Mössbauer spectroscopic studies on tetra(sulphonaphthyl)porphine iron(II) solutions

Mössbauer (78 K) and electronic absorption spectra (room temperature) of tetra(sulphonaphthyl)- porphine iron(II) solutions are reported and discussed. Evidence for only two iron(II) electronic environments, a low spin and a high spin site is found. The nature of each iron(II) environment is deduced with reference to previous work. The influence of the steric bulk of the meso substituents is discussed in comparison to similar studies on protoporphyrin IX iron(II) solutions and tetra(p-sulphophenyl)iron(II) solutions. The presence of the napthyl substituents on the methine carbons stabilises the low spin iron(II) species containing two axial water ligands.     

Mössbauer and spectroscopic studies on substituted tetraphenylporphyrinato iron(III) complexes in aqueous solutions and the formation of the μ-oxo-bridged species

Electronic and ⁵⁷Fe Mössbauer spectra are reported for two new water-soluble porphyrinato iron(III) complexes. Equilibrium constants for μ-oxo bishaem formation are calculated assuming two protons are released.Comparisons are made of the data with other porphyrinato iron(III) systems and it is shown that, in the absence of well-defined fifth ligands, the mononuclear species in acidic solution probably contain two axial water ligands. The μ-oxo bishaems do not contain water or hydroxide coordinated to iron but may hold water by hydrogen-bonding to the oxygen bridge or possibly by aquation of the porphyrin ligands.μ-Oxo bridge formation is controlled by the acid strength of the water coordinated to the iron in the mononuclear species, low pK_a values assisting oxo-bridge formation. Such low pK_a values are assisted by electron-attracting substituents on the porphyrin periphery. It is noted that this same property assists the stabilisation of iron(II) complexes. Steric inhibition of oxo-bridge formation requires large substituents, unsubstituted phenyl groups being apparently not large enough.     

Solution studies on tetra(p-carboxyphenyl)porphyrin iron(II) using Mössbauer spectroscopy and electronic absorption spectroscopies

Mössbauer (78 K) and electronic absorption spectra (298 K) of tetra(p-carboxyphenyl)porphyrin iron(II) solutions are reported and discussed. Evidence for only two iron(II) complexes, the first an intermediate spin and the second a high spin complex, is found in the Mössbauer spectra. Electronic absorption spectra show a low spin complex is present at very low concentrations. It is observed from these results that the carboxy groups on the phenyl rings of this porphyrin greatly influence the chemistry. From the difference in the quadrupole splitting for the intermediate spin complex compared to that found in the tetra(p-sulphophenyl)porphyrin iron(II) system, the substituent on the phenyl ring clearly changes the electron density on the pyrrole nitrogen atoms.     

Evidence for a mu-oxo-bridged binuclear iron cluster in the hydroxylase component of methane monooxygenase. M?ssbauer and EPR studies

M?ssbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The M?ssbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the M?ssbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.     

Mössbauer spectroscopic studies on protoporphyrin IX iron(III) cyanide complexes

The Mössbauer and other spectral data of a dicyanoprotoporphyrin IX iron(III) complex are reported. The low quadrupole splitting is discussed in relation to known low-spin iron(III) porphyrin structures and their Mössbauer parameters.     

Emission M?ssbauer studies of some organocobalamins

Subtle changes in the M?ssbauer parameters are observed while going from methyl- to ethyl- to adenosylcobalamin, and also when the "base" is detached from the cobalt. The observation of these changes demonstrates that the Co-C bond, among others, remains intact after the Auger event, accompanying the electron-capture decay of the cobalt-57. The differences between ethylcobalamin and the other two organocobalamins in the magnitude of the quadrupole splittings have been interpreted on the basis of the sigma-donating tendency of the organic moiety and the Co-C bond length. The latter is presumably determined by the steric hindrance offered to the group in approaching the cobalt atom. The ethyl- and adenosylcobalamins in their "base-off" form exhibit a larger quadrupole splitting than the corresponding "base-on" form. In the "bas-off" form, the cobalt atom is perhaps raised above the mean plane of the four equatorial nitrogen atoms of the corrin ring, which may result in the diminution of the delocalization of the 3dpi electron density. The higher population of dpi orbitals and the enhanced metallic character of the dz2, resulting from shrinkage of the Co-C bond length, enhances the magnitude of the quadrupole splitting.     

M?ssbauer 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 M?ssbauer 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.     

Is Fe deficiency rather than P deficiency the cause of cluster root formation in Casuarina species?

When subjected, directly (through nutritional deficiencies) or indirectly (through alkaline constraints leading to such deficiencies) to nutrient deficiencies, certain plants respond by developing special root structures called cluster roots. This phenomenon can be considered as an ecophysiological response to a specific nutrient deficiency enabling plants to enhance nutrient uptake. Experiments conducted on an alkaline and an acid soil showed that Casuarina glauca (Sieber ex Spreng.) produced cluster roots only in the alkaline soil and not in the acid soil. In addition, iron (Fe) and phosphorus (P) deficiencies were examined separately or together to determine their effect on cluster root formation in C. glauca seedlings grown hydroponically. Results from experiments carried out on three Casuarina species (C. glauca, C. cunninghamiana Miq. and C. equisetifolia L.) indicated that Fe is involved in cluster root formation. In nutrient media lacking P but containing Fe, no cluster roots formed while seedlings receiving P and lacking Fe developed cluster roots. When incubated on chrome-azurol S-agar on blue plates (CAS assay), a technique used routinely to detect the production of siderophores by micro-organisms, the root system of Fe-deficient plants exhibited orange halos around cluster roots, indicating production of a ferric-chelating agent. It is concluded that the capacity of cluster roots of C. glauca to chelate Fe allows the plant to grow normally on alkaline soils.     

M?ssbauer effect and electron paramagnetic resonance studies on yeast aconitase.

M?ssbauer effect and electron paramagnetic resonance (EPR) were measured for yeast aconitase [EC 4.2.1.3] purified from the cells of Candida lipolytica (ATCC 200114). M?ssbauer spectra suggested that yeast aconitase nostly contained two high-spin Fe(III) ions in an antiferromagnetically coupled binuclear complex that resembled oxidized 2 Fe ferredoxins, together with a small amount of high-spin Fe(II). EPR spectra recorded no signal at 77degreesK, but showed a slightly asymmetric signal centered at g=2.0 at 4.2degreesK, presumably due to the small amount of Fe(II) Fe(III) pairs.     

Mössbauer spectroscopy on respiratory complex I: the iron-sulfur cluster ensemble in the NADH-reduced enzyme is partially oxidized

In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-cluster chain extending from the flavin) are (at least partially) reduced by NADH. The other three clusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyme: they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use M?ssbauer spectroscopy on (57)Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent clusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential clusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential clusters. The implications for intramolecular electron transfer along the extended chain of cofactors in complex I are discussed.     

M?ssbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates

Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using M?ssbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic M?ssbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room temperature before freezing). The measurements made at 4.1 and 90 K showed that for samples with n less than or equal to 40 at pH greater than or equal to 6.25 all iron was trivalent at tf = 3 min. Four different Fe(III) species were identified: solitary Fe(III) atoms giving relaxation spectra, which can be identified with the species observed before by EPR and UV difference spectroscopy; oxo-bridged dimers giving doublet spectra with large splitting, observed for the first time in ferritin; small Fe(III) clusters giving doublets of smaller splitting and larger antiferromagnetically coupled Fe(III) clusters, similar to those found previously in larger ferritin iron cores, which, for samples with n greater than or equal to 40, gave magnetically split spectra at 4.1 K. Both solitary Fe(III) and dimers diminished with time, suggesting that they are intermediates in the formation of the iron core. Two kinds of divalent iron were distinguished for n = 480, which may correspond to bound and free Fe(II).     

Mössbauer spectrum and magnetic behavior of the iron(II)-saccharinate complex

The room temperature ⁵⁷Fe-Mössbauer spectrum and magnetic susceptibility measurements, in a wide temperature range, demonstrate that the iron-tetraaquabis(saccharinate) complex contains Fe(II) as a high spin species in an octahedral environment. The isostructural Ni(II) complex, measured for comparative purposes, shows a similar magnetic behavior.     

M?ssbauer and electron paramagnetic resonance studies of the cytochrome bf complex.

The (57)Fe-enriched cytochrome bf complex has been isolated from hydrocultures of spinach. It has been studied at different redox states by optical, EPR, and M?ssbauer spectroscopy. The M?ssbauer spectrum of the native complex at 190 K with all iron centers in the oxidized state reveals the presence of four different iron sites: low-spin ferric iron in cytochrome b [with an isomer shift (delta) of 0.20 mm/s, a quadrupole splitting (DeltaE(Q)) of 1.77 mm/s, and a relative area of 40%], low-spin ferric iron of cytochrome f (delta = 0.26 mm/s, DeltaE(Q) = 1.90 mm/s, and a relative area of 20%), and two high-spin ferric iron sites of the Rieske iron-sulfur protein (ISP) with a bis-cysteine and a bis-histidine ligated iron (delta(1) = 0.15 mm/s, DeltaE(Q1) = 0.70 mm/s, and a relative area of 20%, and delta(2) = 0.25 mm/s, DeltaE(Q2) = 0.90 mm/s, and a relative area of 20%, respectively). EPR and magnetic M?ssbauer measurements at low temperatures corroborate these results. A crystal-field analysis of the EPR data and of the magnetic M?ssbauer data yields estimates for the g-tensors (g(z)(), g(y)(), and g(x)()) of cytochrome b (3.60, 1.35, and 1.1) and of cytochrome f (3.51, 1.69, and 0.9). Addition of ascorbate reduces not only the iron of cytochrome f to the ferrous low-spin state (delta = 0.43 mm/s, DeltaE(Q) = 1.12 mm/s at 4.2 K) but also the bis-histidine coordinated iron of the Rieske 2Fe-2S center to the ferrous high-spin state (delta(2) = 0.73 mm/s, DeltaE(Q2) = -2.95 mm/s at 4.2 K). At this redox step, the M?ssbauer parameters of cytochrome b have not changed, indicating that the redox changes of cytochrome f and the Rieske protein did not change the first ligand sphere of the low-spin ferric iron in cytochrome b. Reduction with dithionite further reduces the two hemes of cytochrome b to the ferrous low-spin state (delta = 0.49 mm/s, DeltaE(Q) = 1.08 mm/s at 4.2 K). The spin Hamiltonian analysis of the magnetic M?ssbauer spectra at 4.2 K yields hyperfine parameters of the reduced Rieske 2Fe-2S center in the cytochrome bf complex which are very similar to those reported for the Rieske center from Thermus thermophilus [Fee, J. A., Findling, K. L., Yoshida, T., et al. (1984) J. Biol. Chem. 259, 124-133].     

M?ssbauer 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 M?ssbauer 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 M?ssbauer 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 M?ssbauer 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 M?ssbauer spectrum of peroxo Delta 9D, showing that the released O(2) was effectively sequestered within the active site.     

M?ssbauer studies of cytochrome c' from Rhodospirillum rubrum.

Cytochrome c' from Rhodospirillum rubrum has been investigated in the ferric form with M?ssbauer and EPR spectroscopy. In the pH range from 6 to 9.5, three species are observed which belong to two pH-dependent equilibria with pK values near 6 and 8.5. The pK = 6 transition is resolved only with high-field M?ssbauer spectroscopy. For the three species we have determined the zero-field splitting parameters and the hyperfine coupling constants. The data were fitted to a spin Hamiltonian which takes into account a weak mixing of excited S = 3/2 states into the sextet ground manifold. The low temperature spectra clearly show that the quadruple coupling constant deltaEQ is positive for ferricytochrome c' and thus in accord with all other high-spin ferric heme proteins.     

M?ssbauer studies on the active Fe ... [2Fe-2S] site of putidamonooxin, its electron transport and dioxygen activation mechanism

Putidamonooxin, the oxygenase of a 4-methoxybenzoate monooxygenase enzyme system, catalyzes the oxidative O-demethylation of the substrate 4-methoxybenzoate in conjunction with the NADH:putidamonooxin oxidoreductase. Putidamonooxin is a conjugated iron-sulfur protein which needs iron ions as cofactors for its enzymatic activity. Putiamonooxin was isolated from Pseudomonas putida, which was grown on a 57Fe-enriched culture medium. Thus putidamonooxin was enriched in vivo with 57Fe up to about 80%. During our M?ssbauer study of putidamonooxin a number of parameters have been varied: (a) the oxidation state of putidamonooxin (oxidized, reduced and aerobically reoxidized); (b) the substrate bound to putidamonooxin (4-methoxybenzoate, benzoate, 4-tert-butylbenzoate); (c) the temperature between 2.7 K and 245 K; (d) the applied magnetic field between 0 and 0.1 T and (e) the amount of iron cofactor. From our M?ssbauer results it is obvious that the iron-sulfur centers of putidamonooxin are [2 Fe-2S] clusters similar to those of the plant-type ferredoxins. Further, we have evidence for the existence of iron ions (one per [2 Fe-2S] cluster), which serve as cofactors for the dioxygen activation, functioning as the dioxygen binding site and mediating the electron flow from the [2 Fe-2S] cluster to dioxygen.     

M?ssbauer studies of solid thionin-oxidized MoFe protein of nitrogenase

Recently Hagen et al. (Hagen, W. R., Wassink, H., Eady, R. R., Smith, B. E., and Haaker, H. (1987) Eur. J. Biochem. 169, 457-465) reported the observation of S = 7/2 EPR signals for thionin-oxidized nitrogenase MoFe protein. Here we have studied the protein from Azotobacter vinelandii and Klebsiella pneumoniae with M?ssbauer and EPR spectroscopies, with the following results: when the MoFe protein is oxidized by addition of stoichiometric amounts (6-8 equivalents) of dissolved thionin, the well characterized P-cluster state Pox results. Pox has an as yet undetermined, but half-integer electronic spin; however, the state is EPR-silent. In contrast, oxidation by addition of a large excess of solid thionin powder, the method used by Hagen et al., yields mixtures with variable proportions of two oxidized P-cluster forms, namely the familiar Pox and the new state Pox(S = 7/2) observed by Hagen et al. The M?ssbauer data suggest that Pox and Pox(S = 7/2) are isoelectronic. The two states, however, have distinct electronic structures; the M?ssbauer spectra of Pox exhibit the characteristic trapped-valence Fe2+ site, whereas the spectra of Pox(S = 7/2) lack this feature. Hagen et al. have proposed two new P-cluster models. We conclude that one of the models is incompatible with the M?ssbauer data and that the basic assumptions of the other model are not supported by the available data. Finally, the M?ssbauer data show that either oxidation method puts the cofactor centers into the diamagnetic state Mox.