M?ssbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5

The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides. We have studied oxidized samples of the enzyme with M?ssbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01. The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances. One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz. These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. The M?ssbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms. Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated [4Fe-4S] cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a [4Fe-4S] core. The M?ssbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type [4Fe-4S] clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme.     

Unusual features in EPR and M?ssbauer spectra of the 2[4Fe-4Se]+ ferredoxin from Clostridium pasteurianum

The electronic and magnetic properties of the selenium-substituted 2[4Fe-4Se]2+/+ ferredoxin (Fd) from Clostridium pasteurianum have been investigated by EPR and M?ssbauer spectroscopy. The [4Fe-4Se]2+ clusters of oxidized Fd are diamagnetic and the M?ssbauer spectra are nearly identical to those of oxidized 2[4Fe-4S]2+ Fd. The addition of 2e- per molecule of Se-substituted Fd causes the simultaneous appearance of three EPR signals: one (g1,2,3 = 2.103, 1.940, 1.888) is reminiscent of [4Fe-4S]+ EPR spectra and accounts for 0.7 to 0.8 spin/molecule. The two others consist of a broad signal with g = 4.5, 3.5, and approximately 2 (0.7 to 0.8 spin/molecule) and of a narrow peak at g = 5.172 which is observed up to 60 K. Peculiar features are also present in the M?ssbauer spectra of 2[4Fe-4Se]+ Fd below 20 K: a subcomponent with lines near to +/- 4 mm/s and accounting for 20% of the total iron corresponds to two antiferromagnetically coupled sites in approximately a 3:1 ratio and displays fully developed paramagnetic hyperfine interactions at 4.2 K without any applied field. At 77 K, however, the reduced Se-substituted Fd yields a M?ssbauer spectrum similar to that of 2[4Fe-4S]+ Fd. The new EPR and M?ssbauer spectroscopic features of the 2[4Fe-4Se]+ Fd are attributed to S = 3/2 and S = 7/2 spin states which accompany the classical S = 1/2 state of [4Fe-4X]+ (X = S, Se) structures.     

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.     

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 effect in the `super-reduced' form of the high-potential iron–sulphur protein from Chromatium (Short Communication)

Mössbauer-effect studies of the super-reduced form of Chromatium high-potential iron–sulphur protein indicate that the iron atoms are in a similar valency state to those in reduced ferredoxin from Clostridium pasteurianum, with possibly some inequivalence between the iron atoms within the four-iron centre. Mössbauer spectroscopy also shows magnetic differences between the four-iron centres in the two proteins.     

M?ssbauer study of CO dehydrogenase from Clostridium thermoaceticum

We have studied with M?ssbauer spectroscopy the metal clusters of CO dehydrogenase from Clostridium thermoaceticum. At potentials greater than -200 mV, all of the approximately 12 irons reside in diamagnetic environments and contribute a quadrupole doublet characteristic of [Fe4S4]2+ clusters. At lower potentials a variety of components are observed. About 40% of the Fe appears to belong to one [Fe4S4]1+ cluster. We have also observed the M?ssbauer spectrum (approximately 18% of Fe) of the complex which yields EPR with g = 2.01, 1.81, and 1.65. Also present is a doublet (9% of Fe) with delta EQ = 2.90 mm/s and delta = 0.70 mm/s, values typical of a ferrous FeS4 complex. This component seems to interact with a nickel site to form an EPR-silent complex with half-integral electronic spin. We have also characterized the iron environments of the S = 1/2 NiFeC complex. This complex contributes approximately 20% of the total M?ssbauer absorption when the EPR signal has approximately 0.35 spins/12 Fe. From isomer shift comparisons in the oxidized and CO-reacted states of this center, we speculate that the NiFeC complex may consist of a nickel site exchange-coupled to a [Fe4S4]2+ cluster. Finally, the M?ssbauer and EPR data, taken together, force us to conclude that current preparations, while homogeneous according to purifications standards, are spectroscopically heterogeneous, thus rendering the development of a model of the cluster types and compositions in this enzyme premature.     

M?ssbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins

M?ssbauer spectroscopy has indicated significant differences in the iron-containing cores of various haemosiderins. In the present study, haemosiderin was isolated from a number of animal species including man. In addition, haemosiderin was isolated from patients with primary idiopathic haemochromatosis or with secondary (transfusional) iron-overload. The iron cores of the animal and normal human haemosiderin appear to be very similar by M?ssbauer spectroscopy, and the electron diffraction data indicate a ferrihydrite structure similar to that of ferritin cores. The haemosiderin isolated from secondary iron-overload shows anomalous behaviour in its temperature-dependent M?ssbauer spectra. This can be understood in terms of the microcrystalline goethite structure of the cores as indicated by electron diffraction. The haemosiderin cores obtained in the case of primary haemochromatosis have an amorphous Fe(III) oxide structure and show M?ssbauer spectra characteristic of a magnetically disordered material, which only orders at very low temperatures.     

EPR and M?ssbauer studies of nucleotide-bound nitrogenase iron protein from Azotobacter vinelandii

We have recently shown (Lindahl, P. A., Day, E. P., Kent, T. A., Orme-Johnson, W. H., and Münck, E. (1985) J. Biol. Chem. 260, 11160-11173) that the [4Fe-4S]1+ cluster of the native Fe protein can exist in two forms characterized by different cluster spin: an S = 1/2 state exhibiting a g = 1.94 type EPR signal and an S = 3/2 state yielding signals at g approximately 5.8 and 5.1. We have now extended our study of the Fe protein to include the MgATP- and MgADP-bound forms. The [4Fe-4S]1+ cluster of the nucleotide-bound Fe protein exists in a similar S = 1/2, S = 3/2 spin mixture. The S = 3/2 cluster exhibits a broad EPR signal at g approximately 4.8. In spectra of the MgATP-bound protein, we have also observed a g = 4.3 signal from an S = 5/2 state (D = 1 - 3 cm-1, E/D approximately 0.32). Various experiments strongly suggest that this signal does not originate from adventitiously bound Fe3+. The g = 4.3 signal may arise from approximately 2% of the [4Fe-4S]1+ clusters when MgATP is protein-bound. We have also discovered substoichiometric amounts of what appears to be ADP in some nominally nucleotide-free Fe protein samples. MgATP binds to Fe protein in the presence of perturbing solvents, resulting in EPR spectra similar to those of MgATP-bound samples in aqueous solutions; MgADP binding, on the other hand, results in signals more typical of the solvent state. Spectra of samples frozen during turnover of the nitrogenase system exhibit a mixture of spin states. Characterization of the Fe protein EPR signature described here should aid future mechanistic and nucleotide-binding studies.     

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 effect in Scenedesmus and spinach ferredoxins. The mechanism of electron transfer in plant-type iron–sulphur proteins

1. The Mössbauer spectra of Scenedesmus ferredoxin enriched in ⁵⁷Fe were measured and found to be identical with those of two other plant-type ferredoxins (from spinach and Euglena) that had been previously measured. Better resolved Mössbauer spectra of spinach ferredoxin are also reported from protein enriched in ⁵⁷Fe. All these iron–sulphur proteins are known to contain two iron atoms in a molecule that takes up one electron on reduction. 2. The Mössbauer spectra at 195°K have electric hyperfine structure only and show that on reduction the electron goes to one of the iron atoms, the other appearing to remain unchanged. 3. In the oxidized state, both iron atoms are in a similar chemical state, which appears from the chemical shift and quadrupole splitting to be high-spin Fe³⁺, but they are in slightly different environments. In the reduced state the iron atoms are different and the molecule appears to contain one high-spin Fe²⁺ and one high-spin Fe³⁺ atom. 4. At lower temperatures (77 and 4.2°K) the spectra of both iron atoms in the reduced proteins show magnetic hyperfine structure which suggests that the iron in the oxidized state also has unpaired electrons. This provides experimental evidence for earlier suggestions that in the oxidized state there is antiferromagnetic exchange coupling, which would result in a low value for the magnetic susceptibility. 5. In a small magnetic field the spectrum of the reduced ferredoxin shows a Zeeman splitting with hyperfine field (H_n) of 180kG at the nuclei. On application of a strong magnetic field H the spectrum splits into two spectra with effective fields H_n±H, thus confirming the presence of the two antiferromagnetically coupled iron atoms. 6. These results are in agreement with the model proposed by Gibson, Hall, Thornley & Whatley (1966); in the oxidized state there are two Fe³⁺ atoms (high spin) antiferromagnetically coupled and on reduction of the ferredoxin by one electron one of the ferric atoms becomes Fe²⁺ (high spin).     

Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labelsm-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl)-m-(fluorosulfonyl)benzamide] andm-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl)benzamide], which are sensitive to differences betweenα- andγ-thrombin, were quite similar to that ofα-thrombin. That is, no major conformational differences between meizothrombin(desF1) andα-thrombin were observed in this region of the extended active site. On the other hand,p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide],p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl)benzamide], andm-VII [N-[m-(fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl-piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility betweenα-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labelsp-IV,p-V, andm-VII, which corresponds to the apolar binding region, differs in conformation fromα-thrombin.     

M?ssbauer effect in rubredoxin. Determination of the hyperfine field of the iron in a simple iron–sulphur protein

1. Rubredoxin isolated from the green photosynthetic bacterium Chloropseudomonas ethylica was similar in composition to those from anaerobic fermentative bacteria. Amino acid analysis indicated a minimum molecular weight of 6352 with one iron atom per molecule. 2. The circular-dichroism and electron-paramagnetic-resonance spectra of Ch. ethylica rubredoxin showed many similarities to those of Clostridium pasteurianum, but suggested that there may be subtle differences in the protein conformation about the iron atom. 3. Mössbauer-effect measurements on rubredoxin from Cl. pasteurianum and Ch. ethylica showed that in the oxidized state the iron (high-spin Fe³⁺) has a hyperfine field of 370±3kG, whereas in the reduced state (high-spin Fe²⁺) the hyperfine field tensor is anisotropic with a component perpendicular to the symmetry axis of the ion of about −200kG. For the reduced protein the sign of the electric-field gradient is negative, i.e. the ground state of the Fe²⁺ is a [unk] orbital. There is a large non-cubic ligand-field splitting (Δ/k=900°K), and a small spin-orbit splitting (D~+4.4cm⁻¹) of the Fe²⁺ levels. 4. The contributions of core polarization to the hyperfine field in the Fe³⁺ and Fe²⁺ ions are estimated to be −370 and −300kG respectively. 5. The significance of these results in interpretation of the Mössbauer spectra of other iron–sulphur proteins is discussed.     

Hyperfine structure of [57Fe]iron in the M?ssbauer spectrum of the high-potential iron protein from Chromatium

The magnetic hyperfine structure observed in the ⁵⁷Fe Mössbauer spectra of the high-potential iron protein from Chromatium shows that the iron atoms are inequivalent in pairs, with hyperfine fields of 121 and 90kG.     

Photoreducible high spin iron electron paramagnetic resonance signals in dark-adapted Photosystem II: are they oxidised non-haem iron formed from interaction of oxygen with PSII electron acceptors?

An electron paramagnetic resonance (EPR) signal near g=6 in Photosystem II (PSII) membranes has been assigned to a high spin form of cytochrome (Cyt) b(559) (R. Fiege, U. Schreiber, G. Renger, W. Lubitz, V.A. Shuvalov, FEBS Lett. 377 (1995) 325-329). Here we have further investigated the origin of this signal. A slow formation of the signal during storage in the dark is observed in oxygen-evolving PSII membranes, which correlate with the oxidation of Fe(2+) by plastosemiquinone or oxygen. Removal of oxygen inhibits formation of the high spin iron signal. The g=6 EPR signal is photoreduced at cryogenic temperatures and is restored slowly by subsequent dark storage at 77 K. The amplitude of the photoreduced signal increases as the pH is lowered, which shows that the origin is not the hydroxyl ligated Cyt b(559) species proposed previously. Different cryoprotectants also influence the amplitude and lineshape of the high spin iron signal in a manner suggesting that smaller cryoprotectants can penetrate the iron environment. A correlation between the high spin iron and g=1.6 EPR signal assigned to an interaction involving the semiquinones of Qa and Qb is shown. It is concluded that the appearance of the high spin iron signal in oxygen-evolving PSII membranes involves reduced PSII electron acceptors and oxygen and suggests that the signal is from the non-haem iron of PSII.     

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MCD 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 δ=0.24±0.01?mm/s at 4.2?K rather than δ=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 δ=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.     

A Mössbauer spectroscopic study of the form of iron in iron overload

There are major differences in the temperature dependence of the Mössbauer spectra of ferritin and haemosiderin extracted from the organs of humans suffering from transfusional iron overload. Iron overload can also occur in animal systems as a result of artificial treatments or dietary factors. None of the animal systems which were investigated in the present study showed evidence in their Mössbauer spectra for the presence of the haemosiderin found in transfusional iron overload in humans. This suggests that the haemosiderin which occurs in the case of human transfusional iron overload may be specific to that situation.     

Mössbauer and EPR study of iron in vacuoles from fermenting Saccharomyces cerevisiae

Vacuoles were isolated from fermenting yeast cells grown on minimal medium supplemented with 40 μM (57)Fe. Absolute concentrations of Fe, Cu, Zn, Mn, Ca, and P in isolated vacuoles were determined by ICP-MS. M?ssbauer spectra of isolated vacuoles were dominated by two spectral features: a mononuclear magnetically isolated high-spin (HS) Fe(III) species coordinated primarily by hard/ionic (mostly or exclusively oxygen) ligands and superparamagnetic Fe(III) oxyhydroxo nanoparticles. EPR spectra of isolated vacuoles exhibited a g(ave) ~ 4.3 signal typical of HS Fe(III) with E/D ~ 1/3. Chemical reduction of the HS Fe(III) species was possible, affording a M?ssbauer quadrupole doublet with parameters consistent with O/N ligation. Vacuolar spectral features were present in whole fermenting yeast cells; however, quantitative comparisons indicated that Fe leaches out of vacuoles during isolation. The in vivo vacuolar Fe concentration was estimated to be ~1.2 mM while the Fe concentration of isolated vacuoles was ~220 μM. M?ssbauer analysis of Fe(III) polyphosphate exhibited properties similar to those of vacuolar Fe. At the vacuolar pH of 5, Fe(III) polyphosphate was magnetically isolated, while at pH 7, it formed nanoparticles. This pH-dependent conversion was reversible. Fe(III) polyphosphate could also be reduced to the Fe(II) state, affording similar M?ssbauer parameters to that of reduced vacuolar Fe. These results are insufficient to identify the exact coordination environment of the Fe(III) species in vacuoles, but they suggest a complex closely related to Fe(III) polyphosphate. A model for Fe trafficking into/out of yeast vacuoles is proposed.     

Identification of FeS clusters in the glycyl-radical enzyme benzylsuccinate synthase via EPR and M?ssbauer spectroscopy

The anaerobic degradation pathway of toluene is initiated by the addition of the methyl group of toluene to the double bond of fumarate. This reaction is catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase (BSS). The enzyme consists of three subunits, α, β, and γ, and differs from most other glycyl-radical enzymes in having additional cofactors. We have purified a Strep-tagged nonactivated BSS from recombinant Escherichia coli and identified the additional cofactors as FeS clusters by UV/vis, EPR, and M?ssbauer spectroscopy. Analysis of the metal content as well as the EPR and M?ssbauer spectra indicated that BSS contains magnetically coupled low-potential [4Fe–4S] clusters. Several enzyme preparations showed differing amounts of [3Fe–4S] clusters that could be reconstituted to [4Fe–4S] clusters, indicating that they arise from partial decay of the initial [4Fe–4S] clusters. The most likely location of these FeS clusters in the enzyme are the small β and γ subunits, which are unique for the BSS subfamily of glycyl-radical enzymes and contain conserved cysteines as potential ligands.     

The initial characterization of the iron environment in lipoxygenase by M?ssbauer spectroscopy

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using M?ssbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in M?ssbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect M?ssbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(II) M?ssbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The M?ssbauer spectra (4.2-250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm/s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these M?ssbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III).diethylenetriaminepentaacetic acid.     

M?ssbauer studies of adrenodoxin. The mechanism of electron transfer in a hydroxylase iron–sulphur protein

1. Mössbauer spectra were measured of adrenodoxin purified from porcine adrenal glands. They show similarities to the spectra of the plant ferredoxins. All of these proteins contain two atoms of iron and two of inorganic sulphide per molecule, and on reduction accept one electron. 2. As with the plant ferredoxins the adrenodoxin for these measurements was enriched with ⁵⁷Fe by reconstitution of the apo-protein, and subsequently was carefully purified and checked by a number of methods to ensure that it was in the same conformation as the native protein and contained no extraneous iron. 3. The Mössbauer spectra of oxidized adrenodoxin at temperatures from 4.2°K to 197°K show that the iron atoms are probably high-spin Fe³⁺, and in similar environments, and experience little or no magnetic field from the electrons. 4. Mössbauer spectra of reduced adrenodoxin showed magnetic hyperfine structure at all temperatures from 1.7°K to 244°K, in contrast with the reduced plant ferredoxins, which showed it only at lower temperatures. This is a consequence of a longer electron-spin relaxation time in reduced adrenodoxin. 5. At 4.2°K in a small magnetic field the spectrum of reduced adrenodoxin shows a sixline Zeeman pattern due to Fe³⁺ superimposed upon a combined magnetic and quadrupole spectrum due to Fe²⁺. 6. In a large magnetic field (30kG) each hyperfine pattern is further split into two. Analysis of these spectra at 4.2°K and 1.7°K shows that the effective fields at the Fe³⁺ and Fe²⁺ nuclei are in opposite directions. This agrees with the proposal, first made for the ferredoxins, that the iron atoms are antiferromagnetically coupled. 7. In accord with the model for the ferredoxins, it is proposed that the oxidized adrenodoxin contains two high-spin Fe³⁺ atoms which are antiferromagnetically coupled; on reduction one iron atom becomes high-spin Fe²⁺.