Non-heme Iron Center Research Articles

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine.

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.

A Highly Conserved Glutamate Residue (Glu-270) Is Essential for Plant Alternative Oxidase Activity

We have previously demonstrated that expression of a Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe confers cyanide-resistant respiratory activity on these cells (Albury, M. S., Dudley, P., Watts, F. Z., and Moore, A. L. (1996) J. Biol. Chem. 271, 17062-17066). Using this functional expression system we have investigated the active site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center. Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand within the active site, to asparagine abolishes catalytic activity because mitochondria containing the E270N mutant protein do not exhibit antimycin A-resistant respiration. Western blot analysis, using antibodies specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed by S. pombe mitochondria in a manner similar to that of the wild-type protein. It is possible that lack of antimycin A-insensitive respiration observed in mitochondria containing the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane. However, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lack of insertion is unlikely. These results provide the first experimental evidence to support the structural model in which the active site of the alternative oxidase contains a coupled binuclear iron center.

Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase.

Pseudomonas sp. NCIB 9816-4 utilizes a multicomponent enzyme system to oxidize naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme component catalyzing this reaction, naphthalene 1,2-dioxygenase (NDO), belongs to a family of aromatic-ring-hydroxylating dioxygenases that oxidize aromatic hydrocarbons and related compounds to cis-arene diols. These enzymes utilize a mononuclear non-heme iron center to catalyze the addition of dioxygen to their respective substrates. The present study was conducted to provide essential structural information necessary for elucidating the mechanism of action of NDO. The three-dimensional structure of NDO has been determined at 2.25 A resolution. The molecule is an alpha 3 beta 3 hexamer. The alpha subunit has a beta-sheet domain that contains a Rieske [2Fe-2S] center and a catalytic domain that has a novel fold dominated by an antiparallel nine-stranded beta-pleated sheet against which helices pack. The active site contains a non-heme ferrous ion coordinated by His208, His213, Asp362 (bidentate) and a water molecule. Asn201 is positioned further away, 3.75 A, at the missing axial position of an octahedron. In the Rieske [2Fe-2S] center, one iron is coordinated by Cys81 and Cys101 and the other by His83 and His104. The domain structure and iron coordination of the Rieske domain is very similar to that of the cytochrome bc1 domain. The active-site iron center of one of the alpha subunits is directly connected by hydrogen bonds through a single amino acid, Asp205, to the Rieske [2Fe-2S] center in a neighboring alpha subunit. This is likely to be the main route for electron transfer.

Effect of Carboxamido N Coordination to Iron on the Redox Potential of Low-Spin Non-Heme Iron Centers with N,S Coordination: Relevance to the Iron Site of Nitrile Hydratase.

The redox parameters of [Fe(PyPepS)2]-, the first low-spin Fe(III) complex with both carboxamido N and thiolato S donors in the first coordination sphere, indicate significant stabilization of the +3 oxidation state of iron. Such stabilization could account for the reactivity of the iron center in nitrile hydratase.

Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771: EVIDENCE OF A NOVEL POST-TRANSLATIONAL MODIFICATION IN THE CYSTEINE LIGAND

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.

Split-soret cytochrome [formula omitted] from desulfovibrio desulfuricans ATCC 27774, a novel protein that contains both heme and non-heme iron centers

Split-soret cytochrome [formula omitted] from desulfovibrio desulfuricans ATCC 27774, a novel protein that contains both heme and non-heme iron centers

Photodissociation of nitric oxide from the non-heme iron center activates nitrile hydratase from Rhodococcus sp. N-771: FTIR and resonance Raman evidence

Photodissociation of nitric oxide from the non-heme iron center activates nitrile hydratase from Rhodococcus sp. N-771: FTIR and resonance Raman evidence

Nitric Oxide Prevents Oxidative Damage Produced bytert-Butyl Hydroperoxide in Erythroleukemia Cells via Nitrosylation of Heme and Non-heme Iron: ELECTRON PARAMAGNETIC RESONANCE EVIDENCE

We studied protective effects of NO against tert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations. t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitive cis-parinaric acid was induced by t-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-iu m-1, 2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected against t-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2, 2'-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species.

Activity Regulation of Photoreactive Nitrile Hydratase by Nitric Oxide

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a novel enzyme that possesses a non-heme iron(III) center binding endogenous nitric oxide (NO). It is inactivated by aerobic incubation of cells in the dark, whereas the inactive form is converted to the active one by light irradiation. To clarify the mechanism of activity regulation in the NHase, we investigated the role of NO. When the inactive NHase was irradiated in the presence of Fe(II) ions and a spin trap, N-methyl-d-glucamine dithiocarbamate (MGD), three hyperfine lines from the nitrogen atom of the [(MGD)2-FeII-NO] complex were resolved, indicating that NO was released from the enzyme upon photoactivation. The amount of NO release was obtained as 0.99 ± 0.12 per enzyme (n = 4) by using an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. The active NHase was completely inactivated by exogenous NO, and upon photoirradiation 86% of the original activity was restored. FTIR, ESR, and UV−VIS absorption measurements...

Highly purified D1/D2/cyt b559 preparations from spinach do not contain the non-heme iron center

57Fe enriched D1/D2/cyt b559 preparations were isolated from spinach grown hydroponically on a 57Fe containing medium. In terms of polypeptide and pigment composition these samples are of high purity and functional integrity of P680. Mössbauer spectra measured in D1/D2/cyt b559 complexes revealed that these preparations are completely deprived of the non-heme iron center. Possible implications of this finding are discussed for the electron transfer from Pheo −⋅ to exogenous electron acceptors. © 1997 Federation of European Biochemical Societies.

Analysis, Design and Engineering of Simple Iron-Sulfur Proteins: Tales from Rubredoxin and Desulforedoxin

Abstract The most thoroughly characterized non-heme iron center in biology is Rubredoxin, the simplest member of the iron-sulfur class of metalloproteins. Rubredoxin contains a highspin iron atom with tetrahedral coordination by four cysteinyl sulfur atoms. A structural variant of this center is found in Desulforedoxin, the smallest known Rubredoxin type protein. The 3D structure of both Rd and Dx has been determined at high resolution. These two proteins can therefore be used as case studies in which structural control by the polypeptide chain over the metal site can be discussed in detail.

Location of the Non-Heme Iron Center on the α Subunit of Photoreactive Nitrile Hydratase fromRhodococcussp. N-771

Nitrile hydratase (NHase) fromRhodococcussp. N-771, which possesses a non-heme iron center binding nitric oxide (NO), is activated by light irradiation. To localize the iron center in the protein, we quantified Fe atoms and performed FTIR measurements of the isolated α and β subunits. The native NHase and the isolated α subunit contained about 1.0 and 0.8 mol Fe per mol protein, respectively, whereas the β subunit contained only a trace of Fe. An NO stretching band was observed at 1852 cm−1in the FTIR spectrum of the α subunit, but not in that of the β subunit. Upon light irradiation of the α subunit, the affinity of the Fe atom decreased and the NO band disappeared from the FTIR spectrum. These observations indicate that the non-heme iron center, which is responsible for the photoreaction, is present on the α subunit.

Resonance Raman evidence that photodissociation of nitric oxide from the non-heme iron center activates nitrile hydratase from Rhodococcus sp. N-771.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771, which contains a non-heme iron center in the catalytic site, has been known to be activated by light illumination. Recently, endogenous nitric oxide (NO) was found in this enzyme by FTIR spectroscopy [Noguchi et al. (1995) FEBS Lett. 358, 9-12]. In order to directly detect the bonding between NO and the iron atom and the reaction of NO upon photoactivation, resonance Raman spectra of the NHase were measured with 413 nm excitation at 85 K. Two prominent bands at 592 and 570 cm-1 were observed in the inactive from, and both of them were completely lost upon photoactivation. Upon subsequent introduction of 15NO, the active NHase was converted to the inactive form again and the above two bands were restored with downshifts by 10 and 12 cm-1, respectively. Also, the excitation profiles of these bands in the 350-500 nm region mostly followed the absorption spectrum arising from the iron center. From these isotopic shifts and the excitation profiles, the two Raman bands were assigned to the Fe-NO stretching and bending vibrations that are probably coupled with each other. The results provided solid evidence that NO is bound to the non-heme iron in the inactive NHase and its photodissociation activates the enzyme.

Characterization of the arachidonate and ATP binding sites of human 5-lipoxygenase using photoaffinity labeling and enzyme immobilization

The arachidonic acid and the ATP binding sites of human 5-lipoxygenase were characterized using photoaffinity labeling and immobilization of the enzyme on ATP-agarose. Photoaffinity labeling of the active site of 5-lipoxygenase was achieved with a novel thiopyranoindole inhibitor containing a 4-azido-3-iodobenzenesulfonyl moiety (L-708,714). This probe was found to inhibit the activity of 5-lipoxygenase (IC50 = 0.3 microM) and to covalently label the enzyme after UV light irradiation. The labeling was inhibited by arachidonic acid, N-hydroxyurea, and dihydrobenzofuranol inhibitors which have been shown to reduce the non-heme iron center of 5-lipoxygenase. Photoaffinity labeling of 5-lipoxygenase by L-708,714 was dependent on the presence of both Ca2+ ions and phospholipids and was independent of ATP. It occurred at similar levels using native (Fe2+), oxidized (Fe3+), or H2O2-inactivated enzyme, but was abolished by heat inactivation of the enzyme. Competition of the labeling by various thiopyranoindoles and other inhibitors such as L-697,198,ZD-2138, and zileuton was found to be related to their inhibitory potency. Immobilized 5-lipoxygenase on ATP-agarose was found to be selectively eluted by adenine nucleotides (ATP > ADP > AMP) but not by solutions containing high salt concentrations, mild detergents, arachidonic acid, or inhibitors. 5-Lipoxygenase inhibitors were selectively retained on the immobilized enzyme and eluted by buffer containing arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the extraction of the non-heme iron centre from photosystem II of higher plants

Studies on the extraction of the non-heme iron centre from photosystem II of higher plants

Nonheme Iron Centers in Oxygen Activation: Characterization of an Iron(III) Hydroperoxide Intermediate

——————— [*] Prof. L. Que, Jr., E. C. Wilkinson Department of Chemistry, University of Minnesota Minneapolis, MN 55455 (USA) Telefax: Int. code + (612)624-7029 Prof. B. Feringa, Dr. M. Lubben, Dr. A. Meetsma Department of Organic and Molecular Inorganic Chemistry Groningen Center for Catalysis and Synthesis University of Groningen Nijenborgh 4, NL-9747 AG Groningen (The Netherlands) [**] Financial support of this research by Unilever Research (Vlaardingen, Nether-lands) to B. L. F. and the National Institutes of Health (USA) to L. Q. (GM-33162) is gratefully acknowledged. E. C. W. thanks the NIH for a predoctoral traineeship (GM-07323).

Mutations at the C‐Terminal Isoleucine and Other Potential Iron Ligands of 5‐Lipoxygenase

The non-heme iron centre in human 5-lipoxygenase was studied. Recombinant enzyme was expressed in Escherichia coli, purified and assayed for iron content and enzyme activity. For non-mutated enzyme, the iron content was 1.01 +/- 0.19 mol/mol. Deletion of the C-terminal Ile673 resulted in an iron content of 0.03 +/- 0.07 mol/mol and undetectable lipoxygenase activity. Mutations at His367, Glu376 and Asn554 led to drastically decreased enzyme activity (< 2% of non-mutated control) but iron was still present. In addition to Glu376, eight other conserved acidic residues (Asp/Glu) in 5-lipoxygenase were replaced, none of which was crucial for enzyme activity. We conclude that Ile673 is an iron ligand in 5-lipoxygenase, while our results do not support that Glu376 or Asn554 have this function. The possible role of His367 as a replaceable iron ligand is discussed.

Mutations at the C-Terminal Isoleucine and Other Potential Iron Ligands of 5-Lipoxygenase

The non-heme iron centre in human 5-lipoxygenase was studied. Recombinant enzyme was expressed in Escherichia coli, purified and assayed for iron content and enzyme activity. For non-mutated enzyme, the iron content was 1.01±0.19 mol/mol. Deletion of the C-terminal Ile673 resulted in an iron content of 0.03±0.07 mol/mol and undetectable lipoxygenase activity. Mutations at His367, Glu376 and Asn554 led to drastically decreased enzyme activity (<2% of non-mutated control) but iron was still present. In addition to Glu376, eight other conserved acidic residues (Asp/Glu) in 5-lipoxygenase were replaced, none of which was crucial for enzyme activity. We conclude that Ile673 is an iron ligand in 5-lipoxygenase, while our results do not support that Glu376 or Asn554 have this function. The possible role of His367 as a replaceable iron ligand is discussed.

Photosensitive nitrile hydratase intrinsically possesses nitric oxide bound to the non-heme iron center: evidence by Fourier transform infrared spectroscopy

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a photosensitive enzyme that catalyzes hydration of nitriles to the corresponding amides. Light-induced Fourier transform infrared difference spectra between the inactive and active forms of NHase were measured with both the natural ( 14N) and 15N-labeled NHases. The results showed, for the first time, that NHase intrinsically possesses nitric oxide (NO) molecules bound to the non-heme iron center. The possible role of NO in the photoactivation process of NHase is discussed.

Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase.

Mononuclear non-heme iron centers are found in the active sites of a variety of enzymes that require molecular oxygen for catalysis. The mononuclear non-heme iron is believed to be the active site for catalysis, and is presumed to bind and activate molecular oxygen. The mechanism of this reaction is not understood. Phthalate dioxygenase is one such enzyme. Because it also contains a second iron site, the Rieske site, it is difficult to obtain information on the structure of the active site. We therefore used magnetic circular dichroism (MCD) spectroscopy to probe the mononuclear, non-heme Fe2+ site in this biodegradative enzyme. The MCD spectrum of the resting enzyme shows features indicative of one six-coordinate Fe2+ site; substrate binding converts the site to two different five-coordinate species, opening up a coordination position for O2 binding. MCD spectra of the corresponding apoenzyme have been subtracted to account for temperature-independent contributions from the Rieske site. Azide binds both to the resting enzyme to produce a new six-coordinate species, showing that one of the ferrous ligands is exchangeable, and also to the enzyme-substrate complex to form a ternary species. The low azide binding constant for the substrate-enzyme species relative to the resting enzyme indicates steric interaction and close proximity between exogenous ligand and the substrate. We have been able to provide some detailed structural insight into exogenous ligand and substrate binding to the non-heme Fe2+ site, even in the presence of the enzyme's [2Fe-2S] Rieske center. Further mechanistic studies are now required to maximize the molecular-level detail available from these spectroscopic studies.