Preliminary X-ray diffraction studies on a ferredoxin from the thermophilic archaebacterium,Thermoplasma acidophilum

A ferredoxin from the thermophilic archaebacterium, Thermoplasmaacidophilum, is supposed to contain two (4Fe-4S) active centers; one center could be linked by four cysteine residues to the protein and the other bonded with three cysteines and an unknown group. This ferredoxin has been crystallized by salting-out against 2.3 m-ammonium sulfate solution. The space group is P2₁2₁2 with cell dimensions of $\text{a = 59.20}\text{A}\text{?}\text{, b = 52.77}\text{A}\text{?}\text{and}\text{c = 41.28}\text{A}\text{?}$. Four molecules pack in the unit cell with $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.03}\text{A}\text{?}{}^3\text{/dalton}$.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

In 80% dimethyl sulfoxide/H₂O, $\text{Azotobacter}$ ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the $\text{Azotobacter}$ ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.     

Electron transport components from methanogenic bacteria: The ferredoxin from Methanosarcinabarkeri (strain Fusaro)

A ferredoxin has been isolated from the methanogenic organism $\text{Methanosarcina}\text{barkeri}$ (strain Fusaro). The protein appears to be constituted by two identical subunits of molecular weight approx. 6000 daltons. The UV-visible spectrum of the protein is characterized by two broad absorption peaks centered at 410 and 300 nm and an absorbance ratio $\text{A}{}_{410}\text{A}{}_{300}\text{= 0.8}$. The molar extinction coefficients at 410 and 300 nm are 36,500 and 45,625 M^?1 cm^?1, respectively. The amino acid compsition of $\text{M.}\text{barkeri}$ ferredoxin shows a preponderance of acidic residues and lacks five amino acids. The protein contains 8 cysteine residues and approx. 7 iron atoms and 7–8 acid-labile sulfide groups per molecule which are indicative of the presence of two iron-sulfur clusters in the molecule. The N-terminal sequence shows a high degree of homology with the sequences of ferredoxins from $\text{Clostridium}\text{pasteurianum}\text{,}\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{africanus}\text{.}\text{M.}\text{barkeri}$ ferredoxin functions as an electron carrier in the pyruvate dehydrogenase system. Its possible role in a variety of electron transfer reactions is discussed.     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.     

Amino acid sequence of ferredoxin II from the phototroph Rhodospirillum rubrum: Characteristics of a 7Fe ferredoxin

The complete sequence of amino acids of ferredoxin II (FdII) from Rhodospirillum rubrum was determined by repetitive Edman degradation using pyridylethylated-ferredoxin and oxidized, denatured ferredoxin. Peptides derived from trypsin, pepsin, Glu-C endoproteinase, Arg-C endoproteinase, tryptophan specific cleavage and partial acid hydrolysis and C-terminal sequence from carboxypeptidase digestion were used to construct the total sequence. RrFdII is a polypeptide of 104 amino acids having a calculated molecular weight of 11556 excluding the iron and sulfur atoms. The complete amino acid sequence was: PYVVTENCIKCKYQDCVEVCPVDCFYEGENFLVINPDECIDCGVCNPECPAEAIAGKWLEINRKFADLWPNITRKGPAL ADADDWKDKPDKTGLLSENPGKGTV. Sequence comparisons, EPR characteristics and iron analyses indicate that RrFdII has structural features in common with ferredoxins containing [3Fe-4S], [4Fe-4S] centers. Of 104 amino acids, 60 (58%) including all 9 cysteines, are found in identical locations in the 7Fe ferredoxin prototype, Azotobacter vinelandii FdI.The protein sequence data reported in this paper will appear in the SWISS-PROT database and EMBL Data Library under the accession number P80448.     

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans

A two cluster (4Fe4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins.The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an absorbance ratio of $\text{A}{}_{385}\text{A}{}_{283}\text{= 0.74}$. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52°C.The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.     

The biosynthesis of phosphorylated tyrosine hydroxylase by organ cultures of rat adrenal medulla and superior cervical ganglia: a correction.

Determination of the complete amino acid sequence of the rubredoxin isolated from the sulfate reducing bacterium $\text{Desulfovibrio}$$\text{gigas}$ showed that the molecule consists of a single polypeptide chain of 52 residues. The sequence of the first 42 residues was determined using an automatic Protein Sequencer. Peptides derived from tryptic hydrolysis and from specific cleavage at tryptophan residue were used to construct the total sequence. Compared with the sequence of $\text{Desulfovibrio}$$\text{vulgaris}$ rubredoxin, 37 positions are identical, and with the sequences of $\text{Clostridium}$$\text{pasteurianum}$, $\text{Peptostreptococcus}$$\text{elsdenii}$, $\text{Micrococcus}$$\text{aerogenes}$ and $\text{D.}$$\text{vulgaris}$ rubredoxins, 20 matching residues occur. A crystallographic study of the $\text{D.}$$\text{gigas}$ rubredoxin is in progress.     

Structure of rhodotorucine A, a novel lipopeptide,inducing mating tube formation in Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ is a peptidyl factor which induces mating tube formation in $\text{Rhodosporidium}\text{toruloides}$. The amino acid sequence of the factor was determined by Edman degradation and enzymatic hydrolysis. Rhodotorucine $\text{A}$ was shown to contain a lipophilic amino acid, S-farnesyl cysteine, at C-terminus by proton magnetic resonance, mass spectrometry and chemical synthesis. We proposed the following structure for rhodotorucine $\text{A}$. H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-farnesyl)-OH     

An immune complex assay for SV40 T antigen.

The two forms of ferredoxin from $\text{Desulfovibrio gigas}$, Fd I and Fd II, are studied by differential pulse polarography 1. Fd I and Fd II give one well defined peak corresponding to $\text{E}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= ?0.33}\text{and}\text{?0.35}$ V (vs. the hydrogen electrode) respectively, at c > 5 μM. The influence of the concentration on the peak potentials Ep and the peak heights i_p is examined. The denaturation of the two forms of ferredoxin is studied by polarography in conjunction with UV spectrophotometry. Two new peaks at negative potentials before the reduction of the solvent are observed in denaturated proteins.     

Properties of the primary electron acceptor complex of photosystem I in the blue green alga Chlorogloea fritschii.

Photooxidation of P700 at low temperatures in membrane fractions from the blue-green alga $\text{Chlorogloea fritschii}$ may be coupled irreversibly to the reduction of a bound ferredoxin. If this ferredoxin is reduced before freezing, P700 photooxidation at low temperatures becomes reversible. This reversible photooxidation is coupled to the reduction of a component with an EPR signal at g = 2.08, 1.88 and 1.78. A complete spectrum of this component has been obtained for the first time. We propose that as in higher plants this component is the primary electron acceptor of Photosystem I, the bound ferredoxin is a secondary electron acceptor. Using ⁵⁷Fe enriched preparations we have shown that the ERP signals attributed to the bound ferredoxin are due to iron containing centres. This experiment did not show the presence of iron in the primary electron acceptor.     

The amino acid sequence of desulforedoxin, a new type of non heme iron protein from Desulfovibrio gigas

A new type of non heme iron protein called desulforedoxin has been isolated from the sulfate reducing bacterium, $\text{Desulfovibrio gigas}$. The complete amino acid sequence has been established. The 36 amino acid residues of the sequence are aligned with the aid of peptides obtained by cyanogen bromide cleavage and by hydrolysis with a peptidase isolated from $\text{Staphylococcus aureus}$. Desulforedoxin has been described as a non heme iron protein of molecular weight 7,600 with 2 iron atoms linked to eight cysteine residues. In fact, sequence elucidation shows that it consists of a dimer of a peptide containing 36 aminoacids. We do not know whether if each monomer contains 1 iron atom linked to 4 cysteine residues or whether the two iron cross link the two monomers. Additional studies on the elucidation of the structure of this new cluster are presently under study.     

The detection of a bound ferredoxin in the photosynthetic lamellae of blue-green algae and other oxygen evolving photosynthetic organisms

The presence of an electron transport component with an EPR spectrum similar to that of a ferredoxin has been demonstrated in the blue-green alga $\text{Anabaena}$$\text{cylindrica}$, the green alga $\text{Euglena}$$\text{gracilis}$, and in chloroplasts from sorghum ($\text{Sorghum}$$\text{bicolour}$) and beans ($\text{Phaseolus}$$\text{vulgaris}$). The component is photoreduced at 77°K and is very similar to that previously reported in spinach. It seems likely that this component is a primary electron acceptor in photosynthesis in all of these organisms.     

Relationship between P700 turnover (m second) and NADP reduction as a function of ferredoxin concentration in isolated broken chloroplasts

Steady-state electron flux through P₇₀₀ (t $\text{1}\text{2}$ 20 msec) and concomitant rate of NADP reduction have been measured under weak actinic illumination as a function of concentration of ferredoxin added to broken chloroplasts isolated from peas. At suboptimal concentrations of ferredoxin this P₇₀₀ is not sufficient to account for the NADP reduction. At high concentrations ferredoxin inhibits the rate of NADP reduction without affecting the P₇₀₀ flux under short wavelength illumination. Under far red illumination P₇₀₀ flux is also inhibited by ferredoxin at high concentrations. Addition of 5 mM Mg⁺⁺ increases the rate of NADP reduction at all concentrations of ferredoxin under both kinds of illumination, while P₇₀₀ flux is inhibited under short wavelength illumination and remains unchanged under far red illumination. The results indicate that the observed (20 msec) P₇₀₀ is not involved in NADP reduction.     

Chemical modification of spinach ferredoxin: evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction.

Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome $\text{c}$. The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.     

Low-temperature magnetic circular dichroism evidence for the conversion of four-iron-sulphur clusters in a ferredoxin from Clostridium pasteurianum into three-iron-sulphur clusters

Oxidation of the 8Fe ferredoxin from Clostridium pasteurianum with potassium ferricyanide, followed by purification on Sephadex G-25 and DE-23 cellulose columns, gives a protein with an intense EPR signal at g 2.01. The low-temperature magnetic circular dichroism (MCD) spectra of this species are different from those of the oxidized high-potential iron protein from Chromatium but identical with the spectra of ferredoxin II from Desulphovibrio gigas. On reduction of the ferricyanide-treated ferredoxin with sodium dithionite only a weak EPR signal with g factors of 2.05, 1.94 and 1.89 is obtained. The low-temperature MCD spectra are strongly temperature dependent with a form similar to those of dithionite-reduced D. gigas ferredoxin II. The MCD magnetization curves are dominated by a species with ground-state effective g factors of $\text{g}{}_{\mathrm{?}}$ 8.0 and g_⊥ 0.0, which are also similar to those determined recently by low-temperature MCD spectroscopy for D. gigas ferredoxin II. The MCD characteristics are quite different from those of dithionite-reduced ferredoxin from Cl. pasteurianum, untreated with ferricyanide. This establishes the close similarity of the iron-sulphur clusters in ferricyanide-treated Cl. pasteurianum ferredoxin and in D. gigas ferredoxin II. The latter is known to contain a single 3Fe centre, similar to that observed in ferredoxin I from Azotobacter vinelandii by X-ray crystallography. Therefore, it is concluded that the [4Fe-4S] clusters of Cl. pasteurianum ferredoxin are converted to 3Fe clusters on oxidation with ferricyanide.     

Preliminary X-ray diffraction studies on a [4Fe4S] ferredoxin from Bacillus thermoproteolyticus

A [4Fe4S] ferredoxin from Bacillus thermoproteolyticus has been crystallized. The space group is P1 with two molecules in the unit cell, with the dimensions $\text{a = 32.96}\text{A}\text{?}\text{, b = 37.83}\text{A}\text{?}\text{, c = 39.82}\text{A}\text{?}\text{, α = 118.1 °, β = 104.2 °}\text{and}\text{γ = 89.7 °}$. The Bijvoet-difference Patterson map of the native crystal shows up a prominent peak of [4Fe4S] cluster.     

Reaction of p-nitrophenyl trimethylacetate with yeast carboxypeptidase. Evidence for an acyl-enzyme intermediate

The kinetics of the hydrolysis of $\text{p}$-nitrophenyl trimethylacetate catalyzed by yeast carboxypeptidase have been measured under conditions of substrate in excess and indicate that the release of $\text{p}$-nitrophenol in two discrete stages can be observed. A fast release of $\text{p}$-nitrophenol in a concentration approximating that of the enzyme is seen initially, followed by a slow release, corresponding to the “turnover” reaction of the ester. These observations provide strong support for the postulation of a three step reaction sequence including the formation and decomposition of not only a Michaelis complex but also an acyl-enzyme species.