Ferredoxin from Halobacterium of the Dead Sea — Mössbauer and EPR spectra and comparison with mössbauer spectrum of whole cells

Recoil-free measurements were carried out on a 2 Fe-ferredoxin, which was isolated and purified from an extreme halophile, Halobacterium of the Dead Sea. The spectrum of this ferredoxin in the oxidized state at 82 K is a superposition of two quadrupole doublets, representing two non-equivalent Fe³⁺ sites of equal intensity. The spectrum of the reduced ferredoxin is consistent with the presence of two pure classes of iron atoms, ferric (lower isomer shift) and ferrous (higher isomer shift). Interpretations of the recoil-free spectra are discussed. Mössbauer measurements were also carried out on frozen whole bacterial cells and the resulting spectrum was found to be quite different from that observed in the isolated ferredoxin. Tentative conclusions are reached concerning the localization of this ferredoxin in the cytosol of the Halobacteria.The EPR spectrum of the reduced ferredoxin obtained at 24 K exhibits rhombic symmetry with the following g values: 1.894, 1.984 and 2.07. These values are similar to those obtained with 2 Fe-ferredoxins of the plant type, except that the g _y and g _z values are somewhat higher. Both from the EPR and Mössbauer data, it is deduced that the spin relaxation times in reduced halophilic ferredoxins are faster than in the reduced plant ferredoxins.     

EPR and Mössbauer studies of benzoyl-CoA reductase

Benzoyl-CoA reductase catalyzes the two-electron transfer from a reduced ferredoxin to the aromatic ring of benzoyl-CoA; this reaction is coupled to stoichiometrical ATP hydrolysis. A very low reduction potential (less than -1 V) is required for the first electron transfer to the aromatic ring. In this work the nature of the redox centers of purified benzoyl-CoA reductase from Thauera aromatica was studied by EPR and M?ssbauer spectroscopy. The results obtained indicated the presence of three [4Fe-4S] clusters. Redox titration studies revealed that the reduction potentials of all three clusters were below -500 mV. The previously reported S = 7/2 state of the enzyme during benzoyl-CoA-independent ATPase activity (Boll, M., Albracht, S. J. P., and Fuchs, G. (1997) Eur. J. Biochem. 244, 840-851) was confirmed by M?ssbauer spectroscopy. Inactivation by oxygen was associated with the irreversible conversion of part of the [4Fe-4S] clusters to [3Fe-4S] clusters. Acetylene stimulated the benzoyl-CoA-independent ATPase activity and induced novel EPR signals with g(av) >2. The presence of simple cubane clusters in benzoyl-CoA reductase as the sole redox-active metal centers demonstrates novel aspects of [4Fe-4S] clusters since they adopt the role of elemental sodium or lithium which are used as electron donors in the analogous chemical Birch reduction of aromatic rings.     

Dynamics of Hemoglobin Investigated by Mössbauer Spectroscopy

Abstract

Mössbauer Spectra of ⁵⁷Fe enriched horse hemoglobin and sperm whale myoglobin were measured in the temperature range from 80 K to 260 K. An analysis of the temperature dependence of the recoiless fraction (the Lamb-Mössbauer factor) shows it to be sensitive to conformational fluctuations which affect the mean square displacement of the iron. We have found that the protein conformation has a dramatic effect on these measurements. For hemoglobin greater conformational fluctuations at lower temperatures are observed for carbonmonoxyhemoglobin in the liganded conformation than for deoxyhemoglobin in the unliganded conformation. On the other hand, the Lamb-Mössbauer factor is insensitive to the binding of ligands to myoglobin and shows conformational fluctuations similar to deoxyhemoglobin even in the liganded state. It is also shown that a reversible complex with the distal histidine is formed in frozen deoxyhemoglobin solutions above 200 K where the Lamb-Mössbauer factor shows the excitation of new modes of conformational fluctuations. This complex is not formed with carbonmonoxyhemoglobin which already has a sixth ligand and with deoxymyoglobin which appears to undergo much more limited conformational fluctuations. A possible relationship between the formation of the distal histidine complex and the cooperative ligand binding reaction is suggested by results with partially liganded hemoglobin which indicate increased formation of the distal histidine complex.     

Mössbauer effect study of gamma-irradiated human oxyhemoglobin

Summary Preliminary results of the Mössbauer effect study of human adult oxyhemoglobin in erythrocytes exposed to gamma-irradiation with doses of 100, 300 and 600 kGy are presented. Mössbauer spectra measured at 87 K have been analyzed in two ways. At first, to fit these spectra we used the four components oxyhemoglobin, deoxyhemoglobin, hemochromes and nonheme Fe(III) compound which had been obtained earlier from Mössbauer spectra of X-irradiated oxyhemoglobin by Chevalier et al. (1983). However, this approximation was not satisfactory. Then a new model of spectral fitting with five components was used. These were oxyhemoglobin, deoxyhemoglobin and components marked1, 2 and3. Using Mössbauer hyperfine parameters of each component the valence/spin states of iron ions were determined and possible complexes were considered. The most probable compounds for components1, 2 and3 were hematin and/orµ-oxodimers, methemoglobin hydroxide and/or hemichromes, and the high spin Fe(III) complex, respectively. Changes of the relative areas of Mössbauer subspectra of all components (its content in samples) versus doses were evaluated and the presence of the high and low spin aquomethemoglobin was indicated.     

Mössbauer studies of ferrihemequinidine complexes

The system ferriprotoporphyrin IX-(+)-quinidine (FPQd) was investigated by Mössbauer spectroscopy at both 4.1 and 90 K. FPQd complexes were prepared by interaction of 10⁻² to 10⁻³ M aqueous solutions of the components at pH 11–12 and 26 °C. Previous investigations of analogous complexes showed characteristic and unusually large circular dichroism bands near 400 nm at alkaline pH values. The present Mössbauer data obtained for FP either in the presence or absence of Qd at both pH 11–12 and 9 indicate identical isomeric shifts in all cases. Both free and complexed FP iron is in a high-spin state. The temperature dependence of the FPQd complex indicates slow spin-spin relaxation at 90 K and fast relaxation at 4.1 K. Qd appears to increase the iron-iron distance of FP in the complexes with references to FP alone, in agreement with previous suggestions on the structure of the complex.     

Mössbauer and NMR studies of protonated acyl diphosphaferrocenes

The acyl derivatives of 3,3′,4,4′-tetramethyldi- phosphaferrocene (TMDPF) have been examined in strong acids by ⁵⁷Fe Mössbauer, ¹H and ³¹P NMR spectroscopy. As with ferrocenyl ketones, protonation was found to occur at the keto function, the diphosphaferrocenyl ketones having comparable or, in some cases, reduced basicities compared to ferrocenyl ketones. [p ]Trends in the ⁵⁷Fe Mössbauer parameters are not as additive as in ferrocene systems due to steric crowding. The keto derivatives show some unusual deuteriation patterns and these have been compared with those of ferrocenyl ketones. The ¹³C spectra of several derivatives have been reported to illustrate the rather complex stereochemistry found in these derivatives.     

125Te Mössbauer spectra of the intercalation compound (Te2)2(I2)

The quadrupole split asymmetric ¹²⁵Te Mössbauer spectrum recorded from the compound (Te₂)₂(I₂), in which monomolecular planar layers of iodine molecules are intercalated between layers of tellurium, is a reflection of the distorted environment of tellurium atoms in a two-dimensional layered compound in which the elongated flat crystals are preferentially orientated. The differences between the Mössbauer parameters recorded from (Te₂)₂(I₂) and those recorded from elemental tellurium and the tellurium(0) species in the compound Te₃Cl₂ are associated with small differences between the environments of tellurium in the three compounds. The Mössbauer spectra recorded from (Te₂)₂(I₂) are consistent with a recently proposed model on which the electronic band structure of (Te₂)₂(I₂) has been derived.     

Investigation of two deoxygenated haemoglobin-Haptoglobin complexes by Mössbauer spectroscopy

Haemoglobin Haptoglobin complexes formed when [Hp⁺]/[Hb] = 1/1 and [Hp]/[Hb] = 2/1 were investigated by ⁵⁷Fe Mössbauer spectroscopy. Both samples gave a spectrum consisting of a single quadrupole doublet. The temperature dependence of the quadrupole splitting was also identical for both samples. This proves that in both samples the nearest neighbour environment of the iron atom must be the same. A comparison with earlier investigations on myoglobin and haemoglobin indicates that the electronic structure of iron in the HbHp-complexes is similar to that in myoglobin.This work was supported by the Deutsche Forschungsgemeinschaft...     

Low temperature study of myoglobin-ligand rebinding kinetics with Mössbauer spectroscopy

We have studied the recombination kinetics of carboxymyoglobin (after photodissociation of the CO ligand) by Mössbauer spectroscopy for temperatures in the range 4.2 – 60 K. The observed kinetics display non-exponential behaviour which was monitored over periods of a few days. It is shown that the time dependence of the kinetics can be reduced to a single universal function of the temperature-dependent variable (t/τ _1/2(T)) ^β(T) . The half-decay time τ _1/2(T) and the scaling parameter β(T) are analysed for the presence of tunneling effects. The non-Arrhenius temperature dependence of the half-decay time below 60 K is interpreted as activated tunneling in models with an Eckart barrier or a fluctuating barrier.     

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.     

The purification and Mössbauer parameters of the haem undecapeptide of cytochrome c

A new method of preparing and purifying the haem undecapeptide of cytochrome c is reported. The Mössbauer spectra of solid samples, lyophilized at pH 7 from water, show mainly the presence of low-spin ferric iron, in contrast with earlier reports. No evidence of temperature dependent spin-spin equilibria was observed. A small proportion of the haem (～ 15%) inhabits an environment distinctly different from that of the majority. These observations are discussed.     

Synthesis,vibrational and mössbauer spectra studies of complexes of organotin chlorides and 2-pyridinecarboxaldehyde thiosemicarbazone

The complex formation between organotin chlorides and 2-pyridinecarboxaldehyde thiosemicarbazone (PT) has been investigated. In only one case is a substitution reaction observed whereas in all other cases, 1:1 addition complexes are formed. The solid state configurations of the complexes have been studied by ^119mSn Mössbauer and far infrared spectroscopy. The chelating ligand (PT) functions as a bidentate ligand towards diorganotin chlorides giving octahedral coordination geometry around the tin atom.     

The fractionation of iron-phosphorus compounds in sediments studied by Mössbauer spectroscopy

Iron compounds of phosphorus form a large part of the phosphorus bound in sediments. Mössbauer spectroscopy is a technique that enables us to study, directly, chemical forms of iron in solid samples. Mössbauer spectroscopy allowed us to check, directly, the selectivity of the extraction scheme for soil phosphorus proposed by Chang &; Jackson (1957), but only as far as the iron compounds are concerned. It appears that selectivity of the extraction method leaves much to be desired.

     

Iron-sulfur clusters of biotin synthase in vivo: a Mössbauer study

Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe M?ssbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.     

Mössbauer and EPR study of recombinant acetyl-CoA synthase from Moorella thermoacetica

M?ssbauer and EPR spectroscopies were used to study the electronic structure of the A-cluster from recombinant acetyl-CoA synthase (the alpha subunit of the alpha2beta2 acetyl-CoA synthase/CO dehydrogenase). Once activated with Ni, these subunits have properties mimicking those associated with the alpha2beta2 tetramer, including structural heterogeneities. The Fe4S4 portion of the A-cluster in oxidized, methylated, and acetylated states was in the 2+ core oxidation state. Upon reduction with dithionite or Ti3+ citrate, samples of Ni-activated alpha developed the ability to accept a methyl group. Corresponding M?ssbauer spectra exhibited two populations of A-clusters; roughly, 70% contained [Fe4S4]1+ cubanes, while approximately 30% contained [Fe4S4]2+ cubanes, suggesting an extremely low [Fe4S4](1+/2+) reduction potential for the 30% portion (perhaps <-800 mV vs NHE). The same population ratio was observed when Ni-free unactivated alpha was used. The 70% fraction exhibited paramagnetic hyperfine structure in the absence of an applied magnetic field, excluding the possibility that it represents an [Fe4S4]1+ cluster coupled to a (proximal) Ni(p)1+. EPR spectra of dithionite-reduced, Ni-activated alpha exhibited features at g = 5.8 and g(ave) approximately 1.93, consistent with a physical mixture of {S = 3/2; S = 1/2} spin-states for A-clusters containing [Fe4S4]1+ clusters. Incubation of Ni-activated alpha with dithionite and CO converted 25% of alpha subunits into the S = 1/2 A(red)-CO state. Previous correlation of this state to functional A-clusters suggests that only the 30% fraction not reduced by dithionite or Ti3+ citrate represents functional A-clusters. Comparison of spin states in oxidized and methylated states suggests that two electrons are required for reductive activation, starting from the oxidized state containing Ni(p)2+. Refitting published activity-vs-potential data supports an n = 2 reductive activation. Enzyme starting in the methylated state exhibited catalytic activity in the absence of an external reductant, suggesting that the two electrons used in reductive activation are retained by the enzyme after each catalytic cycle and that the enzyme does not have to pass through the A(red)-CO state during catalysis. Taken together, our results suggest that a Ni(p)0 state may form upon reductive activation and reform after each catalytic cycle.     

Iron components in PS II particles of spinach by Mössbauer spectroscopy

Mössbauer spectrum measured for the iron components of photosystem II (PS II) particles of spinach is a superposition of 4 doublets. Quadrupole splitting and chemical shifting of doublets I–IV are characteristics of proteins with oxidized cytochrome b-559, reduced cytochrome b-559, Fe³⁺-Q complex and Fe²⁺ -Q complex respectively. After the PS II particles are treated with La³⁺, two doublets of Fe²⁺ disappear and Fe²⁺ is converted into Fe³⁺, indicating that the reduced cytochrome b-559 has been converted into the oxidized cytochrome b-559, and Fe²⁺ -Q complex into Fe³⁺ -Q complex. The Mössbauer spectrum of PS II particles treated with La³⁺ and Ca²⁺ shows that Ca²⁺ can weaken the inhibitory effect of La³⁺ in part, and a portion of the reduced cytochrome b-559 and Fe-Q complex still exist.     

A Mössbauer spectroscopy study of cellular acquisition of iron from pyoverdine byPseudomonas aeruginosa

Summary Mössbauer spectroscopy was used to investigate the cellular acquisition of iron byPseudomonas aeruginosa which had been incubated with ferripyoverdine for 20, 40, 60, 120 or 360 min. Studies revealed that no ferripyoverdine accumulated in the cells at any of these times and that the amounts and kinds of iron complexes produced by cellular metabolism vary with time. At 20 and 40 min a ferric species, with isomer shift =0.38–0.42 mm/s and quadrupole splitting E _Q=0.94–0.92 mm/s, was the major iron metabolite comprising approximately 80% of the iron. At later times at least three other ferric species appeared with =0.54 0.72, E _Q = 0.84 1.07 mm/s. Ferrous species, =1.43 1.77 mm/s and E _Q = 2.69 1.82 mm/s, were also seen at times as early as 20 min and comprised as much as 17% of the total iron at 20 and 40 min. The parameters of all these species identify them as being six-coordinated high-spin complexes. In addition a low-spin species, =0.19 mm/s E _Q=0.67 0.91 mm/s, never before reported in cells, appeared at 60, 120, and 360 min as one of the major iron metabolites (50% or more). All isomer shifts are measured with respect to natural iron.     

Synthesis and reactivity of perchlorate bis(tetrahydrothiophen)gold(I). 197Au Mössbauer spectra of three-coordinate gold(I) complexes

The displacement of tetrahydrothiophen (tht) in [Au(tht)₂]ClO₄ by neutral ligands gives bi-, tri- or tetra-coordinated complexes of the type [Au- (py)₂]ClO₄, [(tht)AuPPh₂NHPPh₂Au(tht)] (ClO₄)₂, [LAu(LL)]ClO₄ (L=tht, PPh₃; LL=bipy, phen) or [AuL₄]ClO₄ (L=PPh₃, AsPh₃ or SbPh₃). The reaction between [Au(tht)₂]ClO₄ and (Bu₄N)[AuR₂] (R=C₆F₅, C₆Cl₅, C₆F₃H₂) yields RAu(tht). The ¹⁹⁷Au Mössbauer spectrum of [LAu(LL)]ClO₄ establishes it as a tri-coordinated species, albeit with an asymmetrically linked bidentate ligand.     

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.