Absence Of Ferredoxin Research Articles

Ferredoxin and flavodoxin in eastern Antarctica pack ice

The presence, concentration and distribution of the iron regulated proteins, ferredoxin and flavodoxin, was investigated in pack ice off eastern Antarctica using SDS-PAGE gels. Bands corresponding to ferredoxin and/or flavodoxin were observed in all but eight of the 102 core sections analysed. Flavodoxin was found in most of the ice samples and was strongly correlated with chlorophyll a standing stock. The widespread distribution of flavodoxin here is not thought to indicate iron-limitation as many of the dominant species, such as Fragilariopsis cylindrus, Cylindrotheca closterium, are known to produce this protein under iron-replete conditions and thus the significant correlation between flavodoxin and biomass is likely to be the result of widespread constitutive flavodoxin expression among the diatoms that commonly inhabit sea ice. High concentrations of ferredoxin were predominantly derived from core sections on the floes closest to the continent and also in the upper portion of these floes. There was a consistent lack of ferredoxin expression in the high biomass bottom communities. The absence of ferredoxin is likely to indicate a reduced supply of iron but the significance of this reduced iron supply cannot be inferred on the basis of protein expression alone. Furthermore, in the present study the observed variability in the flavodoxin:ferredoxin ratio may not simply reflect the iron nutritional status of the community, but probably results from changes in the abundance of species capable of expressing ferredoxin.

Metabolic activation of mitomycin C by NADPH-ferredoxin reductase in vitro

Mammalian NADPH-ferredoxin reductase (EC 1.18.1.2) functions in the mitochondrial electron transport chain for cytochrome P-450-dependent steroid hydroxylation. Significant homology of three-dimensional structure exists in the surroundings of FAD between NADPH-ferredoxin reductase and NADH-cytochrome b 5 reductase. The latter is involved in the bioreduction of mitomycin C (MC), a prototype antitumor agent. In this study, we assessed the capacity of NADPH-ferredoxin reductase to activate MC. Mitomycin C increased the NADPH oxidase activity of NADPH-ferredoxin reductase. In the absence of ferredoxin, the K m value of NADPH-ferredoxin reductase for MC was 73.5 ± 2.3 μM. While in the presence of 500 nM ferredoxin, a Lineweaver-Burk plot exhibited a biphasic curve. NADPH-ferredoxin reductase-mediated reduction of MC resulted in the formation of an alkylated complex of 4-(p-nitrobenzyl) pyridine and an increase in plasmide DNA single-strand breaks under hypoxic conditions. With the addition of 500 nM ferredoxin, the amount of the alkylated complex of 4-(p-nitrobenzyl) pyridine and the plasmide DNA single-strand breaks increased by 40% and 37%, respectively. However, neither alkylated complex of 4-(p-nitrobenzyl) pyridine nor DNA strand breaks was observed in the presence of SOD and catalase under aerobic conditions. These findings demonstrate that NADPH-ferredoxin reductase is capable of catalyzing the bioactivation of mitomycin C under hypoxic conditions in vitro.

Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates.

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5alphaF'(pOD22) and DH5alphaF'(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (beta-ISP) and 48,243 Da (alpha-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.

PsaC Subunit of Photosystem I Is Oriented with Iron-Sulfur Cluster F B as the Immediate Electron Donor to Ferredoxin and Flavodoxin

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F A and F B, functioning as electron carriers between F X and soluble ferredoxin. To resolve the issue whether F A or F B is proximal to F X, we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F A and F B present) and HgCl 2-treated (F B-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700 + reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700 + to be reduced from PMS. In F B-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700 + was reduced from PMS only on the first flash and was reduced from F X − on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F B-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700 + and F A − in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F A − reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F A − could be reoxidized by methyl viologen in F B-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F B cluster. Using rate versus distance estimates for electron transfer rates from F X to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F A as the cluster proximal to F X and F B as the distal cluster that donates electrons to ferredoxin.

Mössbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum.

We have purified to homogeneity the 88-kDa corrinoid protein from Clostridium thermoaceticum which acts as a methyl carrier in the synthesis of acetyl-CoA. As shown here, this protein contains a [4Fe-4S]1+/2+ cluster in addition to a corrinoid. The corrinoid is 5-methoxybenzimidazolylcobamide, with an OH- group probably present as the upper axial ligand. Co+ is present in the reduced form, Co2+ in the as-isolated form, and Co3+ in the methylated form of the protein. The as-isolated corrinoid/Fe-S protein exhibits a Co2+ EPR signal lacking nitrogen superhyperfine splittings, indicating that the benzimidazole base is uncoordinated ("base-off") in the Co2+ state. Optical studies suggest that the Co3+-CH3 corrinoid is also base-off. In the as-isolated and methylated forms, the iron-sulfur cluster is diamagnetic, with quadrupole splittings and isomer shifts characteristic of [4Fe-4S]2+ clusters. The protein can be reduced by CO and CO dehydrogenase in the absence of ferredoxin. The EPR spectra of the reduced cluster exhibit two components: one with principal g-values at 2.07, 1.93, and 1.82 and the other at 2.02, 1.94, and 1.86. The Mössbauer data show that these signals result from [4Fe-4S]1+ clusters. Chemical analysis shows that the iron:cobalt atomic ratio is close to 4:1, suggesting that a single [4Fe-4S]1+ cluster occurs in two distinct S = 1/2 spin states in the reduced state. Treatment with 1-2.5 M urea converts the two cluster forms into a single one, with EPR and Mössbauer spectra of typical [4Fe-4S]1+ clusters. A 27-kDa corrinoid protein (Ljungdahl, L.G., LeGall, J., and Lee, J.P. (1973) Biochemistry 12, 1802-1808) also was purified and found to be inactive in the synthesis of acetyl-CoA, contrary to the suggestion of Ljungdahl et al. (1973).

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.

Oxygen evolution by a reconstituted spinach chloroplast system in the presence ofl-glutamine and 2-oxoglutarate

Intact chloroplasts prepared from summer-grown spinach plants supported (aspartate plus 2-oxoglutarate)-dependent O2 evolution but not (glutamine plus 2-oxoglutarate)-dependent O2 evolution. The former activity, which was sensitive to amino oxyacetate, was attributed to transaminase activity and reduction of the resulting oxalo-acetate to malate using H2O as eventual electron donor. A reconstituted chloroplast system which included chloroplast stroma, thylakoid membranes, ferredoxin and NADP(H) supported O2 evolution in the presence ofL-glutamine and 2-oxoglutarate at rates of 15-22 μmol mg(-1) chlorophyll h(-1) although lower rates were obtained with material from winter-grown plants. Activity was not observed in the absence of ferredoxin and omission of NADP(H) decreased activity by 40%. The reaction was associated with the production of 0.49 mol O2 mol(-1) 2-oxoglutarate consumed and up to 0.46 mol O2 mol(-1) glutamine supplied. The reaction, which was inhibited by azaserine but not by methionine sulphoximine or amino oxyacetate, was attributed to light-coupled glutamate synthase (EC 1.4.1.13) with H2O serving as eventual electron donor. Activity was not affected significantly byL-malate. The reconstituted system also supported O2 evolution in the presence of nitrite, oxaloacetate, (aspartate plus 2-oxoglutarate) and oxidised glutathione.

Oxidation-reduction potentials of the hemes in cytochrome c3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.

Oxygen Activation in Isolated Chloroplasts. Mechanism of Ferredoxin-Dependent Ethylene Formation from Methionine

Low-potential electron acceptors of photosystem I of chloroplast lamellae produce superoxide anions (0-2) and hydrogen peroxide by autoxidation, but have no effect on ethylene formation from methionine; equimolar amounts of ferredoxin are less active in photosynthetic O-2 and H2O2 production but strongly stimulate ethylene production from methionine. 2. Ten to fifty units of superoxide dismutase inhibit fifty to two hundred units of superoxide dismutase stimulate ethylene formation from methionine by chloroplast lamellae in the presence of ferredoxin. This stimulation is stronger at pH 7.0 than at pH 7.8. Catalase inhibits ethylene formation from methionine. 3. Pulse-radiolytic production of nitrite (NO-2) from hydroxylamine, initiated by hydroxyl radicals (.OH) or O-2, shows no difference in the presence or absence of ferredoxin, nor do the decay kinetics of O2. 4. From the above observations and from model reactions (xanthine/xanthine oxidase; iron salts in the presence of H2O2), it is concluded that reduced ferredoxin in the presence of H2O2 forms a Fenton-type oxidizing species for methionine, generating ethylene in the presence of pyridoxal phosphate. 5. Inhibitory effects of both superoxide dismutase and catalase in oxygen-dependent reactions need not necessarily indicate the participation of the 'Haber-Weiss' reaction.

Purification and Properties of Reduced Ferredoxin: CO2 Oxidoreductase from Clostridium pasteurianum, a Molybdenum Iron‐Sulfur‐Protein

Reduced ferredoxin: CO2 oxidoreductase from Clostridium pasteurianum, a ferredoxin‐dependent soluble formate dehydrogenase, was purified 3000‐fold with an overall yield ranging between 10 and 20%. The enzyme was at least 85% pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by the molybdenum content. The enrichment was performed in the presence of 10 mM azide, a competitive inhibitor of the enzyme, and under strictly anaerobic conditions (E∼–400 mV) in order to protect the CO2 reductase from rapid inactivation. The purification procedure included a heat step, a precipitation by polyethyleneimine and by ammonium sulfate, a sedimentation by ultracentrifugation, and a chromatography on DEAE‐cellulose in 39% ammonium sulfate.The highly purified CO2 reductase contained 7.2 nmol of molybdenum, 170 nmol of non‐heme iron and 150 nmol of acid‐labile sulfur per mg of protein. The spectrum of the enzyme was typical of an iron‐sulfur protein. Selenium, tungsten, flavins or heme could not be detected in significant amounts. The enzyme consisted of a number of aggregated forms with molecular weights near 700000, 460000, and 240000 which could be partially dissociated in the presence of 1% Triton X‐100 into an enzymatically active species of molecular weight 118000. Treatment with sodium dodecl sulfate led to a dissociation of the enzyme into two enzymatically inactive polypeptide chains of molecular weights near 34000 and 86000. The purified enzyme will catalyze the following reactions at the same relative rates as the cell‐free extracts: (a) the reduction of CO2 to formate with reduced ferredoxin, (b) the oxidation of formate to CO2 with oxidized ferredoxin, (c) an isotopic exchange between 14CO2 and formate in the absence of ferredoxin, and (d) the reduction of methyl viologen with formate.It is concluded that CO2 reductase from C. pasteurianum is a molybdenum iron‐sulfur protein containing 1 mol of molybdenum, and 24 mol of non‐heme iron and acid‐labile sulfur per mol enzyme of molecular weight 118000.

Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.

The Active Species of ‘CO2’ Utilized by Reduced Ferredoxin: CO2 Oxidoreductase from Clostridium pasteurianum

Reduced ferredoxin:CO2 oxidoreductase (CO2 reductase) from Clostridium pasteurianum catalyzes the reduction of 'CO2' to formate with reduced ferredoxin, an isotopic exchange between 'CO2' and formate in the absence of ferredoxin, and the oxidation of formate to 'CO2' with oxidized ferredoxin. The active species of 'CO2', i.e. CO2 or HCO3 (H2CO3), utilized by the enzyme was determined. The method employed for the species identification was that of Copper et al. (1968). Both 'CO2' reduction to formate and the exchange reaction were studied. Data were obtained which are compatible with those expected if CO2 is the active species. The V and the dissociation constant Ks of the enzyme - CO2 complex in dependence of pH were determined from initial velocity studies of the exchange reaction. V was found to be only slightly affected by pH between 5.5 and 7.5. Ks was markedly dependent on pH; the constant increased with decreasing pH from 0.2 mM at pH 7.5 to 3 mM at pH 5.5.

Reduced Ferredoxin:CO2Oxidoreductase FromClostridium pasteurianum.Effect of Ligands to Transition Metals on the Activity and the Stability of the Enzyme

Reduced ferredoxin: CO2 oxidoreductase (CO2-reductase) from Clostridium pasteurianum catalyzes the reduction of CO2 to formate at the expense of reduced ferredoxin, an isotopic exchange between CO2 and formate in the absence of ferredoxin, and the oxidation of formate to CO2 with oxidized ferredoxin. The three activities were found to be equally affected by monovalent anions known to be ligands to transition metals: The enzyme was reversibly inhibited by azide (Ki = 0.004mM), cyanate (Ki = 0.3 mM), thiocyanate (Ki = 1mM), nitrite (Ki = 0.4mM), nitrate (Ki = 6mM), chlorate (Ki = 3mM), fluoride (Ki = 5mM), and by chloride, bromide, iodide (Ki greater than 5mM). There was no observable effect of pH on the inhibition constants. The enzyme was not inhibited by carbon monoxide. The enzyme was irreversibly inactivated by low concentrations (10muM) of cyanide. The rate of inactivation increased with increasing pH with an inflection point near pH 9.5. Reduced ferredoxin and formate rather than oxidized ferredoxin or CO2 protected the enzyme from inactivation by cyanide. The enzyme was protected by azide and cyanate from inactivation. In the presence of high concentrations of the monovalent anions the rate of inactivation by heat (55 degrees C), by molecular oxygen, and by cyanide was decreased by a factor of more than 100. Half maximal protection was observed at the Ki concentrations of the two reversible inhibitors. The data are interpreted to indicate that a transition metal of weak "a class" character and a disulfide are catalytically significant groups of CO2-reductase from C. pasteurianum.

Studies on Aggregated Multienzyme Systems: STIMULATION OF OXYGEN UPTAKE OF FERREDOXIN-NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE REDUCTASE-FERREDOXIN COMPLEX BY CYTOCHROME c

Abstract The rate of oxygen uptake of spinach ferredoxin-NADP reductase-ferredoxin complex is increased up to 20-fold by the addition of cytochrome c. Initiation of epinephrine and sulfite oxidation indicated an involvement of the superoxide anion radical in the stimulated oxidase reaction. The final product of the reaction was shown to be H2O instead of H2O2, which is the product of the flavoprotein-catalyzed oxidase reaction in the absence of ferredoxin. In the stimulated oxidase reaction, the electron-donating site to molecular oxygen, accordingly the site for the superoxide anion radical generation, has been ascribed to the iron-sulfur moiety of the reductase-ferredoxin complex rather than to the flavoprotein or cytochrome c. Ferredoxin-NADP reductase was found to possess a cytochrome c reductase activity in the absence of ferredoxin, and this activity was increased under anaerobic conditions. Cytochrome c reduction mediated by the superoxide anion radical was negligible both in the absence and presence of ferredoxin. The stimulation was independent of the oxidation-reduction state of cytochrome c, and has been concluded to be a result of an interaction of cytochrome c with the ferredoxin component of the complex with a Michaelis constant of approximately 0.8 µm. These results suggest that the binding of cytochrome c molecule to the reductase-ferredoxin complex dramatically alters its catalytic activity toward molecular oxygen.