Non-heme Iron Atom Research Articles

Purification and Properties of Rat Liver Microsomal Stearyl Coenzyme A Desaturase

The terminal enzyme of the NADH-dependent stearyl coenzyme A desaturase system has been isolated from rat liver microsomes. This desaturase is a single polypeptide of 53,000 daltons containing 62% nonpolar amino-acid residues and one atom of non-heme iron. The purified protein forms high molecular weight aggregates that can be dispersed by detergent procedures. Desaturase activity requires NADH, stearyl coenzyme A, oxygen, lipid, and the three enzymes, cytochorme b(5) reductase (EC 1.6.2.2), cytochrome b(5), and desaturase. Cytochrome b(5) is the direct electron donor to the desaturase, which appears to utilize the iron in the oxidation-reduction sequence during desaturation of stearyl coenzyme A.

Ribonucleoside Diphosphate Reductase Induced by Bacteriophage T4: I. PURIFICATION AND CHARACTERIZATION

Abstract Ribonucleoside diphosphate reductase induced in Escherichia coli after infection with bacteriophage T4 was prepared in an essentially pure state by a method involving among other things affinity chromatography on dATP-Sepharose. The enzyme has a molecular weight of approximately 225,000 as determined by a combination of gel filtration and velocity sedimentation techniques. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates the presence of equimolar amounts of two polypeptide chains, α and β, with molecular weights of about 85,000 and 35,000, respectively. It is suggested that the native enzyme has the subunit structure α2β2. This structure is analogous to that of the active form of E. coli ribonucleotide reductase. α2 corresponds in size to protein B1 and β2 to protein B2. In contrast to the bacterial enzyme the phage enzyme does not dissociate into α2 and β2 on purification. T4 ribonucleotide reductase contains 2.3 atoms of non-heme iron per molecule. The light absorption spectrum shows a sharp peak at 410 nm, and the electron spin resonance spectrum shows a doublet structure centered at g = 2.00. Both spectra are very similar to those of protein B2 of the E. coli ribonucleotide reductase. The reduction of ribonucleoside diphosphates shows absolute requirements for NADPH, T4 thioredoxin, and E. coli thioredoxin reductase. The reaction is stimulated 2- to 3-fold by MgCl2.

Structure of the Iron-Sulfur Cluster in the Chromatium Iron Protein at 2.25 Resolution

The high-potential iron-sulfur protein (HiPIP or HiPISP) of Chromatium vinosum, strain D, is representative of a widely occurring and highly diverse class of oxidation-reduction proteins, each of which contains one or more non-heme iron atoms and a variable complement of inorganic sulfide and cysteine. HiPIP consists of an 86 residue polypeptide chain (Dus et al., 1971) with four cysteines, and an inorganic prosthetic group composed of four sulfides and four iron atoms bound to the apoprotein by the cysteine sulfur atoms.

Purification and properties of adenylyl sulfate reductase from the phototrophic sulfur bacterium, Thiocapsa roseopersicina.

Adenylyl sulfate reductase was purified from Thiocapsa roseopersicina 60- to 80- fold, and the properties were studied. The molecular weight is 180,000. The enzyme contains, per molecule; one flavine group, two heme groups of cytochrome c character, four atoms of nonheme iron, and six labile sulfide groups. Cytochrome c and ferricyanide serve as electron acceptors. With ferricyanide as the electron acceptor, the pH optimum of the enzyme is at 8.0; with cytochrome c, the pH optimum is at 9.0. Of the nucleotides studied, adenosine 5'-monophosphate is most effective. The influence of substrate concentrations on the activity of the enzyme was studied, and the K(m) values for sulfite, adenosine 5'-monophosphate, ferricyanide, and cytochrome c were determined. The properties of the enzyme are compared with those of adenylyl sulfate reductases purified from sulfate-reducing bacteria and thiobacilli.

Ferredoxin aus Bumilleriopsis filiformis Vischer

Ferredoxin from the alga Bumilleriopsis filiformis Vischer (Xanthophyceae) is characterized by comparing some of its properties with spinach ferredoxin.It is similar to the known plant ferredoxins in the following points: a) Absorption spectrum. b) Molecular weight. c) Amino acid composition (approx. 100 amino acid residues). d) Uptake of 1 electron upon complete reduction and a 50 per cent decrease of the optical density at 420 nm. e) 2 non-heme iron atoms per molecule, which do not exchange with an iron isotope. f) SH-groups of the native protein do not react with DTNB. Bumilleriopsis ferredoxin differs from known plant ferredoxins in the following points: a) Greater stability towards atmospheric oxygen when gently heated. b) Only 1 detectable acid-labile sulfur atom per molecule. c) Dialysis against EDTA: by this procedure a part of the iron but none of the acid-labile sulfur is removed. With spinach ferredoxin a part of both elements is removed simultaneously. d) Greater reactivity with ferredoxin-NADP reductase from Bumilleriopsis than with that from spinach. With spinach ferredoxin no substantial difference between the reaction rates of the two reductases is observed (see Böger, 1969 b). The results may indicate an active center in Bumilleriopsis ferredoxin consisting of 1 atom iron and 1 atom acid-labile sulfur.

Properties of azoferredoxin purified from nitrogen-fixing extracts of Clostridium pasteurianum

Azoferredoxin has been purified 34–35 fold from N 2-fixing extracts of Clostridium pasteurianum by three steps of protamine sulfate fractionation and Sephadex G-100 gel filtration. The protein contains two nonheme iron atoms and two acid-labile sulfide groups per molecular weight of 40 000. It is decolorized by the sodium salt of O-((3-hydroxymercuri-2-methoxypropyl)carbamyl)phenoxyacetic acid (mersalyl) and the effect of mersalyl on its spectrum is similar to the effect of mersalyl on the spectrum of ferredoxin. Azoferredoxin is cold labile and the rate of cold inactivation is decreased in the presence of 10% ethanol or 10% acetone. Exposure of azoferredoxin to O 2 for 5 min causes complete inactivation. The effect of cold inactivation on azoferredoxin is different from that of oxidation as shown by distinct differences in spectral changes, measurable Fe 2+ content and mercury titratable sulfhydryl and sulfide groups of the protein.

Glutamine Phosphoribosylpyrophosphate Amidotransferase: Purification, substructure, amino acid composition, and absorption spectra

Abstract A purification procedure has been developed for the isolation of phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14, ribosylamine 5-phosphate:pyrophosphate phosphoribosyltransferase) from pigeon liver. It has been shown that the enzyme molecule of molecular weight 200,000 dissociates readily into 100,000 mol wt subunits and that these, in the presence of thiols, dissociate into 50,000 mol wt subunits. These basic 50,000 mol wt subunits, or monomers, are identical electrophoretically. Each 100,000 mol wt subunit, or dimer, contains 6 nonheme iron atoms some of which are involved in the catalytic reaction, whereas others serve primarily a structural function. The visible absorption spectrum of the enzyme reveals a maximum at 415 mµ, which disappears with the removal of iron from the protein. The tetramer, dimer, and monomer forms, as isolated, are enzymatically active and subject to purine ribonucleotide inhibition. The molecular weight of the functionally active unit, however, is unknown.

Acid-labile sulfide of clostridial ferredoxin and its enzymatic exchange with sodium sulfide- 35S

Ferredoxin (Fd) from Clostridium pasteurianum contains 8 non-heme iron atoms and from 6–8 acid-labile sulfide groups ( Lovenberg et al , 1963 ; Mortenson, 1964; Malkin and Rabinowitz, 1967). Malkin and Rabinowitz (1966a) have concluded that the sulfide released by acid treatment is an independent component of Fd rather than a byproduct of acid degradation of its cysteinyl residues, a suggestion of Bayer et al , (1964) , Bayer & Parr (1966) and Gersonde & Druskeit (1968). Evidence presented in this paper supports the conclusion that the acid-labile sulfide released from Fd is a different entity from the -SH group of the cysteinyl residues. This paper also reports on a new enzymatic reaction for the exchange of sodium sulfide- 35S with acid-labile sulfide of clostridial Fd.

Protocatechuate 3,4-Dioxygenase: I. Crystallization and Characterization

A procedure is described for the purification and crystallization of protocatechuate 3,4-dioxygenase from p-hydroxybenzoate-induced cells of Pseudomonas aeruginosa. Overall purification was about 30-fold, with 23% yield. Crystallization of the enzyme was achieved when it was incubated with a sulfhydryl reagent such as mercaptoethanol for over 1 week prior to the addition of ammonium sulfate. The crystalline enzyme was homogeneous as judged by ultracentrifugation, starch gel electrophoresis, and immunoelectrophoresis. The molecular weight was estimated to be approximately 700,000 and the molecular activity was calculated to be 45,500 at 24°. The enzyme had a deep red color with a broad absorption between 400 and 650 mµ, and contained about 7 g atoms of nonheme iron per mole of enzyme. Crystallization was also achieved by treating the enzyme with iodoacetamide prior to the addition of ammonium sulfate, or by dialyzing it against distilled water. Crystalline enzyme obtained by these methods showed the same enzymic activity and physicochemical properties as described above. Polymerization of the enzyme appeared to occur under certain conditions and to prevent the formation of crystals. The mode of polymerization and certain other properties of the crystalline enzyme are also reported.

Studies on yeast sulfite reductase I. Purification and characterization

1. NADPH-sulfite reductase, catalyzing the reduction of sulfite to sulfide by NADPH, was purified from baker's yeast to an almost homogeneous state. The purified enzyme showed a sedimentation coefficient of 14.8 S. A minimal molecular weight of 350 000 was estimated from its flavin content. 2. The oxidized enzyme was greenish yellow in color, showing absorption peaks at 386, 455 and 587 mμ. The intensity of these peaks was lowered considerably by NADPH or dithionite. 3. The enzyme contained 1 mole each of FMN and FAD per 350 000 g of protein. About 5 atoms of non-heme iron were detected per mole of FMN or FAD. The enzyme also contained a non-flavin chromophore (or chromophores) absorbing at 587 mμ and at about 386 mμ. The 587-mμ chromophore was unstable to treatment causing alterations in protein conformation. Five to six moles of p-chloromercuribenzoate combined with 350 000 g of the enzyme protein. 4. The enzyme catalyzed, besides the NADPH-sulfite reductase activity, the reduction of sulfite by reduced viologen dyes, the reductions by NADPH of nitrite, hydroxylamine, ferricyanide, cytochrome c, quinones, and 2,6-dichlorophenolindophenol, and the reduction of NADP + by reduced methyl viologen. 5. All the NADPH-linked activities were inhibited by NADP +, 2′-AMP and p-chloromercuribenzoate. NADP + and 2′-AMP, however, did not affect the reduction of sulfite by reduced viologen dyes. 6. Cyanide strongly inhibited the NADPH-sulfite reductase activity only when the reaction was started by adding sulfite to the enzyme which had been incubated with NADPH and cyanide. The addition of cyanide to the NADPH-reduced (but not the oxidized) enzyme caused an irreversible conversion to a reddish violet form. This conversion was prevented by the presence of sulfite.

Ferrous iron oxidation by Ferrobacillus ferrooxidans. Purification and properties of Fe++-cytochrome c reductase.

The electron transport chain and Fe++-cytochrome c reductase of Ferrobacillus ferrooxidans were studied to elucidate the mechanism of iron oxidation by this autotrophic bacterium.The iron oxidation involved the cytochrome c and a type cytochrome of F. ferrooxidans in the electron transport system. The initial enzyme of the oxidation system was found to be Fe++-cytochrome c reductase. The iron oxidase system was labile to freezing or sonication; either treatment disrupted some link between the cellular cytochromes c and a. Fe++-cytochrome c reductase and cytochrome oxidase retained their individual activities after either treatment.Fe++-cytochrome c reductase was purified 60-fold to 70-fold. The enzyme was judged to be approximately 90% pure by disc electrophoresis, sedimentation, and DEAE-cellulose chromatography. A suitable assay system with a veronal–acetate buffer was developed for the determination of enzyme activity. The effects of inhibitors and potential activators were studied. No specific inhibitor or cofactor was found, although the enzyme was inhibited by various ionic compounds.Fe++-cytochrome c reductase was dissociated into two subunits, one protein and the other ribonucleic acid (RNA). Neither of the subunits had enzymatic activity and efforts to reconstitute the holoenzyme from the two subunits were unsuccessful. The molecular weights of the holoenzyme, protein subunit, and RNA subunit were determined as 100,000–110,000, 27,000–30,000, and 315,000–330,000, respectively. The protein subunit contained one non-heme iron atom per protein molecule. It was concluded that RNA is an essential component of the enzyme and the failure to recover the activity from subunits is due to the aggregation of RNA after dissociation.

The amino acid sequence of [formula omitted] rubredoxin

In the past few years, several non-heme iron proteins have been isolated and characterized (San Pietro, 1965). Among others, the ferredoxins have come under extensive investigation. The amino acid sequences of Clostridium pasteurianum and C. butyricum ferredoxins have been reported from this laboratory ( Tanaka, et al., 1964; Benson, et al., 1966). Another non-heme iron protein, rubredoxin, was first obtained in the crystalline state from C. pasteurianum and was shown to replace ferredoxin in certain enzymatic reactions (Lovenberg and Sobel, 1965). The protein was reported to contain one gram atom of non-heme iron per mole of protein and to have a molecular weight around 6,000. The present report is concerned with the determination of the complete amino acid sequence of M. aerogenes rubredoxin.

Studies on the terminal electron transport system VIII. Conversion of succinic dehydrogenase complex to soluble succinic dehydrogenase

A method for the extraction of soluble succinic dehydrogenase from the particulate succinic dehydrogenase complex (SDC) is described. The enzyme, as extracted from SDC, catalyzes the dehydrogenation at 38° of 10 μmoles of succinate per min per mg with ferricyanide as the electron acceptor, or μmoles of succinate per min per mg with phenazine methosulfate as electron acceptor. It contains 3−5·10 −3 μmoles of flavin as well as 5−10·10 −3 μm atoms of non-heme iron per mg of protein.