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E Münck

Publications and source records attributed to E Münck.

At least 19 recordsLinked to original sources
PubMed2004

Initial structure modification of tetrahedral to planar nickel(II) in a nickel-iron-sulfur cluster related to the C-cluster of carbon monoxide dehydrogenase.

A method has been devised that creates a planar Ni(II) site from a tetrahedral site in a NiFe(3)S(4) cubane-type cluster. Reaction of [(Ph(3)P)NiFe(3)S(4)(LS(3))](2)(-) (2) with 1,2-bis(dimethylphosphino)ethane affords [(dmpe)NiFe(3)S(4)(LS(3))](2)(-) (3), isolated in ca. 45% yield as (Et(4)N)(2)[3a].2.5MeCN and (Et(4)N)(2)[3b].0.25MeCN, both of which occur in triclinic space group P. Each crystalline form contains two crystallographically inequivalent clusters with the same overall structure but slightly different dimensions. The cluster is bound by three thiolate terminal ligands to semirigid cavitand ligand LS(3). The NiFe(3)S(4) core contains three tetrahedral sites, one Fe(micro(3)-S)(3)(SR) and two Fe(micro(3)-S)(2)(micro(2)-S)(SR) with normal metric features, and one distorted square planar Ni(micro(3)-S)(2)P(2) site in a Ni(micro(3)-S)(2)Fe face with mean bond lengths Ni-P = 2.147(9) A and Ni-S = 2.29(2) A. The opposite Fe(2)(micro(3)-S)(micro(2)-S) face places the micro(2)-S atom at nonbonding and variable distances (2.60-2.90 A) above the nickel atom. Binding of the strong-field ligand dmpe results in a planar Ni(II) site and deconstruction of the full cubane geometry. The structure approximates that established crystallographically in the C-cluster of C. hydrogenoformans carbon monoxide dehydrogenase whose NiFe(4)S(4) core contains a planar NiS(4) site and three tetrahedral FeS(4) sites in a fragment that is bridged by sulfide atoms to an exo iron atom. Mössbauer studies of polycrystalline samples containing both clusters 3a and 3b reveal the presence of at least two cluster types. The spectroscopically best defined cluster accounts for ca. 54% of total iron and exhibits hyperfine interactions quite similar to those reported for the S = (5)/(2) state of the protein-bound cubane-type cluster [ZnFe(3)S(4)](1+), whose Mössbauer spectrum revealed the presence of a high-spin Fe(2+) site and a delocalized Fe(2.5+)Fe(2.5+) pair. Development of reactions leading to a planar nickel and a sulfide-bridged iron atom is requisite to attainment of a synthetic analogue of this complex protein-bound cluster. This work demonstrates a tetrahedral (2) --> planar (3) Ni(II) stereochemical conversion can be effected by binding of ligands that generate a sufficiently strong in-plane ligand field (dmpe = 1,2-bis(dimethylphosphino)ethane, LS(3) = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-)).

Aldehyde Oxidoreductases↗
PubMed2001

Influence of extrinsic factors on electron transfer in a mixed-valence Fe(2+)/Fe(3+) complex: experimental results and theoretical considerations.

The crystal structure of the mixed-valence complex (NEt(4))[Fe(2)(salmp)(2)].xMeCN(crystal) (x = 2,3) [1].xMeCN(crystal) was determined at temperatures between 153 and 293 K. The complex shows distinct Fe(2+) and Fe(3+) sites over this temperature interval. Variable temperature Mössbauer spectra confirm the valence-localized character of the complex. In contrast, spectroscopic investigation of powder samples generated from [1].xMeCN(crystal) indicate the presence of a valence-averaged component at temperatures above 150 K. To elucidate this apparent contradiction we have conducted a variable-temperature Mössbauer investigation of different forms of 1, including [1].xMeCN(crystal), [1].2DMF(crystal), [1].yMeCN(powder), and solution samples of 1 in acetonitrile. The low-temperature Mössbauer spectra of all forms are virtually identical and confirm the valence-localized nature of the S = (9)/(2) ground state. The high-temperature spectra reveal a subtle control of electron hopping by the environment of the complexes. Thus, [1].xMeCN(crystal) has valence-localized spectra at all explored temperatures, [1].2DMF(crystal) exhibits a complete collapse into a valence-averaged spectrum over a narrow temperature range, the powder exhibits partial valence averaging over a broad temperature interval, and the solution sample shows at 210 K the presence of a valence-averaged component in a minor proportion. The spectral transformations are characterized by a coexistence of valence-localized and valence-averaged spectral components. This phenomenon cannot be explained by intramolecular electron hopping between the valence-localized states Fe(A)(2+)Fe(B)(3+) and Fe(A)(3+)Fe(B)(2+) in a homogeneous ensemble of complexes, but requires relaxation processes involving at least three distinguishable states of the molecular anion. Hopping rates for [1].2DMF(crystal) and [1].yMeCN(powder) have been determined from spectral simulations, based on stochastic line shape theory. Analysis of the temperature dependences of the transfer rates reveals the existence of thermally activated processes between (quasi) degenerate excited states in both forms. The preexponential factors in the rate law for the hopping processes in the [1].yMeCN(powder) and [1].2DMF(crystal) differ dramatically and suggest an important influence of the asymmetry of the complex environment (crystal) on intramolecular electron hopping. The differences between the spectra for the crystalline sample [1].xMeCN and those for powders generated under vacuum from these crystals indicate that solvate depletion has a profound effect on the dynamic behavior. Finally, two interpretations for the three states involved in the relaxation processes in 1 are given and critically discussed (salmp = bis(salicyledeneamino)-2-methylphenolate(3-)).

Crystallography, X-Ray↗
PubMed2001

Mössbauer and EPR studies of the photoactivation of nitrile hydratase.

The alphabeta dimer of active nitrile hydratase from Rhodococcus sp. R312 contains one low-spin ferric ion that is coordinated by three Cys residues, two N-amide groups from the protein backbone, and one OH(-). The enzyme isolated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NH(dark). The active state can be obtained from the dark state by photolysis of the Fe-NO bond at room temperature. Activation is accompanied by the conversion of NH(dark) to a low-spin ferric complex, NH(light), exhibiting an S = (1)/(2) EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NH(dark) and NH(light) with Mössbauer spectroscopy. The z-axis of the 57Fe magnetic hyperfine tensor, A, of NH(light) was found to be rotated by approximately 45 degrees relative to the z-axis of the g tensor (g(z) = 1.97). Comparison of the A tensor of NH(light) with the A tensors of low-spin ferric hemes indicates a substantially larger degree of covalency for nitrile hydratase. We have also performed photolysis experiments between 2 and 20 K and characterized the photolyzed products by EPR and Mössbauer spectroscopy. Photolysis at 4.2 K in the Mössbauer spectrometer yielded a five-coordinate low-spin ferric species, NH(A), which converted back into NH(dark) when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.

Darkness↗
PubMed2000

Iron-sulfur center of biotin synthase and lipoate synthase.

Biotin synthase and lipoate synthase are homodimers that are required for the C-S bond formation at nonactivated carbon in the biosynthesis of biotin and lipoic acid, respectively. Aerobically isolated monomers were previously shown to contain a (2Fe-2S) cluster, however, after incubation with dithionite one (4Fe-4S) cluster per dimer was obtained, suggesting that two (2Fe-2S) clusters had combined at the interface of the subunits to form the (4Fe-4S) cluster. Here we report Mössbauer studies of (57)Fe-reconstituted biotin synthase showing that anaerobically prepared enzyme can accommodate two (4Fe-4S) clusters per dimer. The (4Fe-4S) cluster is quantitatively converted into a (2Fe-2S)(2+) cluster upon exposure to air. Reduction of the air-exposed enzyme with dithionite or photoreduced deazaflavin yields again (4Fe-4S) clusters. The (4Fe-4S) cluster is stable in both the 2+ and 1+ oxidation states. The Mössbauer and EPR parameters were DeltaE(q) = 1.13 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and DeltaE(q) = 0.51 mm/s, delta = 0.85 mm/s, g(par) = 2.035, and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+). Considering that we find two (4Fe-4S) clusters per dimer, our studies argue against the early proposal that the enzyme contains one (4Fe-4S) cluster bridging the two subunits. Our study of lipoate synthase gave results similar to those obtained for BS: under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit [DeltaE(q) = 1.20 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and g(par) = 2.039 and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+)], which reacts with oxygen to generate a (2Fe-2S)(2+) center.

Bacterial Proteins↗
PubMed2000

Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the [4Fe-4S](2+) cluster to oxygen.

To understand the role of the [4Fe-4S](2+) cluster in controlling the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) during changes in O(2) availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions. The His-28 substitution appears to stabilize the [4Fe-4S](2+) cluster of FNR-L28H in the presence of O(2) because air-exposed FNR-L28H did not undergo the rapid [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions. This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S](2+) cluster-deficient preparations of FNR-L28H. The Mössbauer spectra of FNR-L28H indicated that the coordination environment of the [4Fe-4S](2+) cluster did not differ from that of WT-FNR. Whole cell Mössbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the [4Fe-4S](2+) cluster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [4Fe-4S](2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.

Aerobiosis↗
PubMed2000

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MDC studies of the wild-type protein.

Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D = 1.2 cm(-1), that are about 40% smaller than the D-value of the wild-type protein. The 57Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has delta=0.24+/-0.01 mm/s at 4.2 K rather than delta = 0.32 mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts delta = 0.79 mm/s at 4.2 K, consistent with tetrahedral Fe2+(Cys)3(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D = -7+/-1 cm(-1), E/D = 0.09 for form A and D = +6.2+/-1.3 cm(-1), E/D = 0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained g(z) = 2.08+/-0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77 K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe2+ site. Studies in different buffers in the pH 6-9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30-50 % v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700 cm(-1). Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300 cm(-1); a third d-d transition, if present, must occur with energy lower than 3300 cm(-1). The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100 cm(-1) in form A and 5350, 6380 cm(-1) in form B.

Bacterial Proteins↗
PubMed1999

Mössbauer studies of the formation and reactivity of a quasi-stable peroxo intermediate of stearoyl-acyl carrier protein Delta 9-desaturase.

Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:0-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O(2) yields a blue chromophore (lambda(max) approximately 700 nm) that exhibits a vibrational spectrum indicative of a micro-1,2-peroxo complex; this species has been designated peroxo Delta9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) approximately 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H(2)O(2) or product (18:1-ACP). In this work, optical, transient kinetic, and Mössbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O(2)-equilibrated 18:0-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-)(1) at 6 degrees C). The Mössbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E(Q)(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E(Q)(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-)(1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E(Q)(1) = 1. 91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E(Q)(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:0-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the Mössbauer cryostat caused the release of O(2) and the reappearance of a diferrous Delta 9D.18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the Mössbauer spectrum of peroxo Delta 9D, showing that the released O(2) was effectively sequestered within the active site.

Fatty Acid Desaturases↗
PubMed1999

Unusual NMR, EPR, and Mössbauer properties of Chromatium vinosum 2[4Fe-4S] ferredoxin.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its Mössbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.

Bacterial Proteins↗
PubMed1998

Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli.

The global regulator FNR (for fumarate nitrate reduction) controls the transcription of >100 genes whose products facilitate adaptation of Escherichia coli to growth under O2-limiting conditions. Previous Mössbauer studies have shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster that, on exposure to oxygen, is converted into a [2Fe-2S]2+ cluster, a process that decreases DNA binding by FNR. Using 57Fe Mössbauer spectroscopy of E. coli cells containing overexpressed FNR, we show here that the same cluster conversion also occurs in vivo on exposure to O2. Furthermore, the data show that a significant amount of the [4Fe-4S]2+ cluster is regenerated when the cells are shifted back to an anaerobic environment. The present study also demonstrates that 57Fe Mössbauer spectroscopy can be employed to study the in vivo behavior of (overexpressed) proteins. The use of this technique to study other iron-containing cell components is discussed.

Bacterial Proteins↗
PubMed1998

An all-ferrous state of the Fe protein of nitrogenase. Interaction with nucleotides and electron transfer to the MoFe protein.

The MoFe protein of nitrogenase catalyzes the six-electron reduction of dinitrogen to ammonia. It has long been believed that this protein receives the multiple electrons it requires one at a time, from the [4Fe-4S]2+/+ couple of the Fe protein. Recently an all-ferrous [4Fe-4S]0 state of the Fe protein was demonstrated suggesting instead a series of two electron steps involving the [4Fe-4S]2+/0 couple. We have examined the interactions of the [4Fe-4S]0 Fe protein with nucleotides and its ability to transfer electrons to the MoFe protein. The [4Fe-4S]0 Fe protein binds both MgATP and MgADP and undergoes the MgATP induced conformational change and then binds properly to the MoFe protein, as evidenced by the fact that the behavior of the 0 and +1 oxidation states in the chelation and chelation protection assays are indistinguishable. Nucleotide binding does not effect the distinctive UV/Vis, CD, or Mössbauer spectra exhibited by the [4Fe-4S]0 Fe protein; however, because the intensity of the g = 16.4 EPR signal of the [4Fe-4S]0 Fe protein is extremely sensitive to minor variations of the rhombicity parameter E/D, the EPR signal is sensitive to the binding of nucleotides. A 50:50 mixture of [4Fe-4S]2+ and [4Fe-4S]0 Fe protein results in electron self-exchange and 100% production of [4Fe-4S]+ Fe protein, demonstrating that the +1/0 couple is fully reversible. MgATP is absolutely required for electron transfer from the [4Fe-4S]0 Fe protein to the reduced state of the MoFe protein. In that reaction both electrons are transferred and are used to reduce substrate.

Adenosine Diphosphate↗
PubMed1997

Assembly of a [2Fe-2S]2+ cluster in a molecular variant of Clostridium pasteurianum rubredoxin.

The rubredoxin from Clostridium pasteurianum contains a single iron atom bound to the polypeptide chain by cysteines 6, 9, 39, and 42. The C42A variant of this protein has been prepared by site-directed mutagenesis and heterologous expression of the gene in Escherichia coli. The mutated protein was found to contain an unexpected chromophore that has been characterized by a variety of techniques. UV-visible absorption and resonance Raman spectra were strongly reminiscent of those of [2Fe-2S] proteins. Mössbauer spectra of the oxidized chromophore isolated in oxygen-free conditions indicated low-temperature diamagnetism resulting from antiferromagnetically coupled high-spin ferric ions. Analysis of X-ray absorption fine structure spectra yielded an Fe-Fe distance of 2.68 A. Colorimetric assays of iron and inorganic sulfide showed that the two elements are present in a 1:1 ratio. Electrospray-ionization mass spectra displayed a major component at M = 6190 Da, i.e. the molecular mass of the C42A apoprotein plus two atomic masses of iron and two atomic masses of sulfur. Taken together, these data show that a mere point mutation allows the stabilization of a binuclear [2Fe-2S] cluster in a protein that normally accommodates a mononuclear Fe(Scys)4 site. Assembly of a [2Fe-2S] cluster may occur because rubredoxin assumes a similar fold around its metal center as the [2Fe-2S] Rieske protein. Alternatively, a more extensive structural rearrangement of the polypeptide chain of the C42A rubredoxin variant may be considered as well.

Binding Sites↗
PubMed1997

Iron-sulfur clusters: nature's modular, multipurpose structures.

Iron-sulfur proteins are found in all life forms. Most frequently, they contain Fe2S2, Fe3S4, and Fe4S4 clusters. These modular clusters undergo oxidation-reduction reactions, may be inserted or removed from proteins, can influence protein structure by preferential side chain ligation, and can be interconverted. In addition to their electron transfer function, iron-sulfur clusters act as catalytic centers and sensors of iron and oxygen. Their most common oxidation states are paramagnetic and present significant challenges for understanding the magnetic properties of mixed valence systems. Iron-sulfur clusters now rank with such biological prosthetic groups as hemes and flavins in pervasive occurrence and multiplicity of function.

Catalysis↗
PubMed1997

Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O2: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity.

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]2+ cluster (delta = 0.45 mm/s; DeltaEQ = 1.22 mm/s) and that the [4Fe-4S]2+ cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow-green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]2+ cluster was converted in about 60% yield to a [2Fe-2S]2+ cluster (delta = 0.28 mm/s; DeltaEQ = 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]2+ cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]2+ cluster could be largely restored from the [2Fe-2S]2+ form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]2+ cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein.

Air↗
PubMed1997

Mössbauer studies of alkane omega-hydroxylase: evidence for a diiron cluster in an integral-membrane enzyme.

The gene encoding the alkane omega-hydroxylase (AlkB; EC 1.14.15.3) from Pseudomonas oleovorans was expressed in Escherichia coli. The integral-membrane protein was purified as nearly homogeneous protein vesicles by differential ultracentrifugation and HPLC cation exchange chromatography without the detergent solubilization normally required for membrane proteins. Purified AlkB had specific activity of up to 5 units/mg for octane-dependent NADPH consumption. Mössbauer studies of AlkB showed that it contains an exchange-coupled dinuclear iron cluster of the type found in soluble diiron proteins such as hemerythrin, ribonucleotide reductase, methane monooxygenase, stearoyl-acyl carrier protein (ACP) delta9 desaturase, rubrerythrin, and purple acid phosphatase. In the as-isolated enzyme, the cluster contains an antiferromagnetically coupled pair of high-spin Fe(III) sites, with an occupancy of up to 0.9 cluster per AlkB. The diferric cluster could be reduced by sodium dithionite, and the diferrous state was found to be stable in air. When both O2 and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluster occupies the active site. Mossbauer data on reduced AlkB are consistent with a cluster coordination rich in nitrogen-containing ligands. New sequence analyses indicate that at least 11 nonheme integral-membrane enzymes, including AlkB, contain the 8-histidine motif required for catalytic activity in stearoyl-CoA desaturase. Based on our Mössbauer studies of AlkB, we propose that the integral-membrane enzymes in this family contain diiron clusters. Because these enzymes catalyze a diverse range of oxygenation reactions, this proposal suggests a greatly expanded role for diiron clusters in O2-activation biochemistry.

Iron↗
PubMed1997

An Fe2IVO2 diamond core structure for the key intermediate Q of methane monooxygenase.

A new paradigm for oxygen activation is required for enzymes such as methane monooxygenase (MMO), for which catalysis depends on a nonheme diiron center instead of the more familiar Fe-porphyrin cofactor. On the basis of precedents from synthetic diiron complexes, a high-valent Fe2(micro-O)2 diamond core has been proposed as the key oxidizing species for MMO and other nonheme diiron enzymes such as ribonucleotide reductase and fatty acid desaturase. The presence of a single short Fe-O bond (1.77 angstroms) per Fe atom and an Fe-Fe distance of 2.46 angstroms in MMO reaction intermediate Q, obtained from extended x-ray absorption fine structure and Mössbauer analysis, provides spectroscopic evidence that the diiron center in Q has an Fe2IVO2 diamond core.

Binding Sites↗
PubMed1996

Recombinant toluene-4-monooxygenase: catalytic and Mössbauer studies of the purified diiron and rieske components of a four-protein complex.

Expression of the tmoA-F gene cluster from Pseudomonas mendocina KRI in Escherichia coli BL21(DE3) produces a catalytically active form of the toluene-4-monooxygenase (T4MO) complex. Here we report the purification and characterization of four soluble proteins required for the in vitro reconstitution of T4MO catalytic activity. These proteins are a diiron hydroxylase (T4MOH), a Riesketype ferredoxin (T4MOC), an effector protein (T4MOD), and an NADH oxidoreductase (T4MOF). The T4MOH component is composed of the tmoA, tmoB, and tmoE gene products [quaternary structure (alpha beta epsilon)2, Mr approximately 220 kDa]. The T4MOA polypeptide contains two copies of the amino acid sequence motif (D/E)X(28-37)DEXRH; the same motif provides all of the protein-derived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP delta 9 desaturase. Mössbauer, optical, and EPR measurements show that the T4MOH contains diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase. Mössbauer and EPR measurements also show that the T4MOC contains a Rieske-type iron-sulfur center. This assignment is in accord with the presence of the amino acid sequence motif CPHX(15-17)CX2H, which has also been found in the bacterial, chloroplastic, and mitochondrial Rieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases. While single-turnover catalytic studies confirm the function of the T4MOH as the hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by more than 100-fold in the presence of the T4MOC, which mediates highly specific electron transfer between the T4MOF and the T4MOH. The T4MOD can be purified as an 11.6 kDa monomeric protein devoid of cofactors or redox-active metal ions; this component is also detected as a substoichiometric consitutent of the purified T4MOH. The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separately purified T4MOD to the T4MOH, implying the formation of a high affinity, catalytically competent complex between these two components. These characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotrophic bacteria and the aromatic hydroxylation complexes of the soil pseudomonads.

Amino Acid Sequence↗
PubMed1995

Reaction of NO with the reduced R2 protein of ribonucleotide reductase from Escherichia coli.

The active R2 protein of ribonucleotide reductase from Escherichia coli contains a catalytically essential tyrosine radical at position 122 (Tyr122.) that is formed during the reaction of dioxygen with the nearby diiron(II) center. To gain insight into the mode of dioxygen binding, the reaction of the O2 analog NO with the diiron(II) centers of R2red has been investigated by spectroscopic methods. R2red reacts with NO to form an adduct with visible absorption features at 450 and 620 nm and Mössbauer parameters (delta = 0.75 mm/s, delta EQ = -2.13 and -1.73 mm/s) typical of those observed for S = 3/2 [FeNO]7 complexes of other non-heme iron proteins. However, unlike other non-heme [FeNO]7 complexes, this adduct is EPR silent. Our Mössbauer studies show that each iron site of R2red binds one NO to form local S = 3/2 [FeNO]7 centers which then couple antiferromagnetically (J approximately 5 cm-1, H = JS1.S2) to afford an [FeNO]2 center (77% of total iron). This [FeNO]2 center decomposes with a first-order rate constant of 0.013 min-1 to form R2met, accompanied by the release of N2O. These observations suggest that both iron(II) ions of the two diiron(II) centers of R2red have available sites for NO binding, in agreement with the crystallographic results on R2red, and that the bound NO molecules are sufficiently close to each other to permit N-N bond formation to produce N2O. These observations support the proposal that dioxygen binding may also involve both metal ions of the diiron(II) center to form a (mu-1,1-, or mu-1,2-peroxo)-diiron(III) center. This observed reactivity of R2red with NO may contribute to the in vivo inhibition of ribonucleotide reductase by NO.

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