Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.     

Purification of cytochrome P450 and ferredoxin, involved in bisphenol A degradation, from Sphingomonas sp. strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450bisd), ferredoxin (Fd(bisd)), and ferredoxin reductase (Red(bisd)). We demonstrated that P450bisd and Fd(bisd) are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd(bisd) revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450(bisd), in the presence of Fd(bisd), Red(bisd), and NADH, was able to convert BPA. The K(m) and kcat values for BPA degradation were 85 +/- 4.7 microM and 3.9 +/- 0.04 min(-1), respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450bisd might exhibit strict specificity. Two BPA degradation products of the P450(bisd) system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.     

Electron paramagnetic resonance evidence for a novel interconversion of [3Fe-4S](+) and [4Fe-4S](+) clusters with endogenous iron and sulfide in anaerobic ribonucleotide reductase activase in vitro

We report an EPR study of the iron-sulfur enzyme, anaerobic ribonucleotide reductase activase from Lactococcus lactis. The activase (nrdG gene) together with S-adenosyl-L-methionine (AdoMet) give rise to a glycyl radical in the NrdD component. A semi-reduced [4Fe-4S](+) cluster with an axially symmetric EPR signal was produced upon photochemical reduction of the activase. Air exposure of the reduced enzyme gave a [3Fe-4S](+) cluster. The Fe(3)S(4) cluster was convertible to the EPR-active [4Fe-4S](+) cluster by renewed treatment with reducing agents, demonstrating a reversible [3Fe-4S](+)- to-[4Fe-4S](+) cluster conversion without exogenous addition of iron or sulfide. Anaerobic reduction of the activase by a moderate concentration of dithionite also resulted in a semi-reduced [4Fe-4S](+) cluster. Prolonged reduction gave an EPR-silent fully reduced state, which was enzymatically inactive. Both reduced states gave the [3Fe-4S](+) EPR signal after air exposure. The iron-sulfur cluster interconversion was also studied in the presence of AdoMet. The EPR signal of semi-reduced activase-AdoMet had rhombic symmetry and was independent of which reductant was applied, whereas the EPR signal of the [3Fe-4S](+) cluster after air exposure was unchanged. The results indicate that an AdoMet-mediated [4Fe-4S](+) center is the native active species that induces the formation of a glycyl radical in the NrdD component.     

Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii.

A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.     

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.     

Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6–7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g=2.01 which is typical of [3Fe-4S]¹⁺ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g=1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]¹⁺ clusters at much higher yields (0.4–0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. grisues ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450_soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.     

Identification of FX in the heliobacterial reaction center as a [4Fe-4S] cluster with an S = 3/2 ground spin state

Type I homodimeric reaction centers, particularly the class present in heliobacteria, are not well understood. Even though the primary amino acid sequence of PshA in Heliobacillus mobilis has been shown to contain an F(X) binding site, a functional Fe-S cluster has not been detected by EPR spectroscopy. Recently, we reported that PshB, which contains F(A)- and F(B)-like Fe-S clusters, could be removed from the Heliobacterium modesticaldum reaction center (HbRC), resulting in 15 ms lifetime charge recombination between P798(+) and an unidentified electron acceptor [Heinnickel, M., Shen, G., Agalarov, R., and Golbeck, J. H. (2005) Biochemistry 44, 9950-9960]. We report here that when a HbRC core is incubated with sodium dithionite in the presence of light, the 15 ms charge recombination is replaced with a kinetic transient in the sub-microsecond time domain, consistent with the reduction of this electron acceptor. Concomitantly, a broad and intense EPR signal arises around g = 5 along with a minor set of resonances around g = 2 similar to the spectrum of the [4Fe-4S](+) cluster in the Fe protein of Azotobacter vinelandii nitrogenase, which exists in two conformations having S = (3)/(2) and S = (1)/(2) ground spin states. The M?ssbauer spectrum in the as-isolated HbRC core shows that all of the Fe is present in the form of a [4Fe-4S](2+) cluster. After reduction with sodium dithionite in the presence of light, approximately 65% of the Fe appears in the form of a [4Fe-4S](+) cluster; the remainder is in the [4Fe-4S](2+) state. Analysis of the non-heme iron content of HbRC cores indicates an antenna size of 21.6 +/- 1.1 BChl g molecules/P798. The evidence indicates that the HbRC contains a [4Fe-4S] cluster identified as F(X) that is coordinated between the PshA homodimer; in contrast to F(X) in other type I reaction centers, this [4Fe-4S] cluster exhibits an S = (3)/(2) ground spin state.     

Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus.

We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.     

On the active sites of the [NiFe] hydrogenase from Desulfovibrio gigas. M?ssbauer and redox-titration studies

The [NiFe] hydrogenase isolated from Desulfovibrio gigas was poised at different redox potentials and studied by M?ssbauer spectroscopy. The data firmly establish that this hydrogenase contains four prosthetic groups: one nickel center, one [3Fe-xS], and two [4Fe-4S] clusters. In the native enzyme, both the nickel and the [3Fe-xS] cluster are EPR-active. At low temperature (4.2 K), the [3Fe-xS] cluster exhibits a paramagnetic M?ssbauer spectrum typical for oxidized [3Fe-xS] clusters. At higher temperatures (greater than 20 K), the paramagnetic spectrum collapses into a quadrupole doublet with parameters magnitude of delta EQ magnitude of = 0.7 +/- 0.06 mm/s and delta = 0.36 +/- 0.06 mm/s, typical of high-spin Fe(III). The observed isomer shift is slightly larger than those observed for the three-iron clusters in D. gigas ferredoxin II (Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V., and Münck, E. (1980) J. Biol. Chem. 255, 3242-3244) and in Azotobacter vinelandii ferredoxin I (Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796) and may indicate a different iron coordination environment. When D. gigas hydrogenase is poised at potentials lower than -80 mV (versus normal hydrogen electrode), the [3Fe-xS] cluster is reduced and becomes EPR-silent. The M?ssbauer data indicate that the reduced [3Fe-xS] cluster remains intact, i.e. it does not interconvert into a [4Fe-4S] cluster. Also, the electronic properties of the reduced [3Fe-xS] cluster suggest that it is magnetically isolated from the other paramagnetic centers.     

The adrenodoxin-like ferredoxin of Schizosaccharomyces pombe mitochondria

The single mitochondrial type I [2Fe-2S] ferredoxin of the fission yeast Schizosaccharomyces pombe is produced as the carboxy terminal part of the electron-transfer-protein 1 (etp1) and cleaved off during mitochondrial import [Biochemistry 41 (2002) 2311-2321]. The UV/Vis (UV-visible) spectrum of the purified recombinant ferredoxin domain (etp1(fd)) expressed in Escherichia coli is similar to those of bovine Adx in the oxidized as well as in the reduced state. EPR (electronic paramagnetic resonance) studies revealed a correctly incorporated iron-sulfur cluster of the axial type. The redox potential of this protein was determined to be -353 mV, which is considerably lower than that of adrenodoxin (Adx, -273 mV). Several lines of evidence indicate that the protein forms dimers under physiological and denaturating conditions. Interestingly, the fission yeast ferredoxin could be shown to be active as an electron carrier in heterologous redox systems. It is able to transfer electrons to horse heart cytochrome c and to bovine cytochromes P450(scc) (CYP11A1) and P450(11 beta) (CYP11B1), thereby receiving electrons from bovine NADPH-dependent Adx reductase. The kinetics of substrate conversion in the etp1(fd)-supported CYP11A1 and CYP11B1-dependent systems mediated was studied.     

Structural differences between [2Fe-2S] clusters in spinach ferredoxin and in the "red paramagnetic protein" from Clostridium pasteurianum. A resonance Raman study

The [2Fe-2S] ferredoxin ("Red paramagnetic protein", RPP) from C. pasteurianum has been found to be composed of two identical subunits of 10,000 +/- 2 000 daltons, each containing a [2Fe-2S] cluster. Resonance Raman (RR) spectra of RPP have been obtained at 23 degrees K, and compared to those of spinach ferredoxin (Sp Fd). Ten modes of the [2Fe-2S] chromophore were observed in the 100-450 cm-1 range. Assignments of non fundamental modes in the 500-900 cm-1 range allowed correlations between fundamental stretching modes of RPP and Sp Fd. Although assuming a [2Fe-2S] structure, the chromophore of RPP differs from that of Sp Fd by its conformation and by a slight weakening of Fe-S bonds, involving both the inorganic core and the cysteine ligands.     

Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A(2) heterodimer neurotoxin

Heterodisulfide reductases (HDRs) from methanogenic archaea are iron-sulfur flavoproteins or hemoproteins that catalyze the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). In this work, the ground- and excited-state electronic properties of the paramagnetic Fe-S clusters in Methanothermobacter marburgensis HDR have been characterized using the combination of electron paramagnetic resonance and variable-temperature magnetic circular dichroism spectroscopies. The results confirm multiple S=1/2 [4Fe-4S](+) clusters in dithionite-reduced HDR and reveal spectroscopically distinct S=1/2 [4Fe-4S](3+) clusters in oxidized HDR samples treated separately with the CoM-SH and CoB-SH cosubstrates. The active site of HDR is therefore shown to contain a [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. The catalytic mechanism of HDR is discussed in light of the crystallographic and spectroscopic studies of the related chloroplast ferredoxin:thioredoxin reductase class of disulfide reductases.     

Ferredoxins from the archaeon Acidianus ambivalens: overexpression and characterization of the non-zinc-containing ferredoxin FdB.

Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.     

Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa

APS reductase from Pseudomonas aeruginosa has been shown to contain a [4Fe-4S] cluster. Thiol determinations and site-directed mutagenesis studies indicate that the single [4Fe-4S] cluster contains only three cysteine ligands, instead of the more typical arrangement in which clusters are bound to the protein by four cysteines. Resonance Raman studies in the Fe-S stretching region are also consistent with the presence of a redox-inert [4Fe-4S](2+) cluster with three cysteinate ligands and indicate that the fourth ligand is likely to be an oxygen-containing species. This conclusion is supported by resonance Raman and electron paramagnetic resonance (EPR) evidence for near stoichiometric conversion of the cluster to a [3Fe-4S](+) form by treatment with a 3-fold excess of ferricyanide. Site-directed mutagenesis experiments have identified Cys139, Cys228, and Cys231 as ligands to the cluster. The remaining two cysteines present in the enzyme, Cys140 and Cys256, form a redox-active disulfide/dithiol couple (E(m) = -300 mV at pH 7.0) that appears to play a role in the catalytic mechanism of the enzyme.     

Spectopotentiometric properties and salt-dependent thermotolerance of a [2Fe-2S] ferredoxin-involved nitrate assimilation in Haloferax mediterranei

Haloferax mediterranei is a halophilic archaeon that can grow using nitrate as the sole nitrogen source. A ferredoxin that serves as the physiological electron donor to the nitrate and nitrite reductases in this assimilatory process has been characterized. The ferredoxin was found to contain approximately two atoms of iron and two atoms of sulphur, indicative of the binding of a [2Fe-2S] cluster. The electron paramagnetic resonance spectrum of the reduced form of the protein displayed a rhombic signal, with g(x)=1.91, g(y)=1.98, g(z)=2.07, that shows considerable similarity to plant and algal [2Fe-2S] ferredoxins. UV-visible spectropotentiometric analysis determined a midpoint redox potential for the [2Fe-2S](2+/1+) transition of around -285 mV vs. SHE that was independent of salt concentration. UV-visible spectroscopy was also used to establish that the [2Fe-2S] cluster integrity of this protein was maintained over the pH range 5-11. Significantly, the Haloferax mediterranei ferredoxin was shown to be a highly thermostable protein. It was stable up to 60 degrees C in a low-salt (0.2 M) medium and this increased to 80 degrees C in a high-salt (4 M) medium. This thermostability at high salt concentration is an essential physiological characteristic because haloarchaea are mainly found in environments where high temperatures and concentrated salt water occur.     

Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus

A ferredoxin was purified anaerobically from Rhodobacter capsulatus grown photoheterotrophically with excess ammonia. This ferredoxin, called ferredoxin II (FdII), had a molecular weight of approximatively 15,000 by gel filtration and 14,000 by SDS polyacrylamide gel electrophoresis indicating that it is monomeric. Its absorption spectrum (oxidized form) exhibited maxima at 280 nm and 400 nm; the A400/A280 ratio had a calculated value of 0.55. Chemical determination of its iron and sulfur atom content, the value of the extinction coefficient at 400 nm (epsilon 400 = 26.8 mM-1 cm-1) and EPR spectra indicated that ferredoxin II contained one [3Fe-4S] and one [4Fe-4S] cluster. Upon reduction with excess dithionite only the [3Fe-4S] cluster became reduced. The reduction of both clusters was achieved by using 5-deazaflavin as photocatalyst. Ferredoxin II was also purified from bacteria grown under nitrogen limiting (nif derepressing) conditions. In in vitro assays, ferredoxin II catalyzed electron transport between illuminated chloroplasts and nitrogenase.     

Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.     

Redox chemistry of the Schizosaccharomyces pombe ferredoxin electron-transfer domain and influence of Cys to Ser substitutions

Schizosaccharomyces pombe (Sp) ferredoxin contains a C-terminal electron transfer protein ferredoxin domain (etp^Fd) that is homologous to adrenodoxin. The ferredoxin has been characterized by spectroelectrochemical methods, and Mössbauer, UV-Vis and circular dichroism spectroscopies. The Mössbauer spectrum is consistent with a standard diferric [2Fe-2S]²⁺ cluster. While showing sequence homology to vertebrate ferredoxins, the E°' and the reduction thermodynamics for etp^Fd (− 0.392 V) are similar to plant-type ferredoxins. Relatively stable Cys to Ser derivatives were made for each of the four bound Cys residues and variations in the visible spectrum in the 380-450 nm range were observed that are characteristic of oxygen ligated clusters, including members of the [2Fe-2S] cluster IscU/ISU scaffold proteins. Circular dichroism spectra were similar and consistent with no significant structural change accompanying these mutations. All derivatives were active in an NADPH-Fd reductase cytochrome c assay. The binding affinity of Fd to the reductase was similar, however, V_max reflecting rate limiting electron transfer was found to decrease ~ 13-fold. The data are consistent with relatively minor perturbations of both the electronic properties of the cluster following substitution of the Fe-bond S atom with O, and the electronic coupling of the cluster to the protein.     

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.