4-hydroxybutyryl-CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-dependent 4-hydroxybutyrate dehydrogenase). The genes abfD and abfT were cloned, sequenced and expressed as active enzymes in Escherichia coli. Hence the insertion of the [4Fe-4S]clusters and FAD into the dehydratase required no additional specific protein from C. aminobutyricum. The amino acid sequences of the dehydratase and the CoA-transferase revealed close relationships to proteins deduced from the genomes of Clostridium difficile, Porphyromonas gingivalis and Archaeoglobus fulgidus. In addition the N-terminal part of the dehydratase is related to those of a family of FAD-containing mono-oxygenases from bacteria. The putative assignment in the databank of Cat2 (OrfZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which is thought to be involved in the reductive pathway from succinate to butyrate, was confirmed by sequence comparison with AbfT (57% identity). Furthermore, an acetyl-CoA:4-hydroxybutyrate CoA-transferase activity could be detected in cell-free extracts of C. kluyveri. In contrast to glutaconate CoA-transferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homologues of AbfT, does not form a CoA-ester during catalysis.     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S]⁺ form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S]⁺ form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S]²⁺, [3Fe-4S]^+/0, and to the stable [2Fe-2S]²⁺ form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Futhermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.     

4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate

Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX₃CX₂C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.     

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field ⁵⁷Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.     

The F420-Reducing [NiFe]-Hydrogenase Complex from Methanothermobacter marburgensis, the First X-ray Structure of a Group 3 Family Member

The reversible redox reaction between coenzyme F₄₂₀ and H₂ to F₄₂₀H₂ is catalyzed by an F₄₂₀-reducing [NiFe]-hydrogenase (FrhABG), which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high-resolution cryo-electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7 Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but is different because of the truncation of ca 160 residues that results in similar but modified H₂ and proton transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of an N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F₄₂₀-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°′ ≈ − 400 mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.     

The Thiamine Biosynthetic Enzyme ThiC Catalyzes Multiple Turnovers and Is Inhibited by S-Adenosylmethionine (AdoMet) Metabolites

ThiC (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase; EC 4.1.99.17) is a radical S-adenosylmethionine (AdoMet) enzyme that uses a [4Fe-4S]⁺ cluster to reductively cleave AdoMet to methionine and a 5′-deoxyadenosyl radical that initiates catalysis. In plants and bacteria, ThiC converts the purine intermediate 5-aminoimidazole ribotide to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, an intermediate of thiamine pyrophosphate (coenzyme B1) biosynthesis. In this study, assay conditions were implemented that consistently generated 5-fold molar excess of HMP, demonstrating that ThiC undergoes multiple turnovers. ThiC activity was improved by in situ removal of product 5′-deoxyadenosine. The activity was inhibited by AdoMet metabolites S-adenosylhomocysteine, adenosine, 5′-deoxyadenosine, S-methyl-5′-thioadenosine, methionine, and homocysteine. Neither adenosine nor S-methyl-5′-thioadenosine had been shown to inhibit radical AdoMet enzymes, suggesting that ThiC is distinct from other family members. The parameters for improved ThiC activity and turnover described here will facilitate kinetic and mechanistic analyses of ThiC.     

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)₂ with the catalytic (BchY/BchZ)₂ protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF₄⁻. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)₂. A second [4Fe-4S] cluster was identified on (BchY/BchZ)₂. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.     

SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes

S-Adenosylmethionine (SAM, also known as AdoMet) radical enzymes use SAM and a [4Fe-4S] cluster to catalyze a diverse array of reactions. They adopt a partial triose-phosphate isomerase (TIM) barrel fold with N- and C-terminal extensions that tailor the structure of the enzyme to its specific function. One extension, termed a SPASM domain, binds two auxiliary [4Fe-4S] clusters and is present within peptide-modifying enzymes. The first structure of a SPASM-containing enzyme, anaerobic sulfatase-maturating enzyme (anSME), revealed unexpected similarities to two non-SPASM proteins, butirosin biosynthetic enzyme 2-deoxy-scyllo-inosamine dehydrogenase (BtrN) and molybdenum cofactor biosynthetic enzyme (MoaA). The latter two enzymes bind one auxiliary cluster and exhibit a partial SPASM motif, coined a Twitch domain. Here we review the structure and function of auxiliary cluster domains within the SAM radical enzyme superfamily.     

The synergy of germline C634Y and V292M RET mutations in a northern Chinese family with multiple endocrine neoplasia type 2A

Genetic analysis for germline mutations of RET proto‐oncogene has provided a basis for individual management of medullary thyroid carcinoma (MTC) and pheochromocytoma. Most of compound mutations have more aggressive phenotypes than single point mutations, but the compound C634Y/V292M variant in MTC has never been reported. Thus, we retrospectively investigated synergistic effect of C634Y and V292M RET germline mutations in family members with multiple endocrine neoplasia type 2A. Nine of 14 family members in a northern Chinese family underwent RET mutation screening using next‐generation sequencing and PCR followed by direct bidirectional DNA sequencing. Clinical features of nine individuals were retrospectively carefully reviewed. In vitro, the scratch‐wound assay was used to investigate the difference between the cells carrying different mutations. We find no patients died of MTC. All 3 carriers of the V292M variant were asymptomatic and did not have biochemical or structural evidence of disease (age: 82, 62 and 58). Among 4 C634Y mutation carriers, 2 patients had elevated calcitonin with the highest (156 pg/mL) in an 87‐year‐old male. Two carriers of compound C634Y/V292M trans variant had bilateral MTC with pheochromocytoma or lymph node metastasis (age: 54 and 41 years, respectively). Further, the compound C634Y/V292M variant had a faster migration rate than either single point mutation in vitro (P < .05). In conclusion, the V292M RET variant could be classified as ‘likely benign’ according to ACMG (2015). The compound variant V292M/C634Y was associated with both more aggressive clinical phenotype and faster cell growth in vitro than was either single mutation.     

Cloning and expression of the two genes coding for L-serine dehydratase from Peptostreptococcus asaccharolyticus: relationship of the iron-sulfur protein to both L-serine dehydratases from Escherichia coli.

The structural genes sdhA and sdhB, coding for the alpha- and beta-subunits of the [4Fe-4S] cluster containing L-serine dehydratase from Peptostreptococcus asaccharolyticus, have been cloned and sequenced. Expression of modified sdhB together with sdhA in Escherichia coli led to overproduction of active His6-tagged L-serine dehydratase. E. coli MEW22, deficient in the L-serine dehydratase L-SD1, was complemented by this sdhBA construct. The derived amino acid sequence of SdhBA shares similarities with both monomeric L-serine dehydratases, L-SD1 and L-SD2, from E. coli and with a putative L-serine dehydratase from Haemophilus influenzae, which suggests that these three enzymes are also iron-sulfur proteins.     

Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis: A CHITINASE WITHOUT CHITIN BINDING AND INSERTION DOMAINS*

Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)_n, into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67—Thr-69 and Ile-106–Val-112) to interact with (NAG)_n, generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Å along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)_n reveal (NAG)₂, (NAG)₂, and (NAG)₄ in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)₄; in an intermediate state, E145Q/Y227F with a boat-form NAG at the −1 subsite, −1-(NAG); after hydrolysis, E145Q with a chair form −1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between −1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of −1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of −1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)_n provide new insights into its substrate binding and the mechanistic action.     

Synthesis of (13)C-labeled gamma-hydroxybutyrates for EPR studies with 4-hydroxybutyryl-CoA dehydratase

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the reversible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The enzyme contains one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) molecule per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions followed by freezing at 77K induced the EPR-spectrum of a neutral flavin semiquinone (g=2.005, linewidth 2.1 mT), while at 10K additional signals were detected. In an attempt to characterize these signals, 4-HB-CoA molecules specifically labeled with (13)C have been synthesized. This was achieved via (13)C-labeled gamma-butyrolactones, which were obtained from (13)C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the (13)C-labeled 4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to marked broadening of the signals.     

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S_0.5 value 10³-fold and increased the V_max value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His¹⁷⁹ of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V_max 4-fold but reduced pyruvate S_0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V_max, but 10²-reduced pyruvate S_0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.     

Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme

In nitrogen-fixing cyanobacteria, hydrogen evolution is associated with hydrogenases and nitrogenase, making these enzymes interesting targets for genetic engineering aimed at increased hydrogen production. Nostoc punctiforme ATCC 29133 is a filamentous cyanobacterium that expresses the uptake hydrogenase HupSL in heterocysts under nitrogen-fixing conditions. Little is known about the structural and biophysical properties of HupSL. The small subunit, HupS, has been postulated to contain three iron-sulfur clusters, but the details regarding their nature have been unclear due to unusual cluster binding motifs in the amino acid sequence. We now report the cloning and heterologous expression of Nostoc punctiforme HupS as a fusion protein, f-HupS. We have characterized the anaerobically purified protein by UV-visible and EPR spectroscopies. Our results show that f-HupS contains three iron-sulfur clusters. UV-visible absorption of f-HupS has bands ∼340 and 420 nm, typical for iron-sulfur clusters. The EPR spectrum of the oxidized f-HupS shows a narrow g = 2.023 resonance, characteristic of a low-spin (S = ½) [3Fe-4S] cluster. The reduced f-HupS presents complex EPR spectra with overlapping resonances centered on g = 1.94, g = 1.91, and g = 1.88, typical of low-spin (S = ½) [4Fe-4S] clusters. Analysis of the spectroscopic data allowed us to distinguish between two species attributable to two distinct [4Fe-4S] clusters, in addition to the [3Fe-4S] cluster. This indicates that f-HupS binds [4Fe-4S] clusters despite the presence of unusual coordinating amino acids. Furthermore, our expression and purification of what seems to be an intact HupS protein allows future studies on the significance of ligand nature on redox properties of the iron-sulfur clusters of HupS.