Location of the iron-sulfur clusters FA and FB in photosystem I: an electron paramagnetic resonance study of spin relaxation enhancement of P700+.

Photosystem I (PS I) mediates electron-transfer from plastocyanin to ferredoxin via a photochemically active chlorophyll dimer (P700), a monomeric chlorophyll electron acceptor (A0), a phylloquinone (A1), and three [4Fe-4S] clusters (FX/A/B). The sequence of electron-transfer events between the iron-sulfur cluster, FX, and ferredoxin is presently unclear. Owing to the presence of a 2-fold symmetry in the PsaC protein to which the iron-sulfur clusters F(A) and F(B) are bound, the spatial arrangement of these cofactors with respect to the C2-axis of symmetry in PS I is uncertain as well. An unequivocal determination of the spatial arrangement of the iron-sulfur clusters FA and FB within the protein is necessary to unravel the complete electron-transport chain in PS I. In the present study, we generate EPR signals from charge-separated spin pairs (P700+-FredX/A/B) in PS I and characterize them by progressive microwave power saturation measurements to determine the arrangement of the iron-sulfur clusters FX/A/B relative to P700. The microwave power at half saturation (P1/2) of P700+ is greater when both FA and FB are reduced in untreated PS I than when only FA is reduced in mercury-treated PS I. The experimental P1/2 values are compared to values calculated by using P700-FA/B crystallographic distances and assuming that either FA or FB is closer to P700+. On the basis of this comparison of experimental and theoretical values of spin relaxation enhancement effects on P700+ in P700+ [4Fe-4S]- charge-separated pairs, we find that iron-sulfur cluster FA is in closer proximity to P700 than the FB cluster.     

Iron-sulfur clusters in type I reaction centers.

Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy. The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors. Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC. It is comprised of an interpolypeptide [4Fe-4S] cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal [4Fe-4S] clusters, F(A) and F(B), that are bound to the PsaC subunit. In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs. The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin. The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-). We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.     

Assembly of protein subunits within the stromal ridge of photosystem I. Structural changes between unbound and sequentially PS I-bound polypeptides and correlated changes of the magnetic properties of the terminal iron sulfur clusters

The X-ray structure of Photosystem I (PS I) from Synechococcus elongatus was recently solved at 2.5A resolution (PDB entry 1JB0). It provides a structural model for the stromal subunits PsaC, PsaD and PsaE, which comprise the "stromal ridge" of PS I. In a separate set of studies the three-dimensional solution structures of the unbound, recombinant PsaC (PDB entry 1K0T) and PsaE (PDB entries 1PSF, 1QP2 and 1GXI) subunits were solved by NMR. The PsaC subunit of PS I is a small (9.3 kDa) protein that harbors binding sites for two [4Fe-4S] clusters F(A) and F(B), which are the terminal electron acceptors in PS I. Comparison of the PsaC structure in solution with that in the X-ray structure of PS I reveals significant differences between them which are summarized and evaluated here. Changes in the magnetic properties of [4Fe-4S] centers F(A) and F(B) are related to changes in the protein structure of PsaC, and they are further influenced by the presence of PsaD. Based on experimental evidence, three assembly stages are analyzed: PsaC(free), PsaC(only), PsaC(PS I). Unbound, recombinant PsaD, studied by NMR, has only a few elements of secondary structure and no stable three-dimensional structure in solution. When PsaD is bound in PS I, it has a well-defined three-dimensional structure. For PsaE the three-dimensional structure is very similar in solution and in the PS I-bound form, with the exception of two loop regions. We suggest that the changes in the structures of PsaC and PsaD are caused by the sequential formation of multiple networks of contacts between the polypeptides of the stromal ridge and between those polypeptides and the PsaA/PsaB core polypeptides. The three-dimensional structure of the C(2)-symmetric F(X)-binding loops on PsaA and PsaB were also analyzed and found to be significantly different from the binding sites of other proteins that contain interpolypeptide [4Fe-4S] clusters. The aim of this work is to relate contact information to structural changes in the proteins and to propose a model for the assembly of the stromal ridge of PS I based on this analysis.     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Unifying principles in homodimeric type I photosynthetic reaction centers: properties of PscB and the FA, FB and FX iron-sulfur clusters in green sulfur bacteria

The photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum (CbRC) was solubilized from membranes using Triton X-100 and isolated by sucrose density ultra-centrifugation. The CbRC complexes were subsequently treated with 0.5 M NaCl and ultrafiltered over a 100 kDa cutoff membrane. The resulting CbRC cores did not exhibit the low-temperature EPR resonances from FA- and FB- and were unable to reduce NADP+. SDS-PAGE and mass spectrometric analysis showed that the PscB subunit, which harbors the FA and FB clusters, had become dissociated, and was now present in the filtrate. Attempts to rebind PscB onto CbRC cores were unsuccessful. M?ssbauer spectroscopy showed that recombinant PscB contains a heterogeneous mixture of [4Fe-4S]2+,1+ and other types of Fe/S clusters tentatively identified as [2Fe-2S]2+,1+ clusters and rubredoxin-like Fe3+,2+ centers, and that the [4Fe-4S]2+,1+ clusters which were present were degraded at high ionic strength. Quantitative analysis confirmed that the amount of iron and sulfide in the recombinant protein was sub-stoichiometric. A heme-staining assay indicated that cytochrome c551 remained firmly attached to the CbRC cores. Low-temperature EPR spectroscopy of photoaccumulated CbRC complexes and CbRC cores showed resonances between g=5.4 and 4.4 assigned to a S=3/2 ground spin state [4Fe-4S]1+ cluster and at g=1.77 assigned to a S=1/2 ground spin state [4Fe-4S]1+ cluster, both from FX-. These results unify the properties of the acceptor side of the Type I homodimeric reaction centers found in green sulfur bacteria and heliobacteria: in both, the FA and FB iron-sulfur clusters are present on a salt-dissociable subunit, and FX is present as an interpolypeptide [4Fe-4S]2+,1+ cluster with a significant population in a S=3/2 ground spin state.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S](1+,2+) cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S](1+,2+) clusters termed F(A) and F(B). In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F(B). Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and M?ssbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S](1+,2+) clusters, despite the absence of one of the biological ligands. (19)F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F(B) cluster. The finding that site-modified [4Fe-4S](1+,2+) clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Site-directed conversion of a cysteine to aspartate leads to the assembly of a [3Fe-4S] cluster in PsaC of photosystem I. The photoreduction of FA is independent of FB.

The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-FX core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.     

Low-potential iron-sulfur centers in photosystem I: an X-ray absorption spectroscopy study

We have measured the X-ray absorption spectra of Fe in photosystem I (PS I) preparations from spinach and a thermophilic cyanobacterium, Synechococcus sp., to characterize structures of the Fe complexes that function as electron acceptors in PS I. These acceptors include centers A and B, which are probably typical [4Fe-4S] ferredoxins, and X. The structure of X is not known, but its electron paramagnetic resonance (EPR) spectrum has generated the suggestions that it is either a [2Fe-2S] or [4Fe-4S] ferredoxin or an Fe-quinone species. The iron X-ray absorption K-edge and iron extended X-ray absorption fine structure (EXAFS) spectra reveal that essentially all of the 11-14 Fe atoms present in the reaction center are present in the form of Fe-S centers and that not more than 1 atom out of 12 could be octahedral or oxygen-coordinated Fe. This suggests that, besides A and B, additional Fe-S clusters are present which are likely to be X. Our EXAFS spectra cannot be simulated adequately by a mixture of [4Fe-4S] ferredoxins with typical bond lengths and disorder parameters because the amplitude of Fe backscattering is small; however, excellent simulations of the data are consistent with a mixture of [2Fe-2S] ferredoxins and [4Fe-4S] ferredoxins, or with unusually distorted [4Fe-4S] clusters. We presume that the [2Fe-2S] or distorted [4Fe-4S] centers are X. The X-ray absorption spectra of PS I preparations from Synechococcus and spinach are essentially indistinguishable.     

Three-iron clusters in iron-sulfur proteins

Contents. 1. Introduction and history. 2. Characteristic spectroscopic features of 3Fe clusters. 1. General considerations. 2. M?ssbauer spectroscopy. 3. Magnetic circular dichroism (MCD) spectroscopy. 4. Electron paramagnetic resonance (EPR) spectroscopy. 5. Resonance Raman (RR) spectroscopy. 6. Extended X-ray fine-structure (EXAFS) spectroscopy. 3. Results of X-Ray diffraction studies. 4. Proteins containing or showing features characteristic of 3Fe clusters 1. Overview. 2. Ferredoxin I of Azotobacter vinelandii. 3. Ferredoxin II of Desulfovibrio gigas. 4. Aconitase from beef heart. 5. Other observations and considerations relevant to 3Fe clusters or cluster interconversions 1. Oxidative degradation of [4Fe-4S] clusters to 3Fe clusters. 2. Extrusion studies on 3Fe clusters. 3. Reconstitution of 3Fe clusters. 4. Disposition of iron ligands in cluster interconversions. 6. Do all 3Fe clusters have the same structure? Evidence for [3Fe-4S] clusters. 7. Are 3Fe clusters artifacts or biologically significant structures?     

A proposed role for the Azotobacter vinelandii NfuA protein as an intermediate iron-sulfur cluster carrier

Iron-sulfur clusters ([Fe-S] clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require [Fe-S] clusters for their functions. Previous studies have shown that Azotobacter vinelandii produces at least two [Fe-S] cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other [Fe-S] proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a [4Fe-4S] cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one [4Fe-4S] cluster can be assembled in vitro within a dimeric form of NfuA. The resultant [4Fe-4S] cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate [Fe-S] cluster carriers involved in [Fe-S] protein maturation.     

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.     

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 structure of genetically modified iron-sulfur cluster Fx in photosystem I as determined by X-ray absorption spectroscopy

Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe-4S] iron-sulfur cluster F_X, located on the core subunits PsaA/B to iron-sulfur clusters F_A/B on subunit PsaC. Structure function relations in the native and in the mutant (psaB-C565S/D566E) of the cysteine ligand of F_X cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of F_X was determined in PS I lacking clusters F_A/B by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe-4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe-S and Fe-Fe distances of 2.27 Å and 2.69 Å, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe-O distance of 1.81 Å. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to F_X is discussed.     

Solution structure of the unbound, oxidized Photosystem I subunit PsaC, containing [4Fe-4S] clusters FA and FB: a conformational change occurs upon binding to Photosystem I

This work presents the three-dimensional NMR solution structure of recombinant, oxidized, unbound PsaC from Synechococcus sp. PCC 7002. Constraints are derived from homo- and heteronuclear one-, two- and three-dimensional (1)H and (15)N NMR data. Significant differences are outlined between the unbound PsaC structure presented here and the available X-ray structure of bound PsaC as an integral part of the whole cyanobacterial PS I complex. These differences mainly concern the arrangement of the N- and C-termini with respect to the [4Fe-4S] core domain. In the NMR solution structure of PsaC the C-terminal region assumes a disordered helical conformation, and is clearly different from the extended coil conformation, which is one of the structural elements required to anchor PsaC to the PS I core heterodimer. In solution the N-terminus of PsaC is in contact with the pre-C-terminal region but slides in between the latter and the iron-sulfur core region of the protein. Together, these features result in a concerted movement of the N-terminus and pre-C-terminal region away from the F(A) binding site, accompanied by a bending of the N-terminus. In comparison, the same terminal regions are positioned much closer to F(A) and take up an anti-parallel beta-sheet arrangement in PsaC bound to PS I. The conformational changes between bound and unbound PsaC correlate with the differences reported earlier for the EPR spectra of reduced F(A) and F(B) in bound versus unbound PsaC. The observed different structural features in solution are highly relevant for unraveling the stepwise assembly process of the stromal PsaC, PsaD and PsaE subunits to the PS I core heterodimer. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0321-3.     

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.     

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

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