The electron transport to nitrogenase in Mycobacterium flavum

1. Two ferredoxin-type iron-sulfur proteins have been isolated from Mycobacterium flavum 301 grown under nitrogen-fixing, iron-sufficient conditions. No flavodoxin was observed. 2. These ferredoxins are apparently soluble: they were present in the supernatant fraction after disrupting by decompression. Only small amounts were present in particulate fractions. 3. The two ferredoxins were separated by chromatography on DEAE-cellulose, Sephadex or electrophoresis. 4. Both ferredoxins mediated the transfer of electrons from illuminated spinach chloroplasts to a nitrogenase preparation to reduce acetylene. Ferredoxin II was specifically about five times more active than ferredoxin I. Ferredoxin II was also active in the photosynthetic NADP+-reduction whereas ferredoxin I was not. 5. Both ferredoxins were reversibly reduced by either sodium dithionite, illuminated spinach chloroplasts or hydrogen plus hydrogenase from Clostridium pasteurianum. 6. Attempts to determine the primary electron donor for nitrogen fixation in Mycobacterium flavum were unsuccessful. Acetylene reduction in Mycobacterium extracts was obtained only with sodium dithionite or illuminated spinach chloroplasts as electron donors. The reduction of the electron carrier (e.g. ferredoxin) rather than the transfer of electrons from the reduced carrier to nitrogenase was rate-limiting.     

The stoicheiometry of electron transfer by bacterial and plant ferredoxins

1. The number of electrons carried by ferredoxins from spinach, the blue-green alga Anacystis nidulans, the anaerobic bacterium Clostridium welchii and the photosynthetic bacterium Chromatium was determined. 2. Ferredoxins were reduced by illuminated chloroplasts, and the stoicheiometry of the reoxidation in the dark of the ferredoxins by NADP and benzyl viologen was measured. 3. Spinach and A. nidulans ferredoxins were found to be one-electron carriers, and Cl. welchii and Chromatium ferredoxins were two-electron carriers.     

Electron-transfer reactions between flavodoxin semiquinone and c-type cytochromes: comparisons between various flavodoxins

As an extension of previous work from this laboratory using Clostridium pasteurianum flavodoxin [Tollin, G., Cheddar, G., Watkins, J. A., Meyer, T. E., & Cusanovich, M. A. (1984) Biochemistry 23, 6345-6349], we have measured the rate constants as a function of ionic strength for electron transfer from the semiquinones of Clostridium MP, Anacystis nidulans, and Azotobacter vinelandii flavodoxins to the following oxidants: cytochrome c from tuna and horse, Paracoccus denitrificans cytochrome c2, Pseudomonas aeruginosa cytochrome c-551, and ferricyanide. The rate constants extrapolated to infinite ionic strength (k infinity) for the C. MP flavodoxin are all slightly smaller than for the C. pasteurianum flavodoxin, as would be predicted on the basis of the higher redox potential of the C. MP protein. This indicates that there is a close similarity between the surface topographies of the two proteins in the vicinity of the coenzyme binding site. Moreover, the electrostatic interactions between the two flavodoxins and the various oxidants are also approximately the same. These studies justify our previous use of the crystallographic structure of the C. MP flavodoxin to interpret kinetic results obtained with the structurally uncharacterized C. pasteurianum flavodoxin. Despite their lower redox potentials, both Anacystis and Azotobacter flavodoxins are appreciably less reactive toward all of these oxidants (as much as 2 orders of magnitude in some cases) than are the Clostridium flavodoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the reduction of aliphatic and aromatic nitro compounds by Clostridia, the role of ferredoxin and its stabilization

Crude extracts of a variety of Clostridium species reduce aromatic and aliphatic nitro compounds in the presence of hydrogen gas. Using different Clostridia, the uptake of hydrogen by p-nitrobenzoate is about 5--10 times faster than by 2-nitroethanol. Structurally rather different aliphatic nitro compounds show rates which differ by less than a factor of 3. Hydrogenase from Clostridium kluyveri and ferredoxins from Clostridium spec. La 1 and spinach have been purified. The combinations of the hydrogenase and each one of the ferredoxins catalyse the hydrogen uptake by nitro compounds. Clostridial flavodoxin also transfer electrons onto nitro compounds. Nitroaryl and nitroalkyl compounds behave differently with ferredoxin. The first reduction step (1-electron transfer) of p-nitrobenzoate leads to the nitro radical anion which can be detected by EPR measurements. Nitro alkanes seem to form a rather unstable radical which decomposes partially to form nitrite. Furthermore, 2-(N-hydroxyimino)- and 2-(N-hydroxyamino)ethanol, a nitrogen radical of 2-(N-hydroxyamino)ethanol as well as glycol and 1,4-butanediol were detected as intermediates and side products during the reduction of 2-nitro-ethanol to 2-aminoethanol. While the hydrogenase from Clostridium kluyveri seems not to be affected by any reduction intermediate, the ferredoxin from Clostridium spec. La 1 is inactivated by nitrite in a few minutes. Ferrous and sulfide ions in concentrations substoichiometric to that of nitrite stabilize and even reactivate the ferredoxin in the presence of 2-mercaptoethanol. A mechanism for the reduction of aliphatic nitro compounds catalysed by hydrogenase and ferredoxin is proposed.     

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.     

Ferredoxin from a red alga, Porphyra umbilicalis.

A plant-algal type ferredoxin was isolated from the red alga, Porphyra umbilicalis. In its oxidised form the ferredoxin had absorption maxima at 277, (281), 323, 420 and 462 nm. Two atoms each of non-haem iron and labile sulphur were present per molecule protein. The midpoint potential of the protein was -400 mV and it effectively mediated electron transport in the NADP-photoreduction system of barley. The amino acid composition of Porphyra umbilicalis ferredoxin was determined as (Lys4, His2, Arg1, Asx10, Thr8, Ser7, Glx16-17, Pro3, Gly7, Ala8, Cys5, Val6, Met1, Ile5, Leu8, Tyr5, Phe2). The minimum molecular weight of approximately 11000 was confirmed by sedimentation-equilibrium studies in the analytical ultracentrifuge. Approaching half of the total amino acid sequence was determined by means of an automatic sequencer.     

Apoflavodoxin aggregation following dissociation of flavin

Flavodoxins were isolated for the cyanobacteria Anacystis nidulans and Nostoc strain MAC, and from the red alga Chondrus crispus, and apoflavodoxins prepared by five methods. Gel electrophoretic studies showed that whereas the apoproteins of A. nudulans and Nostoc strain MAC were recovered in monomeric form, the removal of riboflavin 5'-phosphate from C. crispus flavodoxin resulted in extensive aggregation of the apoprotein. In extent and nature this aggregation differed with the dissociating agent used.     

The amino acid sequence of ferredoxin from the red alga Porphyra umbilicalis.

The amino acid sequence of the ferrodoxin of Porphyra umbilicalis was determined by the dansyl-phenyl isothiocyanate method, on peptides obtained by tryptic, chymotryptic and thermolytic digestion of the protein or its CNBr-cleavage fragments. The molecule consists of 98 residues, has an unblocked N-terminus and shows considerable similarity with other plant-type ferredoxins. It is the first reported sequence of a red-algal ferredoxin.     

A constitutive flavodoxin from a eukaryotic alga

We report the isolation and some properties of a flavodoxin from a eukaryotic organism, the naturally occurring red alga $\text{Chondrus crispus}$. Unlike the situation with most other organisms the flavodoxin, under normal growth conditions, is the predominantly formed low-potential electron carrier, an accompanying ferredoxin occurring in only very small amounts. The flavodoxin is of molecular weight 21000 and one mole of FMN is present per mole of protein. Reduction of the flavoprotein proceeds via a blue flavosemiquinone radical. The flavodoxin is active both in photosynthetic NADP reduction by broken chloroplasts, and in phosphoroclastic cleavage of pyruvate by cell-free extracts of $\text{Clostridium pasteurianum}$.     

Properties and structure of the soluble ferredoxin from Synechococcus 6301 (Anacystis nidulans). Relationship to gene sequences.

Photoautotrophic cultures of the unicellular cyanobacterium Synechococcus 6301 (Anacystis nidulans) possessed a single [2Fe-2S] ferredoxin with a midpoint redox potential of -385 mV. Determination of the amino acid sequence of the ferredoxin showed that it consisted of 98 residues, with methionine and tryptophan both absent, and with only the four cysteine residues that are required to co-ordinate the iron-sulphur cluster. Comparisons with other ferredoxin sequences showed that most resemblance was to those from filamentous cyanobacteria, with up to 87% homology. There was less resemblance to the ferredoxins of unicellular cyanobacteria, with 25 differences when compared with that from another Synechococcus sp. However, the sequence of Synechococcus 6301 ferredoxin was identical with that derived for a gene sequence for a putative ferredoxin from the genotypically closely related Synechococcus 7942 (Anacystis nidulans R2). In contrast, the sequence showed substantial differences from that corresponding to a putative ferredoxin gene from Synechococcus 6301 reported by Cozens & Walker [(1988) Biochem. J. 252, 563-569].     

Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC.

Two ferredoxins were isolated from the cyanobacterium Nostoc strain MAC grown autotrophically in the light or heterotrophically in the dark. In either case approximately three times as much ferredoxin I as ferredoxin II was obtained. Both ferredoxins had absorption maxima at 276, 282 (shoulder), 330, 423 and 465 nm in the oxidized state, and each possessed a single 2 Fe-2S active centre. Their isoelectric points were approx. 3.2. The midpoint redox potentials of the ferredoxins differed markedly; that of ferredoxin I was --350mV and that of ferredoxin II was --445mV, at pH 8.0. The midpoint potential of ferredoxin II was unusual in being pH dependent. Ferredoxin I was most active in supporting NADP+ photoreduction by chloroplasts, whereas ferredoxin II was somewhat more active in pyruvate decarboxylation by the phosphoroclastic system of Clostridum pasteurianum. Though the molecular weights of the ferredoxins determined by ultracentrifugation were the same within experimetnal error, the amino acid compositions showed marked differences. The N-terminal amino acid sequences of ferredoxins I and II were determined by means of an automatic sequencer. There are 11--12 differences between the sequences of the first 32 residues. It appears that the two ferredoxins have evolved separately to fulfil different roles in the organism.     

Differential activities of heterocyst ferredoxin,vegetative cell ferredoxin,and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP⁺ reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP⁺, using thylakoids from vegetative cells.Abbreviations BSA bovine serum albumin - FdxH heterocyst ferredoxin - Fld flavodoxin - FNR ferredoxin-NADP⁺ reductase - MV methyl viologen - PetF vegetative cell ferredoxin - PFO pyruvate-ferredoxin oxidoreductase - Pyr piruvate - PS I Photosystem I     

Isoelectric focusing of ferredoxins, flavordoxins and a rubredoxin.

Isoelectric points of ferredoxins, flavodoxins and a rubredoxin from a range of sources were measured by electrofocusing over the pH range between 2.5 and 5.0 on thin layers of polyacrylamide gel. The pH gradient along the gel was measured directly by a surface electrode. The isoelectric points of the plant-type ferredoxins were between approx. 3.15 and 3.35, and those of the flavodoxins close to 3.5. Ferredoxin, rubredoxin and flavodoxin from Clostridium pasteurianum had isolectric points of the of 2.75, 2.9, and 3.1, respectively. The values for the isoelectric points ferredoxins are significantly lower than previous results in the literature suggest.     

Studies on nitrate reductase of Clostridium perfringens. II. Purification and some properties of ferredoxin.

A ferredoxin was purified from Clostridium perfringens by DEAE-cellulose chromatography and Sephadex G-50 gel filtration. It had absorption maxima at 390 and 280 nm. The molecular weight was estimated to be 6,000 by Sephadex gel filtration and from the results of amino acid analysis. The isoelectric point was 3.0. It contained four atoms of iron, four atoms of labile sulfur, and six cysteine residues. This ferredoxin as well as ferredoxin from C. pasteurianum acted as an electron donor for nitrate reductase from C. perfringens. The ferredoxin could also act as an electron donor for the hydrogenase from C. pasteurianum in hydrogen evolution.     

Laser flash photolysis studies of the kinetics of reduction of spinach and Clostridium ferredoxins by a viologen analogue: electrostatically controlled nonproductive complex formation and differential reactivity among the iron-sulfur clusters

We have studied the transient kinetics of electron transfer from a positively charged viologen analogue (propylene diquat), reduced by pulsed laser excitation of the deazariboflavin/EDTA system, to the net negatively charged ferredoxins from spinach and Clostridium pasteurianum. Spinach ferredoxin showed monophasic kinetics over the ionic strength range studied, consistent with the presence of only a single iron-sulfur center. Clostridium ferredoxin at low ionic strength showed biphasic kinetics, which indicates a differential reactivity of the two iron-sulfur centers of this molecule toward the electron donor. The kobsd values for the initial fast phase observed with Clostridium ferredoxin were ionic strength dependent, whereas the slow-phase kinetics were ionic strength independent. This correlates with the highly asymmetric charge distribution on the surface of the bacterial protein relative to the two iron-sulfur clusters. The kinetics corresponding to spinach ferredoxin reduction were also ionic strength dependent, and the results obtained with these kinetics and with the fast phase of the bacterial ferredoxin reduction were consistent with a mechanism involving electrostatically stabilized complex formation. For spinach ferredoxin, the second-order rate constant extrapolated to infinite ionic strength was 2-fold smaller, and the extrapolated limiting first-order rate constant was 10-fold smaller, than for Clostridium ferredoxin, indicating a smaller intrinsic reactivity of the spinach protein toward the electron donor. Differences in the rate constant values and the ionic strength dependencies with both ferredoxins are consistent with differences in cluster structure and environment and protein size and charge distribution. For both proteins, the total amount of ferredoxin reduced increased with the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural similarities between the N-terminal domain of Clostridium pasteurianum hydrogenase and plant-type ferredoxins

An N-terminal domain of Clostridium pasteurianum hydrogenase I, encompassing 76 residues out of the 574 composing the full-size enzyme, had previously been overproduced in Escherichia coli and shown to form a stable fold around a [2Fe-2S] cluster. This domain displays only marginal sequence similarity with [2Fe-2S] proteins of known structure, and therefore, two-dimensional 1H NMR has been implemented to elucidate features of the polypeptide fold. Despite the perturbing presence of the paramagnetic [2Fe-2S] cluster, 57 spin systems were detected in the TOCSY spectra, 52 of which were sequentially assigned through NOE connectivities. Several secondary structure elements were identified. The N terminus of the protein consists of two antiparallel beta strands followed by an alpha helix contacting both strands. Two additional antiparallel beta strands, one of them at the C terminus of the sequence, form a four-stranded beta sheet together with the two N-terminal strands. The proton resonances that can be attributed to this beta2alphabeta2 structural motif undergo no paramagnetic perturbations, suggesting that it is distant from the [2Fe-2S] cluster. In plant- and mammalian-type ferredoxins, a very similar structural pattern is found in the part of the protein farthest from the [2Fe-2S] cluster. This indicates that the N-terminal domain of C. pasteurianum hydrogenase folds in a manner very similar to those of plant- and mammalian-type ferredoxins over a significant part (ca. 50%) of its structure. Even in the vicinity of the metal site, where 1H NMR data are blurred by paramagnetic interactions, the N-terminal domains of hydrogenase and mammalian- and plant-type ferredoxins most likely display significant structural similarity, as inferred from local sequence alignments and from previously reported circular dichroism and resonance Raman spectra. These data afford structural information on a kind of [2Fe-2S] cluster-containing domain that occurs in a number of redox enzymes and complexes. In addition, together with previously published sequence alignments, they highlight the widespread distribution of the plant-type ferredoxin fold in bioenergetic systems encompassing anaerobic metabolism, photosynthesis, and aerobic respiratory chains.     

Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.     

A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.     

Redox potentials of algal and cyanobacterial flavodoxins.

The redox potentials of flavodoxins from the cyanobacteria Synechococcus PCC 6301 (formerly Anacystis nidulans) and Nostoc strain MAC, and from the red alga Chondrus crispus, were determined by potentiometric titration. For the oxidized-semiquinone interconversion the potentials at pH 7.0 of the three flavodoxins were between -210 and -235 mV, and these were pH-dependent over the range pH 6.9-8.2. For the semiquinone-reduced interconversion the potentials of the cyanobacterial flavodoxins were close to -414 mV, and that for the algal flavodoxin, -370 mV, is the highest reported in this group of flavoproteins.