The Radical SAM Superfamily

ABSTRACT

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe–4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe–4S]^{1 +} –SAM complex to [4Fe–4S]^{2 +}–Met and the 5′ -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.     

Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis

Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH₃). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N⁶-isopentenyladenosine (i⁶A) to 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms²i⁶A formation (MiaB activity), and yqeV is required for modification of N⁶-threonylcarbamoyladenosine (t⁶A) to 2-methylthio-N⁶-threonylcarbamoyladenosine (ms²t⁶A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i⁶A from t⁶A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis.     

Viperin is an iron‐sulfur protein that inhibits genome synthesis of tick‐borne encephalitis virus via radical SAM domain activity

Viperin is an interferon‐induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S‐adenosyl‐l ‐methionine (SAM) domain which has been shown in vitro to hold a [4Fe‐4S] cluster. We identified tick‐borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of ⁵⁵Fe, indicative for the presence of an Fe‐S cluster. Mutation of the cysteine residues ligating the Fe‐S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of ⁵⁵Fe. Mutants lacking the extreme C‐terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe‐4S] cluster assembly, and acts through an enzymatic, Fe‐S cluster‐ and SAM‐dependent mechanism to inhibit viral RNA synthesis.     

The ms2io6A37 Modification of tRNA in Salmonella typhimurium Regulates Growth on Citric Acid Cycle Intermediates

The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms²i⁶A) is present in position 37 (adjacent to and 3′ of the anticodon) of tRNAs that read codons beginning with U except tRNA _I,V^Ser in Escherichia coli. In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms²io⁶A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms²i⁶A in E. coli. Mutants (miaE) of S. typhimurium in which ms²i⁶A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms²io⁶A37 and is not due to a second cellular function of tRNA (ms²io⁶A37)hydroxylase, the miaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.     

Parsing redox potentials of five ferredoxins found within Thermotoga maritima

Most organisms contain multiple soluble protein‐based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron–sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the “radical SAM,” or S‐adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron–sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe₄S₄]¹⁺ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side‐by‐side fashion for the first time. While a single [Fe₄S₄]‐cluster bearing Fd (TM0927) has a potential of ?420 mV, the other four 2x[Fe₄S₄]‐bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).     

Proton magnetic resonance studies of 7 Fe ferredoxins: Lower potential of the [4 Fe–4 S] redox center than the [3 Fe–3 S] cluster

Two types of iron-sulfur clusters, [3 Fe–3 S] and [4 Fe–4 S], were identified by ¹H-NMR in ferredoxins from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. The [4 Fe–4 S] clusters always showed the redox couples which had potentials lower than that of the [3 Fe–3 S] clusters.     

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of ?483 ± 1 mV and ?187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.     

Differential activities of heterocyst ferredoxin,vegetative cell ferredoxin,and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP⁺ reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP⁺, using thylakoids from vegetative cells.Abbreviations BSA bovine serum albumin - FdxH heterocyst ferredoxin - Fld flavodoxin - FNR ferredoxin-NADP⁺ reductase - MV methyl viologen - PetF vegetative cell ferredoxin - PFO pyruvate-ferredoxin oxidoreductase - Pyr piruvate - PS I Photosystem I     

NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferredoxin protein from Desulphovibrio gigas, FdI, FdI′, and FdII was carried out. FdI and FdI′ are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted resonances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the “three state hypothesis” terminology it is shown that FdI_ox is predominantly in the C^2? state and changes upon reduction into the C^3? state, while FdII_ox is in the C^? state and changes into the C^2? state. FdI′ does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp.. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.     

Characterization of Two 2[4Fe4S] Ferredoxins from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis>

In vivo hydrogen production in Clostridium acetobutylicum involves electron transfer between ferredoxin and [FeFe]-hydrogenase. Five C. acetobutylicum open reading frames were annotated as coding for putative ferredoxins. We focused our biophysical and biochemical investigations on CAC0303 and CAC3527, which possess the sequence signature and length of classical 2[4Fe4S] clostridial ferredoxins but differ significantly in theoretical pI. After cloning, heterologous expression in E. coli followed by in vitro Fe-S incorporation and purification, CAC0303 was shown to have a regular electron paramagnetic resonance (EPR) signal for a classical 2[4Fe4S] clostridial ferredoxin, while CAC3527 displayed an unusual EPR signal and a quite low reduction potential. Both ferredoxins were reduced in vitro by C. acetobutylicum [FeFe]-hydrogenase, but the CAC3527 reduction rate was 10-fold lower than that of CAC0303. These results are consistent with the efficiency of intermolecular electron transfer being dictated by the redox thermodynamics, the contribution of the ferredoxin global charge being only minor. The physiological function of CAC3527 is discussed.     

Qualitative and semi-quantitative analyses of cytokinins using LC/APCI-MS in combination with ELISA

Application of liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) for the analysis of cytokinins was examined. The fragmentation of cytokinins was studied using authentic trans-zeatin (t-Z), trans-zeatin riboside (t-ZR), isopentenyl adenine (i⁶Ade), isopentenyl adenosine (i⁶Ado), benzyl adenine (BAde), benzyl adenosine (BAdo), and kinetin. These cytokinins were effectively ionized by APCI in aqueous acetonitrile. t-Z, i⁶Ade, BAde, and kinetin showed prominent quasi-molecular ions of [M + H]⁺, and ribosylcytokinins clearly showed both [M + H]⁺ and a characteristic fragment ion ([M + H-ribose]⁺), giving some information about their structures. The qualitative and semi-quantitative analyses of cytokinins by LC/APCI-MS were validated in combination with enzyme-linked immunosorbent assay (ELISA) through the analysis of t-ZR in the teratoma of Nicotiana tobacum. t-ZR was conclusively identified and a semi-quantitative estimate of its endogenous levels were provided by the combination of LC/APCI-MS and ELISA. The quantified values obtained by LC/ APCI-MS (single ion detection) and ELISA are in close agreement.     

Coproporphyrin III excretion identifies the anaerobic coproporphyrinogen III oxidase HemN as a copper target in the Cu+‐ATPase mutant copA− of Rubrivivax gelatinosus

Two genes encoding structurally similar Copper P_1B‐type ATPases can be identified in several genomes. Notwithstanding the high sequence and structural similarities these ATPases held, it has been suggested that they fulfil distinct physiological roles. In deed, we have shown that the Cu⁺‐ATPase CtpA is required only for the activity of cuproproteins in the purple bacterium Rubrivivax gelatinosus; herein, we show that CopA is not directly required for cytochrome c oxidase but is vital for copper tolerance. Interestingly, excess copper in the copA^? mutant resulted in a substantial decrease of the cytochrome c oxidase and the photosystem under microaerobic and anaerobic conditions together with the extrusion of coproporphyrin III. The data indicated that copper targeted the tetrapyrrole biosynthesis pathway at the level of the coproporphyrinogen III oxidase HemN and thereby affects the oxidase and the photosystem. This is the first in vivo demonstration that copper, like oxygen, affects tetrapyrrole biosynthesis presumably at the level of the SAM and [4Fe‐4S] containing HemN enzyme. In light of these results and similar findings in Escherichia coli, the potential role of copper ions in the evolution of [4Fe‐4S] enzymes and the Cu⁺‐ATPases is discussed.     

4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction

4-Hydroxyphenylacetate decarboxylase (4Hpad) is an Fe/S cluster containing glycyl radical enzyme (GRE), which catalyses the last step of tyrosine fermentation in clostridia, generating the bacteriostatic p-cresol. The respective activating enzyme (4Hpad-AE) displays two cysteine-rich motifs in addition to the classical S-adenosylmethionine (SAM) binding cluster (RS cluster) motif. These additional motifs are also present in other glycyl radical activating enzymes (GR-AE) and it has been postulated that these orthologues may use an alternative SAM homolytic cleavage mechanism, generating a putative 3-amino-3-carboxypropyl radical and 5′-deoxy-5′-(methylthio)adenosine but not a 5′-deoxyadenosyl radical and methionine. 4Hpad-AE produced from a codon-optimized synthetic gene binds a maximum of two [4Fe–4S]^2+/+ clusters as revealed by EPR and Mössbauer spectroscopy. The enzyme only catalyses the turnover of SAM under reducing conditions, and the reaction products were identified as 5′-deoxyadenosine (quenched form of 5′-deoxyadenosyl radical) and methionine. We demonstrate that the 5′-deoxyadenosyl radical is the activating agent for 4Hpad through p-cresol formation and correlation between the production of 5′-deoxyadenosine and the generation of glycyl radical in 4Hpad. Therefore, we conclude that 4Hpad-AE catalyses a classical SAM-dependent glycyl radical formation as reported for GR-AE without auxiliary clusters. Our observation casts doubt on the suggestion that GR-AE containing auxiliary clusters catalyse the alternative cleavage reaction detected for glycerol dehydratase activating enzyme.     

Protein Folding: Grand Challenge of Nature

Abstract

Conformational preferences of the hypermodified nucleic acid bases N6-(Δ² -cis-hydroxyisopentenyl)adenine, cis-io⁶Ade also known as cis-zeatin, and N⁶-(Δ² -trans-hydroxyisopentenyl)adenine, trans-io⁶ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms²io⁶Ade or cis-ms²zeatin, and trans-ms²io⁶Ade or trans-ms²zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io⁶Ade, trans-io⁶Ade, cis-ms²io⁶Ade and trans-ms²io⁶Ade are such that in each of these molecules the isopentenyl substituent spreads away (has “dista” conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io⁶Ade as well as cis- ms²io⁶Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io⁶Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms²io⁶Ade, possible O-H…S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms²io⁶Ade as well as trans-ms²io⁶Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i⁶Ade and ms²i⁶Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms²-zeatin (ms²io⁶Ade) modification.     

Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe–4S] protein

The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria. It is also present in the chloroplasts of all phototrophic organisms. Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated. 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate is transformed by GcpE into 4-hydroxy-3-methylbut-2-enyl diphosphate, which is converted by LytB into isopentenyl diphosphate or dimethylallyl diphosphate. Only the bacterial GcpE and LytB enzymes have been investigated to some extent, but nothing is known about the corresponding plant enzymes. In this contribution, the prosthetic group of GcpE from the plant Arabidopsis thaliana and the bacterium Escherichia coli has been fully characterized by Mössbauer spectroscopy after reconstitution with ⁵⁷FeCl₃, Na₂S and dithiothreitol. It corresponds to a [4Fe-4S] cluster, suggesting that both plant and bacterial enzymes catalyze the reduction of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate via two consecutive one-electron transfers. In contrast to the bacterial enzyme, which utilizes NADPH/flavodoxin/flavodoxin reductase as a reducing shuttle system, the plant enzyme could not use this reduction system. Enzymatic activity was only detected in the presence of the 5-deazaflavin semiquinone radical.     

Purification and characterization of a fix ABCX-linked 2[4Fe-4S] ferredoxin from Azotobacter vinelandii

 Ferredoxins that contain 2[4Fe-4S]^2+/+ clusters can be divided into two classes. The "clostridial-type" ferredoxins have two CysXXCysXXCysXXXCysPro motifs. The "photosynthetic bacterial and nif-related" ferredoxins have one motif of that type and one more unusual CysXXCysX_7–9CysXXXCysPro motif. In Azotobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purification of a small anionic [Fe-S] protein with yields of ∼3 mg per 500 g cell paste. NH₂-terminal sequence analysis shows that this protein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now commonly used for a very different cb-type cytochrome oxidase we have renamed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of "photosynthetic bacterial and nif-related" 2[4Fe-4S]^2+/1+ ferredoxins that includes Chromatium vinosum ferredoxin. Studies of the purified protein by Fe analysis, absorption, CD and EPR spectroscopies and electrochemistry confirm this characterization; the reduction potentials of the two clusters are –440 mV vs SHE. The fact that A. vinelandii synthesizes three different proteins with the same sequence motif, each of which is likely to have a different function, shows that although sequence motifs may be used reliably to classify ferredoxins by cluster type they cannot yet be used reliably for classifying ferredoxins by function. Received: 31 January 1997 / Accepted: 9 June 1997     

Redox‐triggered chiroptical switching activity of ruthenium(III)‐bis‐(β‐diketonato) complexes bearing a bipyridine‐helicene ligand

The charged, electroactive bipyridine‐helicene‐ruthenium(III) complex [ 4 ] ^{. +},PF₆^? has been prepared from 3‐(2‐pyridyl)‐4‐aza[6]helicene and a Ru‐bis‐(β‐diketonato)‐bis‐acetonitrile precursor (β‐diketonato: 2,2,6,6‐tetramethyl‐3,5‐heptanedionato). Its chiroptical properties (electronic circular dichroism and optical rotation) were studied both experimentally and theoretically and suggest the presence of 2 diastereoisomers, namely (P,Δ)‐ and (P,Λ)‐[ 4 ] ^{. +},PF₆^? (denoted jointly as (P,Δ*)‐[ 4 ] ^{. +},PF₆^?) and their mirror‐images (M,Λ)‐ and (M,Δ)‐[ 4 ] ^{. +},PF₆^? ((M,Δ*)‐[ 4 ] ^{. +},PF₆^?). The electrochemical reduction of (P,Δ*)‐[ 4 ] ^{. +},PF₆^? to neutral complex (P,Δ*)‐ 4 was performed and revealed strong changes in the UV‐vis and electronic circular dichroism spectra. A reversible redox‐triggered chiroptical switching process was then achieved.     

Structural insights into the enzyme catalysis from comparison of three forms of dissimilatory sulphite reductase from Desulfovibrio gigas

The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr‐I and Dsr‐II, are compared at 1.76 and 2.05 Å resolution respectively. The dimeric α₂β₂γ₂ structure of Dsr‐I contains eight [4Fe–4S] clusters, two saddle‐shaped sirohaems and two flat sirohydrochlorins. In Dsr‐II, the [4Fe–4S] cluster associated with the sirohaem in Dsr‐I is replaced by a [3Fe–4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr‐I and Dsr‐II confirm the co‐factor structures, whereas EPR of a third but inactive form, Dsr‐III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe–4S] cluster replaced by a [3Fe–4S] centre. In Dsr‐I and Dsr‐II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ‐subunit C‐terminus is inserted into a positively charged channel formed between the α‐ and β‐subunits, with its conserved terminal Cysγ104 side‐chain covalently linked to the CHA atom of the sirohaem in Dsr‐I. In Dsr‐II, the thioether bond is broken, and the Cysγ104 side‐chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S₃O₆^2?, S₂O₃^2? and S^2?.     

Di-cluster, seven-iron ferredoxins from hyperthermophilic Sulfolobales

 Seven-iron ferredoxins from the thermoacidophilic archaea Acidianus ambivalens, A. infernus, Metalosphaera prunae and Sulfolobus metallicus were extensively characterised, allowing study of their expression under aerobic and anaerobic growth conditions as well as the putative role in thermal stability of a recently described zinc centre. The archaeon S. metallicus was found to express, under the same growth conditions, two ferredoxins in almost identical amounts, a novelty among Archaea. Most interestingly, these two ferredoxins differ at the N-terminal amino acid sequence in that one has a zinc binding motif (FdA) and the other does not (FdB); in agreement with these findings, FdA contains a zinc ion and FdB does not. These two ferredoxins have identical thermal stabilities, indicating that the zinc atom is not determinant in the protein thermostability. Further, the presence of the additional zinc centre does not interfere with the redox properties of the iron-sulfur clusters since their reduction potentials are almost identical. From the other three archaea, independently of the growth mode in respect to oxygen, only a single zinc-containing ferredoxin was found. EPR studies on the purified proteins, both in the oxidised and dithionite reduced states, allowed the identification of one [3Fe-4S]^1+/0 centre and one [4Fe-4S]^2+/1+ centre in all proteins studied. The complete sequence of A. ambivalens ferredoxin is reported. Together with the data gathered in this study, the properties of the seven-iron ferredoxins from Sulfolobales so far known are re-discussed. Received: 10 June 1998 / Accepted: 25 June 1998