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

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

Electron bifurcation involved in the energy metabolism of the acetogenic bacterium Moorella thermoacetica growing on glucose or H2 plus CO2

Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO(2) molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fd(red)(2-)) molecules. In the reductive part, 2 CO(2) molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO(2) are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO(2) reduction to acetic acid are generated by the reduction of 2 NADP(+) molecules with 1 NADH and 1 Fd(red)(2-) catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD(+) and Fd with 2 H(2) molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H(2)-CO(2)-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H(2)-CO(2)-grown cells additionally contain high specific activities of an NADP(+)-dependent hydrogenase that catalyzes the reduction of NADP(+) with H(2). This activity is hardly detectable in glucose-grown cells.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Structure of the dissimilatory sulfite reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus

Conservation of energy based on the reduction of sulfate is of fundamental importance for the biogeochemical sulfur cycle. A key enzyme of this ancient anaerobic process is the dissimilatory sulfite reductase (dSir), which catalyzes the six-electron reduction of sulfite to hydrogen sulfide under participation of a unique magnetically coupled siroheme-[4Fe-4S] center. We determined the crystal structure of the enzyme from the sulfate-reducing archaeon Archaeoglobus fulgidus at 2-Å resolution and compared it with that of the phylogenetically related assimilatory Sir (aSir). dSir is organized as a heterotetrameric (αβ)₂ complex composed of two catalytically independent αβ heterodimers. In contrast, aSir is a monomeric protein built of two fused modules that are structurally related to subunits α and β except for a ferredoxin domain inserted only into the subunits of dSir. The [4Fe-4S] cluster of this ferredoxin domain is considered as the terminal redox site of the electron transfer pathway to the siroheme-[4Fe-4S] center in dSir. While aSir binds one siroheme-[4Fe-4S] center, dSir harbors two of them within each αβ heterodimer. Surprisingly, only one siroheme-[4Fe-4S] center in each αβ heterodimer is catalytically active, whereas access to the second one is blocked by a tryptophan residue. The spatial proximity of the functional and structural siroheme-[4Fe-4S] centers suggests that the catalytic activity at one active site was optimized during evolution at the expense of the enzymatic competence of the other. The sulfite binding mode and presumably the mechanism of sulfite reduction appear to be largely conserved between dSir and aSir. In addition, a scenario for the evolution of Sirs is proposed.     

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP⁺ with H₂ or formate and the reversible formation of H₂ and CO₂ from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO₂ reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H₂ formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E_0′ = −520 mV).     

Integrated thermodynamic analysis of electron bifurcating [FeFe]-hydrogenase to inform anaerobic metabolism and H2 production

Electron bifurcating, [FeFe]-hydrogenases are recently described members of the hydrogenase family and catalyze a combination of exergonic and endergonic electron exchanges between three carriers (2 ferredoxin_red⁻ + NAD(P)H + 3 H⁺ = 2 ferredoxin_ox + NAD(P)⁺ + 2 H₂). A thermodynamic analysis of the bifurcating, [FeFe]-hydrogenase reaction, using electron path-independent variables, quantified potential biological roles of the reaction without requiring enzyme details. The bifurcating [FeFe]-hydrogenase reaction, like all bifurcating reactions, can be written as a sum of two non-bifurcating reactions. Therefore, the thermodynamic properties of the bifurcating reaction can never exceed the properties of the individual, non-bifurcating, reactions. The bifurcating [FeFe]-hydrogenase reaction has three competitive properties: 1) enabling NAD(P)H-driven proton reduction at pH₂ higher than the concurrent operation of the two, non-bifurcating reactions, 2) oxidation of NAD(P)H and ferredoxin simultaneously in a 1:1 ratio, both are produced during typical glucose fermentations, and 3) enhanced energy conservation (~10 kJ mol⁻¹ H₂) relative to concurrent operation of the two, non-bifurcating reactions. Our analysis demonstrated ferredoxin E°′ largely determines the sensitivity of the bifurcating reaction to pH₂, modulation of the reduced/oxidized electron carrier ratios contributed less to equilibria shifts. Hydrogenase thermodynamics data were integrated with typical and non-typical glycolysis pathways to evaluate achieving the ‘Thauer limit’ (4 H₂ per glucose) as a function of temperature and pH₂. For instance, the bifurcating [FeFe]-hydrogenase reaction permits the Thauer limit at 60 °C if pH ₂ ≤ ~10 mbar. The results also predict Archaea, expressing a non-typical glycolysis pathway, would not benefit from a bifurcating [FeFe]-hydrogenase reaction; interestingly, no Archaea have been observed experimentally with a [FeFe]-hydrogenase enzyme.     

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

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

Exchange integral for a variety of tetranuclear ferredoxins

The temperature dependence of EPR spectra of oxidized [4Fe-4S1]^{(?1, ?2)} ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S1]^(?2,?3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm^?1; Center S-3 from mung bean, 53 cm^?1; the [4Fe-4S1]^(?1,?2) ferredoxin from Thermus thermophilus, 78 cm^?1; Center N-2 of NADH ubiquinone reductase, 83 cm^?1. Increasing axial distortion in the EPR spectra of the [4Fe-4S1]^(?1,?2) ferredoxins was associated with higher energy differences. Center N-2, a [4Fe-4S1]^(?2,?3) iron-sulfur cluster does not fit this relationship.     

The reducing component BoxA of benzoyl-coenzyme A epoxidase from Azoarcus evansii is a [4Fe-4S] protein

BoxA is the reductase component of the benzoyl-coenzyme A (CoA) oxidizing epoxidase enzyme system BoxAB. The enzyme catalyzes the key step of an hitherto unknown aerobic, CoA-dependent pathway of benzoate metabolism, which is the epoxidation of benzoyl-CoA to the non-aromatic 2,3-epoxybenzoyl-CoA. The function of BoxA is the transfer of two electrons from NADPH to the epoxidase component BoxB. We could show recently that BoxB is a diiron enzyme, whereas here we demonstrate that BoxA harbors an FAD and two [4Fe-4S] clusters per protein monomer. The characterization of BoxA was hampered by severe oxygen sensitivity; the cubane [4Fe-4S] clusters degrade already with traces of oxygen. Interestingly, the adventitiously formed [3Fe-4S] centers could be reconstituted in vitro by adding Fe(II) and sulfide to retrieve the native cubane centers. BoxA is the first example of a reductase of this type that has an FAD and two bacterial ferredoxin-type [4Fe-4S] clusters. In other cases within the catalytically versatile family of diiron enzymes, the related reductases have plant-type ferredoxin or Rieske-type [2Fe-2S] centers only.     

Crystal structures of ferricyanide-oxidized [Fe-S] clusters in Azotobacter vinelandii ferredoxin I

Crystals of Azotobacter vinelandii ferredoxin I (FdI) have been soaked in solutions containing K₃Fe(CN)₆ in order to study the oxidation of the [3Fe-4S] and [4Fe-4S] clusters in the protein. Ferricyanide treatment results in partial loss of Fe and S from each cluster accompanied by alteration of Fe-S bonds. The effects of oxidation can be quantitated by crystallographic refinement when each [Fe-S] cluster is modeled as having a single, average structure with non-standard geometry. The oxidized clusters refined at 2.1-Å resolution display statistically significant deviations from geometric ideality. If interpreted in terms of atomic shifts these deviations indicate that each cluster first loses an inorganic S atom. In each case an Fe atom bonded to this S separates from the remaining atoms of the cluster such that the [3Fe-4S] and [4Fe-4S] clusters partially decompose into a single Fe plus 2Fe and 3Fe fragments. The extent of structural changes observed are essentially the same in crystals soaked at 3?:?1, 9?:?1 and 30?:?1 mole ratio of K₃ Fe(CN)₆?:?FdI, suggesting that the crystal lattice permits limited oxidation reactions to occur at a low mole ratio but restricts conformational changes from occurring that may be required for more extensive oxidative reactions at higher mole ratio. The results are relevant to understanding the transformations which may take place when [Fe-S] proteins are deliberately oxidized with ferricyanide.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.     

Generation of adenosyl radical from S-adenosylmethionine (SAM) in biotin synthase

A mechanism of the C―S bond activation of S-adenosylmethionine (SAM) in biotin synthase is discussed from quantum mechanical/molecular mechanical (QM/MM) computations. The active site of the enzyme involves a [4Fe-4S] cluster, which is coordinated to the COO⁻ and NH₂ groups of the methionine moiety of SAM. The unpaired electrons on the iron atoms of the [4Fe-4S]²⁺ cluster are antiferromagnetically coupled, resulting in the S = 0 ground spin state. An electron is transferred from an electron donor to the [4Fe-4S]²⁺-SAM complex to produce the catalytically active [4Fe-4S]⁺ state. The SOMO of the [4Fe-4S]⁺-SAM complex is localized on the [4Fe-4S] moiety and the spin density of the [4Fe-4S] core is calculated to be 0.83. The C―S bond cleavage is associated with the electron transfer from the [4Fe-4S]⁺ cluster to the antibonding σ* C―S orbital. The electron donor and acceptor states are effectively coupled with each other at the transition state for the C―S bond cleavage. The activation barrier is calculated to be 16.0 kcal/mol at the QM (B3LYP/SV(P))/MM (CHARMm) level of theory and the C―S bond activation process is 17.4 kcal/mol exothermic, which is in good agreement with the experimental observation that the C―S bond is irreversibly cleaved in biotin synthase. The sulfur atom of the produced methionine molecule is unlikely to bind to an iron atom of the [4Fe-4S]²⁺ cluster after the C―S bond cleavage from the energetical and structural points of view.     

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.     

A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from<Emphasis Type="Italic"> Acidaminococcus fermentans</Emphasis>

The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S]²⁺ cluster, 1.0 reduced riboflavin-5′-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S]^1+/2+-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg²⁺-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S]^1+/2+-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as ?405 mV and ?340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7–45 μM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 μmol/g protein at 7 μM Fe to 0.02 μmol/g at 45 μM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 μmol/g at 7–13 μM Fe to 1.1±0.1 μmol/g at 17–45 μM Fe.     

2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction

The conversion of [(14)C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying beta-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min(-1) mg(-1). 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ( approximately 10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP(+), and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 +/- 20 kDa and was composed of three subunits of 63 (gamma), 62 (alpha), and 37 (beta) kDa in a 1:1:1 ratio, suggesting an (alphabetagamma)(2) composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP(+) and oxidized viologens as electron acceptors; in contrast NAD(+) was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP(+), depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The alpha-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the beta-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the gamma-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP(+)-specific KGOR. A similar NADP(+)-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.     

Expression,purification and characterization of an active C491G variant of ferredoxin sulfite reductase from Synechococcus elongatus PCC 7942

Recently developed molecular wire technology takes advantage of [4Fe-4S] clusters that are ligated by at least one surface exposed Cys residue. Mutagenesis of this Cys residue to a Gly opens an exchangeable coordination site to a corner iron atom that can be chemically rescued by an external thiolate ligand. This ligand can be subsequently displaced by mass action using a dithiol molecular wire to tether two redox active proteins. We intend to apply this technique to tethering Photosystem I to ferredoxin sulfite reductase (FdSiR), an enzyme that catalyzes the six-electron reduction of sulfite to hydrogen sulfite and nitrite to ammonia. The enzyme contains a [4Fe-4S]^2+/1+ cluster and a siroheme active site. FdSiR_WT and an FdSiR_C491G variant were cloned from Synechococcus elongatus PCC 7942 and expressed along with the cysG gene from Salmonella typhimurium using the pCDFDuet plasmid. UV/Vis absorbance spectra of both FdSiR_WT and the FdSiR_C491G variant displayed characteristic peaks at 278, 392 (Soret), 585 (α) and 714?nm (charge transfer band), and 278, 394 (Soret), 587 (α) and 714?nm (charge transfer band) respectively. Both enzymes in their as-isolated forms displayed an EPR spectrum characteristic of an S?=?5/2 high spin heme. When reduced, both enzymes exhibited the signal of a low spin S?=?1/2 [4Fe-4S]¹⁺ cluster. The FdSiR_WT and FdSiR_C491G variant both showed activity using reduced methyl viologen and Synechococcus elongatus PCC 7942 ferredoxin 1 (Fd1) as electron donors. Based on these results, the FdSIR_C491G variant should be a suitable candidate for wiring to Photosystem I.     

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.     

Molecular Characterization of a Class I P450 Electron Transfer System from Novosphingobium aromaticivorans DSM12444

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via Arx, a [2Fe-2S] ferredoxin, and ArR, a ferredoxin reductase. These systems show fast NADH turnovers (k_cat = 39–91 s⁻¹) that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx, and CYP101D1, which form a physiological class I P450 electron transfer chain, have been resolved by x-ray crystallography. The general structural features of these proteins are similar to their counterparts in other class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx), and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However, significant differences in the proposed protein-protein interaction surfaces of the ferredoxin reductase, ferredoxin, and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity.     

Iron-sulphur cluster composition and redox properties of two ferredoxins from Desulfovibrio desulfuricans Norway strain

Two ferredoxins from Desulfovibrio desulfuricans, Norway Strain, were investigated by EPR spectroscopy. Ferredoxin I appears to be a conventional [4Fe-4S]^2+;1+ ferredoxin, with a midpoint reduction potential of ?374 mV at pH 8. Ferredoxin II when reduced, at first showed a more complex spectrum, indicating an interaction between two [4Fe-4S] clusters, and probably, has two clusters per protein subunit. Upon reductive titration ferredoxin II changed to give a spectrum in which no intercluster interaction was seen. The midpoint potentials of the native and modified ferredoxin at pH 8 were estimated to be ?500 and ?440 mV, respectively.     

Ferredoxin:thioredoxin Reductase: Disulfide Reduction Catalyzed via Novel Site-specific [4Fe–4S] Cluster Chemistry

Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfide reductases that catalyze disulfide reduction in two one-electron steps using a [2Fe–2S] ferredoxin as the electron donor and an active site comprising a [4Fe–4S] cluster and a redox-active disulfide. This review summarizes structural and spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) and a chemically modified form, termed NEM–FTR, which provides a stable analog of the one-electron reduced catalytic intermediate. Detailed spectroscopic characterization of FTR and NEM–FTR using absorption, EPR, electron–nuclear double resonance, variable-temperature magnetic circular dichroism, resonance Raman and Mössbauer spectroscopies indicate that the one-electron reduced catalytic intermediate involves two-electron disulfide reduction coupled with one-electron cluster oxidation of a [4Fe–4S]²⁺ cluster to yield a unique type of S= 1/2 [4Fe–4S]³⁺ cluster with two cysteine residues ligated at a specific Fe site. The results provide the basis for a novel mechanism for disulfide cleavage in two one-electron steps involving site-specific [4Fe–4S] cluster chemistry. A similar mechanism is proposed for direct [4Fe–4S]-mediated cleavage of the CoM–S–S–CoB heterodisulfide in methanogenic archaea by heterodisulfide reductases.