A M?ssbauer analysis of the low-potential iron-sulfur center in photosystem I: spectroscopic evidence that FX is a [4Fe-4S] cluster

We report the results of a M?ssbauer study of the low-potential iron-sulfur cluster FX in the Photosystem I core protein of Synechococcus 6301. The M?ssbauer spectrum of FX in the oxidized state shows an isomer shift of 0.42 mm/s, which is in good agreement with the 0.43 mm/s isomer shift found in [4Fe-4S] proteins but not with the isomer shift of 0.26 mm/s found in [2Fe-2S] proteins. In the reduced state the spectrum is asymmetrically broadened at 80 K, indicating the presence of two very closely spaced doublets with an average isomer shift of 0.55 mm/s, which is also in agreement with [4Fe-4S] proteins. At 4.2 K, the spectrum exhibits broadening and magnetic splitting similar to what is observed for [4Fe-4S] proteins and quite unlike [2Fe-2S] proteins. Given the assumption that the iron atoms of FX are tetrahedrally coordinated with sulfur ligands, the data strongly support the assignment of FX as a [4Fe-4S] cluster.     

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.     

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

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

Vertebrate-type and plant-type ferredoxins: crystal structure comparison and electron transfer pathway modelling

Crystallographic analysis of a fully functional, truncated bovine adrenodoxin, Adx(4-108), has revealed the structure of a vertebrate-type [2Fe-2S] ferredoxin at high resolution. Adrenodoxin is involved in steroid hormone biosythesis in adrenal gland mitochondria by transferring electrons from adrenodoxin reductase to different cytochromes P450. Plant-type [2Fe-2S] ferredoxins interact with photosystem I and a diverse set of reductases.A systematic structural comparison of Adx(4-108) with plant-type ferredoxins which share about 20 % sequence identity yields these results. (1) The ferredoxins of both types are partitioned into a large, strictly conserved core domain bearing the [2Fe-2S] cluster and a smaller interaction domain which is structurally different for both subfamilies. (2) In both types, residues involved in interactions with reductase are located at similar positions on the molecular surface and coupled to the [2Fe-2S] cluster via structurally equivalent hydrogen bonds. (3) The accessibility of the [2Fe-2S] cluster differs between Adx(4-108) and the plant-type ferredoxins where a solvent funnel leads from the surface to the cluster. (4) All ferredoxins are negative monopoles with a clear charge separation into two compartments, and all resulting dipoles but one point into a narrow cone located in between the interaction domain and the [2Fe-2S] cluster, possibly controlling predocking movements during interactions with redox partners. (5) Model calculations suggest that FE1 is the origin of electron transfer pathways to the surface in all analyzed [2Fe-2S] ferredoxins and that additional transfer probability for electrons tunneling from the more buried FE2 to the cysteine residue in position 92 of Adx is present in some.     

Purification and properties of the intact P-700 and Fx-containing Photosystem I core protein

The intact Photosystem I core protein, containing the psaA and psaB polypeptides, and electron transfer components P-700 through FX, was isolated from cyanobacterial and higher plant Photosystem I complexes with chaotropic agents followed by sucrose density ultracentrifugation. The concentrations of NaClO4, NaSCN, NaI, NaBr or urea required for the functional removal of the 8.9 kDa, FA/FB polypeptide was shown to be inversely related to the strength of the chaotrope. The Photosystem I core protein, which was purified to homogeniety, contains 4 mol of acid-labile sulfide and has the following properties: (i) the FX-containing core consists of the 82 and 83 kDa reaction center polypeptides but is totally devoid of the low-molecular-mass polypeptides; (ii) methyl viologen and other bipyridilium dyes have the ability to accept electrons directly from FX; (iii) the difference spectrum of FX from 400 to 900 nm is characteristic of an iron-sulfur cluster; (iv) the midpoint potential of FX, determined optically at room temperature, is 60 mV more positive than in the control; (v) there is indication by ESR spectroscopy of low-temperature heterogeneity within FX; and (vi) the heterogeneity is seen by optical spectroscopy as inefficiency in low-temperature electron flow to FX. The constraints imposed by the amount of non-heme iron and labile sulfide in the Photosystem I core protein, the cysteine content of the psaA and psaB polypeptides, and the stoichiometry of high-molecular-mass polypeptides, cause us to re-examine the possibility that FX is a [4Fe-4S] rather than a [2Fe-2S] cluster ligated by homologous cysteine residues on the psaA and psaB heterodimer.     

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.     

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)     

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.     

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.     

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.     

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.     

The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I

The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I.     

EXAFS studies of the isolated bovine heart Rieske [2Fe-2S]1+(1+,2+) cluster

Recently the involvement of one or, more likely, two nitrogen-ligands in the Rieske-type [2Fe-2S] cluster has been reported based on the chemical assay and various spectroscopic analyses, such as EPR, M?ssbauer, ENDOR, and resonance Raman, of isolated Thermus thermophilus HB-8 protein by Fee and his collaborators. Similarly, the presence of at least one nitrogen ligand was shown in the mitochondrial Rieske [2Fe-2S] cluster. We have conducted EXAFS studies of the Rieske [2Fe-2S] protein isolated from the cytochrome bc1 complex of bovine heart mitochondria. Standard analysis could not distinguish one or two nitrogen ligands per cluster. However, one nitrogen and three cysteine ligands per cluster was found to be, possibly, a better solution in more comprehensive analysis procedures.     

Formation and properties of [4Fe-4S] clusters on the IscU scaffold protein

Rapid and quantitative reductive coupling of two [2Fe-2S]2+ clusters to form a single [4Fe-4S]2+ cluster on the homodimeric IscU Fe-S cluster scaffold protein has been demonstrated by UV-visible absorption, M?ssbauer, and resonance Raman spectroscopies, using dithionite as the electron donor. Partial reductive coupling was also observed using reduced Isc ferredoxin, which raises the possibility that Isc ferredoxin is the physiological reductant. The results suggest that reductive coupling of adjacent [2Fe-2S]2+ clusters assembled on IscU provides a general mechanism for the final step in the biosynthesis of [4Fe-4S]2+ clusters. The [4Fe-4S]2+ center on IscU can be reduced to a S = 1/2[4Fe-4S]+ cluster (g parallel = 2.06 and g perpendicular = 1.92), but the low midpoint potential (< -570 mV) and instability of the reduced cluster argue against any physiological relevance for the reduced cluster. On exposure to O2, the [4Fe-4S]2+ cluster on IscU degrades via a semistable [2Fe-2S]2+ cluster with properties analogous to those of the [2Fe-2S]2+ center in [2Fe-2S]2+ IscU. It is suggested that the ability of IscU to accommodate either [2Fe-2S]2+ or [4Fe-4S]2+ clusters in response to cellular redox status and/or oxygen levels may provide an effective way to populate appropriately cluster-loaded forms of IscU for maturation of different types of [Fe-S] proteins.     

In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein

Genetic experiments have established that IscU is involved in maturation of [Fe-S] proteins that require either [2Fe-2S] or [4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one [2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single [4Fe-4S] cluster. In the present work, it is shown that the [4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the [2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a [4Fe-4S] cluster for enzymatic activity. The rate of [4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a [4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type [4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the [2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the [2Fe-2S] and [4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of [2Fe-2S] and [4Fe-4S] centers in proteins.     

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

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

Evidence for the existence of [2Fe-2S] as well as [4Fe-4S] clusters among FA,FB and FX. Implications for the structure of the Photosystem I reaction center

Core extrusion of the bound iron-sulfur centers from spinach Photosystem I showed the presence of [2Fe-2S] clusters as well as [4Fe-4S] clusters among F_A, F_B and F_X. Extrusion of the iron-sulfur ensemble was not quantitative; however, the presence of [2Fe-2S] clusters correlated with higher concentration of unfolding solvent. Since F_X is highly resistant to denaturation, and since F_A and F_B are known to contain [4Fe-4S] clusters, the [2Fe-2S] clusters are assigned to F_X. The presence of [2Fe-2S] clusters in Photosystem I has significance in the structure and organization of F_X on the reaction center. Since four cysteinyl ligands are assumed to hold an iron-sulfur cluster, a Photosystem I subunit may consist of two approx. 64-kDa proteins bridged by a single [2Fe-2S] cluster. The complete reaction center would consist of two subunits positioned so that two [2Fe-2S] clusters are in magnetic interaction, thereby constituting F_X.     

Circular dichroism and magnetic circular dichroism of Azotobacter vinelandii ferredoxin I

Room temperature circular dichroism (CD) and low temperature magnetic circular dichroism (MCD) spectra of air-oxidized and dithionite-reduced Azotobacter vinelandii ferredoxin I (FdI), a [( 4Fe-4S]2+/1+, [3Fe-4S]1+/0) protein, are reported. Unlike the CD of oxidized FdI, the CD of dithionite-reduced FdI exhibits significant pH dependence, consistent with protonation-deprotonation at or near the cluster reduced: the [3Fe-4S] cluster. The MCD of reduced FdI, which originates in the paramagnetic reduced [3Fe-4S]0 cluster, is also pH-dependent. Detailed studies of the field dependence and temperature dependence of the MCD of oxidized and reduced FdI, in the latter case at pH 6.0 and 8.3, are reported. The low-field temperature dependence of the MCD of oxidized FdI, which originates in the paramagnetic oxidized [3Fe-4S]1+ cluster, establishes the absence of a significant population of excited electronic states of this cluster up to 60 K. The low-field temperature dependence of the MCD of reduced FdI establishes that the ground-state manifold of the reduced [3Fe-4S]0 cluster possesses S greater than or equal to 2 at both pH 6.0 and 8.3. Analysis, assuming S = 2 and an axial zero-field splitting Hamiltonian, leads to D = -2.0 and -3.5 cm-1 at pH 6.0 and 8.3, respectively. The site of the (de)protonation affecting the spectroscopic properties of the [3Fe-4S] cluster remains unknown.