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E Münck

Publications and source records attributed to E Münck.

At least 73 records · Page 4Linked to original sources
PubMed1983

Mössbauer study of beef heart cytochrome oxidase. Comparative study of the bovine enzyme and cytochrome c1aa3 from Thermus thermophilus.

We have studied beef heart cytochrome c oxidase at 4.2 K with Mössbauer spectroscopy using the 57Fe present in natural abundance. The spectra observed are very similar to those of the a- and a3-sites of cytochrome c1aa3 from Thermus thermophilus. Thus, many conclusions derived from studies of the bacterial oxidase (available with enriched 57Fe) also apply to the mammalian enzyme. In the resting (as isolated) state, cytochrome a3 of the mammalian enzyme exhibits a doublet with quadrupole splitting, delta EQ = 1.0 mm/s and isomer shift, delta = 0.48 mm/s. These parameters suggest a high spin ferric heme and rule out an Fe(IV) assignment. The absence of magnetic features in the 4.2 K spectrum is consistent with earlier proposals that cytochrome a3 is spin-coupled to a cupric ion. The absorption lines are rather broad, suggesting that the a3-site is heterogeneous in the resting enzyme. Reduced cytochrome a3 has delta EQ = 1.85 mm/s and delta = 0.93 mm/s, demonstrating that the heme iron is high spin ferrous. The observed value for delta EQ is smaller than those of hemoglobin (2.4 mm/s), myoglobin (2.2 mm/s), and cytochrome a3 from T. thermophilus (2.06 mm/s). The Mössbauer spectra of oxidized cytochrome a3-CN show that the heme iron is low spin ferric and that the ground state has integer spin S greater than or equal to 1, which plausibly results from ferromagnetic coupling of the S = 1/2 heme to an S = 1/2 cupric ion. Reduced cytochrome a is low spin ferrous, with parameters similar to those of cytochrome b5 and cytochrome c.

Animals↗
PubMed1983

Mössbauer study of iron bleomycin and its activation intermediates.

Using Mössbauer spectroscopy, we have examined iron-bleomycin in various oxidation states and in complexes with dioxygen or carbon monoxide. Ferrous bleomycin is a high spin ferrous complex. Addition of O2 converts it into an EPR-silent oxygenated complex. Mössbauer studies in strong applied magnetic fields show that oxygenated bleomycin is diamagnetic. At 4.2 K, the quadrupole splitting delta EQ = -2.96 mm/s and the isomer shift delta = 0.16 mm/s suggest that its electronic structure is best described as low spin ferric iron bound to superoxide anion. A single electron reduction yields activated bleomycin, an EPR-active form that still retains oxygen and which is kinetically competent to initiate DNA cleavage. We have produced this complex by exposing ferrous bleomycin to O2 or by reacting ferric bleomycin with H2O2. The Mössbauer spectra give convincing evidence that the iron of activated bleomycin is low spin ferric. The decay of activated bleomycin yields low spin ferric bleomycin, a complex with Mössbauer parameters nearly identical with those reported for ferric cytochrome P-450. Although iron bleomycin does not have a polyaromatic structure like heme, many features of its electronic structure at the iron are very similar to those produced by the sulfur-coordinated heme iron of ferric cytochrome P-450, a protein that catalyzes a similar oxygen-dependent reaction.

Bleomycin↗
PubMed1983

Mössbauer and EPR studies of activated aconitase: development of a localized valence state at a subsite of the [4Fe-4S] cluster on binding of citrate.

During activation of aconitase a ferrous ion is incorporated into a [3Fe-4S] cluster to yield a structure with a [4Fe-4S] core. Using 57Fe or 56Fe for activation we have studied with Mössbauer spectroscopy the beef heart enzyme in the presence of citrate. Our studies show that the environment of one iron site (Fea) of the [4Fe-4S] cluster is drastically altered in the presence of citrate. Fea is the iron acquired during activation of aconitase. In the oxidized [4Fe-4S]2+ state two species with enzyme-bound substrate are observed, whereas only one is observed in the reduced [4Fe-4S]+ state. The Mössbauer parameters of Fea reveal that the site has acquired substantial high-spin ferrous character. This is most pronounced in the 1+ state where at Fea the cluster exhibits a localized valence state. The dramatic increase of the isomer shift upon substrate binding strongly suggests that the ligand environment of Fea has become at least five-coordinate and that the cluster may function as a Lewis acid. In the absence of citrate the EPR spectra of the active [4Fe-4S]+ enzyme (g1,2,3 = 2.06, 1.93, 1.86) show no hyperfine broadening in the presence of H2 17O. However, in the presence of citrate (g1,2,3 = 2.04, 1.85, 1.78) sizable transferred hyperfine interactions are observed; under the experimental conditions the hydroxyl groups of citrate and isocitrate as well as water are labeled with 17O. We did not detect broadening by 17O-labeled carboxyl groups of citrate in H2 16O. Implications for the mechanism of aconitase are discussed.

Aconitate Hydratase↗
PubMed1982

Mössbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus.

Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase. We have studied 87Fe-enriched samples with Mössbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide. The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes. The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with Mössbauer parameters quite similar to those of deoxymyoglobin. Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes. The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state. We have examined the oxidized enzyme from two different preparations. Both had good activity and identical optical and EPR spectra. The Mössbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations. Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.

Cyanides↗
PubMed1982

Resolution of multiple heme centers of hydroxylamine oxidoreductase from Nitrosomonas. 2. Mössbauer spectroscopy.

Hydroxylamine oxidoreductase (HAO) isolated from Nitrosomas europaea is a complex protein of Mr 220000 with an (alpha beta)3 subunit structure. Each alpha beta subunit contains seven c-type hemes and approximately one unusual prosthetic group termed P-460. We have studied this enzyme in the oxidized and reduced states by using Mössbauer spectroscopy. In the fully reduced enzyme, approximately seven hemes per alpha beta subunit contributed to one spectrum characteristic of low-spin ferrous heme. The remainder of the iron (10-15% of the total) yielded an ill-defined absorption pattern. Carbon monoxide binds to the P-460 as shown by optical spectra. The Mössbauer spectra of reduced hydroxylamine oxidoreductase which had been exposed to CO showed a new spectral component, corresponding to one iron site, with parameters characteristic of a low-spin ferrous heme-carbonyl complex. It appears that this component is derived from the ill-defined spectrum observed in the reduced enzyme. This is the first direct evidence that the P-450 moiety amounts to at least one Fe per alpha beta subunit. Together the Mössbauer results and the optical spectra suggest that the P-460 moiety is a heme. The Mössbauer spectra of the oxidized (as isolated) enzyme suggest the presence of one or two low-spin ferric hemes which might be EPR undetectable because of either fast electronic spin relaxation or participation in a spin-coupled pair. The spectra gave no evidence for the presence of a ferrous site in oxidized HAO.

Carbon Monoxide↗
PubMed1982

Mössbauer studies of beef heart aconitase: evidence for facile interconversions of iron-sulfur clusters.

Beef heart aconitase, isolated under aerobic conditions, has been studied with Mössbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mössbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably of the [3Fe-3S] type, is in a paramagnetic state of interger electronic spin (S = 2); the Mössbauer spectra exhibit al the unique features reported for proteins with 3Fe clusters. On activation of aconitase with ferrous ion, the paramagnetic 3Fe cluster of dithionite-reduced enzyme is converted into a diamagnetic (S = 0) form. Activation studies with iron enriched in either 27 Fe or 56 Fe suggest that activation transforms the 3Fe cluster into a center that has a [4Fe-4S] core. This conclusion is supported by the observation that EPR signals characteristic of reduced [4Fe-4S] clusters can be elicited under appropriate conditions. It has frequently been assumed that the activation of aconitase with Fe2+ produces an active site containing a single ferrous ion. The data reported here suggest that a ferrous ion is used to rebuild a [4Fe-4S] cluster.

Aconitate Hydratase↗
PubMed1981

Mössbauer spectroscopic studies of Escherichia coli sulfite reductase. Evidence for coupling between the siroheme and Fe4S4 cluster prosthetic groups.

Escherichia coli NADPH-sulfite reductase is a complex hemoflavoprotein with an alpha 8 beta 4 subunit structure. The beta-subunits each contain one siroheme and a tetranuclear iron-sulfur center (Fe4S4). Isolated beta-monomers can catalyze the 6-electron reduction of sulfite to sulfide. We have studied the beta-monomers with Mössbauer and EPR spectroscopy. The data show conclusively that the siroheme and the Fe4S4 cluster are strongly exchange-coupled. This is proven by the observations that (a) the two chromophores share a single electronic spin and (b) the addition of 1 electron to oxidized sulfite reductase changes the environments of 5 iron atoms. Spin-sharing is demonstrated in oxidized and 2-electron-reduced sulfite reductase and strongly implicated in 1-electron-reduced material. Thus, sulfite reductase provides the first example of an active site where a heme and an iron-sulfur cluster are closely linked as a functional unit, probably via a common bridging ligand.

Electron Spin Resonance Spectroscopy↗
PubMed1980

Nitrogenase XII. Mössbauer studies of the MoFe protein from Clostridium pasteurianum W5.

We have studied the molybdenum-protein (MoFe protein) from Clostridium pasteurianum with Mössbauer spectroscopy in the temperature range from 1.5 to 200 K in magnetic fields up to 55 kG. Except for some small differences in the hyperfine parameters the results for the C. pasteurianum protein are essentially the same as those published previously for the protein from Azotobacter vinelandii, i.e. (30 +/- 2) Fe atoms partition into two identical cofactor centers M (each center most likely containing six Fe atoms and one Mo atom), four P-clusters (each center containing four Fe atoms), and one iron environment labeled S (about two Fe atoms per holoenzyme). We have analyzed the spectra of the cofactor centers in three distinct oxidation states, Formula: (see test). The diamagnetic (electronic spin S = 0) state MOX is attained by oxidation of the native, EPR-active (S = 3/2) state MN. The reduced state MR is observed in steady state under nitrogen fixing conditions; high-field Mössbauer studies show that the cofactor centers are paramagnetic (integer electronic spin S greater than or equal to 1) in the state MR. We have evaluated the complex high-field spectra resulting from the P-clusters in the oxidized state POX. The analysis shows that one iron site is characterized by a positive hyperfine coupling constant A0 while the other three sites have A0 less than 0. A slightly modified set of parameters also fits the high-field data of the MoFe protein from A. vinelandii. Finally, we will present a discussion summarizing our principle results obtained to date for the proteins from A. vinelandii and C. pasteurianum.

Clostridium↗
PubMed1980

Evidence for a three-iron center in a ferredoxin from Desulfovibrio gigas. Mössbauer and EPR studies.

The tetrameric form of a Desulfovibrio gigas ferredoxin, named Fd II, mediates electron transfer between cytochrome c3 and sulfite reductase. We have studied two stable oxidation states of this protein with Mössbauer spectroscopy and electron paramagnetic resonance. We found 3 iron atoms/monomer and a spin concentration of 0.9 spins/monomer for the oxidized protein. Taken together, the EPR and Mössbauer data demonstrate conclusively the presence of a spin-coupled structure containing 3 iron atoms and labile sulfur. The Mössbauer data show also that this metal center is structurally similar, if not identical, with the low potential center of a ferredoxin from Azotobacter vinelandii, a novel cluster described recently (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796).

Desulfovibrio↗
PubMed1980

Iron-sulfur proteins: spin-coupling model for three-iron clusters.

Recent Mössbauer and EPR studies of two ferredoxins and of aconitase have given evidence for a three-iron cluster, probably of a [3Fe-3S] type. The studies of the oxidized EPR-active centers have shown that the three iron sites are characterized by significantly different magnetic hyperfine coupling constants. For the ferredoxin from Azotobacter vinelandii, for instance, we have observed A1 = -41 MHz, A2 = +18 MHz, and [A3] = 5 MHz. We demonstrate here that the magnetic properties of the clusters can be explained with a simple model of three high-spin ferric ions (S = 5/2) exchange-coupled to a system spin S = 1/2. The model assumes isotropic exchange and different couplings between the iron sites. The results show that the three sites have intrinsic hyperfine interactions similar to those of ferric rubredoxin; the differences in the observed interactions reflect the geometrical features of spin coupling. Furthermore, the three exchange coupling constants are equal within a factor of 2. This implies that the three-iron cluster is a single covalently linked structure and should not be considered as a [2Fe-2S] cluster weakly coupled to a third iron atom.

Azotobacter↗
PubMed1979

Nitrogenase XI: Mössbauer studies on the cofactor centers of the MoFe protein from Azotobacter vinelandii OP.

We have studied the MoFe protein from Azotobacter vinelandii OP with Mössbauer spectroscopy in applied magnetic fields up to 50 kG. The results are as follows. (1) The Mössbauer spectra of the S = 3/2 centers, which reside on the cofactor of nitrogenase, have been decomposed into six subcomponents. This suggests that each center contains 5-7, most probably 6, Fe atoms, thus confirming our earlier conclusions which were based on the quantitation of EPR data and on the assumption that the MoFe protein contains (30 +/- 2) Fe atoms. (2) Analysis of the high-field data shows that three subsites are characterized by a positive magnetic hyperfine coupling constant, A0, while A0 is negative for the other three sites. This observation demonstrates that the S = 3/2 centers are spin-coupled structures. (3) The zero-field splitting parameter D = +(6 +/- 1.5) cm-1 obtained from the Mössbauer data is in good agreement with our earlier EPR results, D approximately +5.5 cm-1. (4) The resolution of the Mössbauer spectra of the MoFe protein can be dramatically increased by employing Fourier transform deconvolution techniques. This allows a clear demonstration of spectral component S.

Azotobacter↗
PubMed1978

Nitrogenase X: Mössbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers.

Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine. Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers. A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal. In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with Mössbauer spectroscopy. Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG. Analysis of the Mössbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms. (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase. The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR. The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen. MR is observed under nitrogen fixing conditions. (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters. Each P-cluster contains four spin-coupled iron atoms. In the native protein the P-clusters are in the diamagnetic state PN, yielding the Mössbauer signature which we have labeled previously 'components D and Fe2+'. Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster. A one-electron oxidation yields the paramagnetic state POX. Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent. Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers.

Azotobacter↗
PubMed1978

Mössbauer study of cytochrome c2 from Rhodospirillum rubrum. Sign of the product gxgygz of some low spin ferric heme proteins.

We have studied cytochrome c2 from Rhodospirllum rubrum with Mössbauer spectroscopy and electron paramagnetic resonance. The Mössbauer data on the ferric protein, taken in external magnetic fields up to 50 kG, were analyzed within the framework of the ligand field model commonly used to evaluate low-spin ferric heme compounds. The data analysis shows that the determinant of the electronic g-tensor, i.e. the product gxgygz, is positive for cytochrome c2. We have reanalyzed published Mössbauer data of some low-spin ferric heme proteins with respect to the sign of the g-tensor determinant. We find that gxgygz is also positive for the cytochromes c, bs, and P-450, and for chloroperoxidase.

Cytochrome c Group↗