On the structure of the iron-sulfur complex in the two-iron ferredoxins

Recent spectroscopic and magnetic susceptibility studies of the iron center in the two-iron ferredoxins provide criteria which any model for the iron-sulfur complex in these proteins must satisfy. These criteria are most stringent for parsley and spinach ferredoxin: the reduced proteins contain a high-spin ferric atom antiferromagnetically exchange-coupled (presumably via sulfide bridging ligands) to a high-spin ferrous atom. In the oxidized proteins the iron atoms are antiferromagnetically spin-coupled, high-spin ferric atoms. Arguments are given to substantiate the claim that the ferrous atom in the reduced protein is ligated by four sulfur atoms in a distorted tetrahedral configuration: two are the bridging sulfides, two are cysteinyl sulfurs. A treatment of proton contact shifts based upon the above model is pertinent to proton magnetic resonance data already available and provides a means to identify directly the ligands at both iron atoms via further PMR experiments.     

Probing magnetic properties of the reduced [2Fe-2S] cluster of the ferredoxin from Arthrospira platensis by 1H ENDOR spectroscopy

The 1H electron nuclear double resonance (ENDOR) spectra in frozen solutions of the reduced [2Fe-2S] cluster in ferredoxin from Arthrospira (Spirulina) platensis have been measured at low temperatures (5-20 K) and simulated using orientational selection methods. The analysis confirmed the existence of a single paramagnetic species with iron valence states II and III connected uniquely to the cluster irons. The experimental ENDOR spectra were fitted to a model including the spin distribution on the centre, the orientation of the g-matrix, and the isotropic and anisotropic hyperfine couplings of the nearest protons in the crystallographically determined structure. In order to partially simulate ENDOR line shapes, a statistical distribution of the corresponding torsion angles between the Fe(III) centre and one of the beta-CH2 protons was introduced. From the analysis, four of the larger hyperfine couplings found were assigned to the cysteine beta-protons near the Fe(III) ion of the cluster, with isotropic hyperfine couplings ranging from 1.6 to 4.1 MHz. The spin distribution on the two iron ions was estimated to be +1.85 for the Fe(III) ion and -0.9 for the Fe(II) ion. The Fe(III) ion was identified as being coordinated to the cysteine ligands Cys49 and Cys79, confirming previous NMR results. The direction of the g-tensor with respect to the cluster was deduced. The g1-g2 plane is parallel to the planes through each iron and its adjacent cysteine sulfurs; the g2-g3 plane is nearly perpendicular to the latter planes and deviates by 25 degrees from the FeSSFe plane. The g1 direction is dominated by the bonding geometry of Fe(II) and does not align with the Fe(II)-Fe(III) vector.     

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.     

Characterization of the [4Fe-4S]+ cluster at the active site of aconitase by 57Fe, 33S, and 14N electron nuclear double resonance spectroscopy

57Fe, 33S, and 14N electron nuclear double resonance (ENDOR) studies have been performed to characterize the [4Fe-4S]+ cluster at the active site of aconitase. Q-band 57Fe ENDOLR of isotopically enriched enzyme, both substrate free and in the enzyme-substrate complex, reveals four inequivalent iron sites. In agreement with M?ssbauer studies [Kent et al. (1985) J. Biol. Chem. 260, 6371-6881], one of the iron ions, Fea, which is easily removed by oxidation to yield the [3Fe-4S]+ cluster of inactive aconitase, shows a dramatic change in the presence of substrate. The remaining iron sites, Feb1,2,3, show minor changes when substrate is bound. Methods devised by us for analyzing and simulating ENDOR spectra of a randomly oriented paramagnet have been used to determine the principal values and orientation relative to the g tensor for the hyperfine tensors of three of the four inequivalent iron sites of the [4Fe-4S]+ cluster, Fea, Feb2, and Feb3, in the substrate-free enzyme and the enzyme-substrate complex. The full tensor for the fourth site, Feb1, could not be obtained because its signal is seen only over a limited range of the EPR envelope. 33S ENDOR data for the enzyme-substrate complex using enzyme reconstituted with 33S show that the four inorganic bridging sulfide ions of the [4Fe-4S]+ cube have isotropic hyperfine couplings of A(S) less than 12 MHz, and analysis indicates that they can be divided into two pairs, one with couplings of A(S1) approximately less than 1 MHz and the other with A(S2) approximately 6-12 MHz; the analysis further places these pairs within the cube relative to the iron sites. 33S data for substrate-free enzyme is qualitatively similar and can be completely simulated by two types of S2- ion, with A(S1) approximately 7.5 and A(S2) approximately 9 MHz; the full hyperfine tensors have been determined. The hyperfine values for the two enzyme forms correspond to surprisingly small unpaired spin density on S2-. 14N ENDOR at Q-band reveals a nitrogen signal that does not change upon substrate binding.     

Magnetic studies of the four-iron high-potential, non-heme protein from Chromatium vinosum.

Extensive EPR studies on high-potential, iron-sulfur protein from Chromatium vinosum indicate that the singular spectrum of this four-iron, non-heme protein consists of a superposition of three distinct signals; namely, two principal signals of equal weight, one reflecting axial and the other rhombic symmetry, and a third nearly isotropic minority component. In addition, magnetic susceptibility experiments on two oxidation states of the protein from 4.2 to approx. 260 degrees K indicate antiferromagnetic exchange coupling between iron atoms. Possible origins of the complex EPR signals are discussed, and a preferred model that is consistent with EPR, magnetic susceptibility, NMR, X-ray, and M?ssbauer data is presented.     

Single-crystal EPR study at 95 GHz of the type 2 copper site of the inhibitor-bound quercetin 2,3-dioxygenase

An electron-spin-echo-detected, electron-paramagnetic-resonance study has been performed on the type 2 copper site of quercetin 2,3-dioxygenase from Aspergillus japonicus. In the protein, copper is coordinated by three histidine nitrogens and two sulfurs from the inhibitor diethyldithiocarbamate. A single crystal of the protein was studied at 95 GHz and the complete g-tensor determined. The electron-paramagnetic-resonance data are compatible with two orientations of the principal g-axes in the copper center, one of which is preferred on the basis of an analysis of the copper coordination and the d-orbitals that are involved in the unpaired-electron orbital. For this orientation, the principal z-axis of the g-tensor makes an angle of 19 degrees with the Cu-N(His112) bond and the N of His112 may be considered the axial ligand. The singly occupied molecular orbital contains a linear combination of copper dxy and dyz-orbitals, which are antibonding with atomic orbitals of histidine nitrogens and diethyldithiocarbamate sulfurs. The orientation of the g-tensor for the quercetin 2,3-dioxygenase is compared with that for type 1 copper sites.     

Redox-dependent structure change and hyperfine nuclear magnetic resonance shifts in cytochrome c

Proton nuclear magnetic resonance assignments for reduced and oxidized equine cytochrome c show that many individual protons exhibit different chemical shifts in the two protein forms, reflecting diamagnetic shift effects due to structure change, and in addition contact and pseudocontact shifts that occur only in the paramagnetic oxidized form. To evaluate the chemical shift differences (delta delta) for structure change, we removed the pseudocontact shift contribution by a calculation based on knowledge of the electron spin g tensor. The g-tensor parameters were determined from the delta delta values of a large set (64) of C alpha H protons at well-defined spatial positions in the oxidized horse protein. The g-tensor calculation, when repeated using only 12 available C alpha H proton resonances for cytochrome c from tuna, proved to be remarkably stable. The largest principal value of the g tensor (gz) falls precisely along the ligand bond between the heme iron and methionine-80 sulfur, while gx and gy closely match the natural heme axes defined by the pyrrole nitrogens. The derived g tensor was then used together with spatial coordinates for the oxidized form to calculate the pseudocontact shift contribution (delta pc) to proton resonances at 400 identifiable sites throughout the protein, so that the redox-dependent chemical shift discrepancy, delta delta-delta pc, could be evaluated. Large residual changes in chemical shift define the Fermi contact shifts, which are found as expected to be limited to the immediate covalent structure of the heme and its ligands and to be asymmetrically distributed over the heme. Smaller chemical shift discrepancies point to a concerted change, involving residues 39-43 and 50-60 (bottom of the protein), and to other changes in the immediate vicinity of the heme ligands. Also, the three internal water molecules are implicated in redox sensitivity. The residues found to change are in good but not perfect agreement with prior X-ray diffraction observations of subangstrom redox-related displacements in the tuna protein. The chemical shift discrepancies observed appear in the main to reflect structure-dependent diamagnetic shifts rather than hyperfine effects due to displacements in the pseudocontact shift field. Although 51 protons in 29 different residues exhibit significant chemical shift changes, the general impression is one of small structural adjustments to redox-dependent strain rather than sizeable structural displacements or rearrangements.     

Ferredoxin from a liverwort, Marchantia polymorpha. Purification and amino acid sequence

Marchantia polymorpha ferredoxin was purified by DE-52 and Sephadex G-75 column chromatographies to homogeneity. The complete amino acid sequence of the carboxymethylated (Cm) ferredoxin was determined by conventional methods to be as follows. Thr-Phe-Lys- Val-Thr-Leu-Asn-Thr-Pro-Thr-Gly-Gln-Ser-Val-Ile-Asp-Val-Glu-Asp- Asp-Glu-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Ser-Leu-Pro- Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Thr- Ala-Gly-Glu-Val-Asp-Gln-Ser-Asp-Glu-Ser-Phe-Leu-Asp-Asp-Asp-Gln- Met-Asp-Glu-Gly-Tyr-Val-Leu-Thr-Cys-Ile-Ala-Tyr-Pro-Thr-Ser-Asp- Leu-Thr-Ile-Asp-Thr-His-Gln-Glu-Glu-Ala-Leu-Ile. The total number of amino acid residues was 95 and the molecular weight was calculated to be 10,174, excluding iron and sulfur atoms. The distribution of the four cysteine residues chelating the two iron atoms was identical to those of other [2Fe-2S] ferredoxins. The relationship between M. polymorpha and other plants was discussed in terms of plant phylogeny.     

Single crystal EPR of myoglobin nitroxide. Freezing-induced reversible changes in the molecular orientation of the ligand

Single crystals of myoglobin nitroxide (MbNO) are examined by the electron paramagnetic resonance spectroscopy at ambient and cryogenic temperatures for both the 14NO and 15NO derivatives. The principal values and the eigenvectors of the g tensor and the hyperfine coupling tensor are determined: g xx = 2.050, g yy = 2.022, and g zz = 1.993; A xi xi = 15.6, A zeta zeta = 21.4, and A eta eta = 26.7 G for the nitrogen in 15NO at ambient temperature. The Fe--N--O bond angle is calculated to be 153 degrees. This result is in good agreement with the x-ray structural result on the six-liganded model compound with the bent Fe--N--O configuration. The principal values and the eigenvectors of the g tensor and the hyperfine coupling tensor are also determined at 77 K for Mb15NO; gxx = 2.076, gyy = 1.979, and gzz = 2.002; A xi xi = 21, A zeta zeta = 24, and A eta eta = 27 G. The Fe--N--O bond angle is calculated to be 109 degrees. The hyperfine splittings attributed to N epsilon atom of proximal histidine are observed in the direction of the gzz at both temperatures. The drastic shift of the EPR spectrum of MbNO single crystal is observed below the freezing point of this crystal. It clearly demonstrates that the conformation of the bonding NO is drastically altered upon freezing. The temperature dependence of the EPR spectra of MbNO below the freezing point cannot be explained only by appropriate combinations of the higher temperature type and the lower temperature type and suggests the contribution from an unpaired electron with the iron dz2 and dyz (or dxz) orbitals. The present EPR results demonstrated that the changes in the molecular orientations are induced by freezing of the biological molecules without disorder of the crystal lattice.     

Isotope dilution mass spectrometry for the quantification of sulfane sulfurs

Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that allows them to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H₂S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfur concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulfide product, PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a ¹³C₃-labeled phosphine sulfide, PS1, as the internal standard. The specificity and efficiency of the method were proven by model reactions. It was also applied to the measurement of sulfane sulfurs in mouse tissues including brain, kidney, lung, liver, heart, spleen, and blood.     

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.     

The structure of the iron complex in metaquohemerythrin

The novel binuclear iron complex in metaquohemerythrin is described. One of the two iron atoms is octahedrally coordinated, the other being penta-coordinate. A number of questions concerning the structure of the metal complex in different forms of this nonheme iron oxygen transport protein have been clarified. The structure of the complex presented here differs from that of metazidohemerythrin in that the Fe atom providing the binding locus for azide ion is five-coordinate with no small molecule ligand bound to it. The coordination polyhedron of this Fe is best described as a trigonal bipyramid.     

Correlations among parameters that describe low-spin ferric heme complexes

The g values from low-spin ferric hemes can be related through the t2g hole model to rhombic (V/lambda) and tetragonal (delta/lambda) ligand field components and to the lowest Kramer's doublet energy (E/lambda). The latter is also a measure of unpaired electron sharing among the iron 3d (t2g) orbitals. For a series of ligands (X), there is a monotonic increase in myoglobin complex (Mb . X) [E/lambda] values with nonheme hexacoordinate metal complex (M . X6) [eg-t2gPg] orbital separations. As the aqueous solution pKa values of the sulfurous or nitrogenous ligands in model heme complexes increase, values of V/lambda and delta/lambda increase linearly, but those of [E/lambda] decrease linearly. The greater the electron-acceptor ability of the ligand, as suggested by its position in the spectrochemical series or its pKa, the more the unpaired electron sharing among the heme t2g orbitals increases. The rate of change of [E/lambda] with V/lambda and the pKa is different with sulfurous and nitrogenous ligands, and the magnitude of both rates increases with two sulfurs less than sulfur and nitrogen less than two nitrogens bound to the heme. The maximum magnitude of this rate with V/lambda for cytochrome P-450 is four times less than that for myoglobin, which may explain, in part, the differences in ligand binding between these two hemeproteins. The perturbation of [E/lambda], V/lambda, and delta/lambda induced by strain of iron-ligand bonds is quantitated for several hemeproteins and heme models. In addition, energy level comparisons suggest that the largest-magnitude g value falls approximately along the iron-chlorin ring normal. This suggestion implies that the electron distribution of the iron at the catalytic sites of cytochrome P-450 and certain chlorin-containing enzymes is in some way similar, but distinct from that at the transport site of myoglobin.     

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).     

A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis

An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis. It has a molecular weight of 85,000 and is composed of four subunits of equal size. The tetramer contains 2.5 g atoms of ferric iron. Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases. The amino acid sequence of the iron superoxide dismutase was determined. Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711. Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%). However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%). The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases.     

31P nuclear magnetic resonance chemical shielding tensors of phosphorylethanolamine, lecithin, and related compounds: Applications to head-group motion in model membranes.

31P nuclear magnetic resonance (NMR) powder spectra have been used to obtain the principal values of the chemical shielding tensors of dipalmitoyellecithin (DPL), dipalmitoylphosphatidylethanolamine, and several related organophosphate mono- and diesters. In addition, the principal values and orientation of the phosphorylethanolamine shielding tensor were determined from 31P NMR spectra of a single crystal. In all compounds studied the shielding tensors were clearly monaxial. The monoester spectra are typified by the spectrum of phosphorylethanolamine with principal values of -67, -13, and 69 ppm relative to H3PO4. The diesters have a larger total anisotrophy, as indicated by the DPL values of -81, -25, and 108 ppm. These data as well as the orientation of the phosphorylethanolamine shielding tensor are correlated with the electron density distribution as determined by the bonding pattern of the phosphate. The spectrum of a DPL-water (1:1) mixture at 52 degrees C has a shift anisotrophy of 30 ppm and displays a shape characteristic of an axial tensor. This change from the rigid lattice DPL pattern is explained in terms of motional narrowing, and the shielding tensor data are used to interpret the motion of the phospholipid head group. Simple rotation about the P-O(glycerol) bond is excluded, and a more complex motion involving rotation about both the P-O (glycerol) and glycerol C(2)-C(3) bonds is postulated.     

Iron and zinc complexation in wild-type and ferritin-expressing wheat grain: implications for mineral transport into developing grain

We have used synchrotron-based X-ray fluorescence and absorption techniques to establish both metal distribution and complexation in mature wheat grains. In planta, extended X-ray absorption fine structure (EXAFS) spectroscopy reveals iron phytate and zinc phytate structures in aleurone cells and in modified aleurone cells in the transfer region of the grain: iron is coordinated octahedrally by six oxygen atoms and fewer than two phosphorous atoms. Zinc is coordinated tetrahedrally by four oxygen atoms and approximately 1.5 phosphorus atoms in an asymmetric coordination shell. We also present evidence of modified complexation of both metals in transgenic grain overexpressing wheat ferritin. For zinc, there is a consistent doubling of the number of complexing phosphorus atoms. Although there is some EXAFS evidence for iron phytate in ferritin-expressing grain, there is also evidence of a structure lacking phosphorus. This change may lead to an excess of phosphorus within the storage regions of grain, and in turn to the demonstrated increased association of phosphorus with zinc in ferritin-expressing grains. Derivative X-ray absorption spectra also suggest that mineral complexation in the transfer region of ferritin-expressing grains is quite different from that in wild-type grain. This may explain why the raised levels of minerals transported to the developing grain accumulate within the crease region of the transgenic grain.     

Crystallization and structure determination to 2.5-A resolution of the oxidized [2Fe-2S] ferredoxin isolated from Anabaena 7120

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.     

A theoretical model to predict distribution of the fabric tensor and apparent density in cancellous bone

 The adaptation of cancellous bone to mechanical forces is well recognized. Theoretical models for predicting cancellous bone architecture have been developed and have mainly focused on the distribution of trabecular mass or the apparent density. The purpose of this study was to develop a theoretical model which can simultaneously predict the distribution of trabecular orthotropy/orientation, as represented by the fabric tensor, along with apparent density. Two sets of equations were derived under the assumption that cancellous bone is a biological self-optimizing material which tends to minimize strain energy. The first set of equations provide the relationship between the fabric tensor and stress tensor, and have been verified to be consistent with Wolff’s law of trabecular architecture, that is, the principal directions of the fabric tensor coincide with the principal stress trajectories. The second set of equations yield the apparent density from the stress tensor, which was shown to be identical to those obtained based on local optimization with strain energy density of true bone tissue as the objective function. These two sets of equations, together with elasticity field equations, provide a complete mathematical formulation for the adaptation of cancellous bone. Received: 25 February 1997/Revised version: 23 September 1997     

A Streptococcus mutans superoxide dismutase that is active with either manganese or iron as a cofactor

The superoxide dismutase produced by Streptococcus mutans OMZ176 during aerobic growth in a chemically defined medium (modified FMC) that was treated with Chelex 100 (to lower trace metal contamination) and supplemented with high purity manganese was purified (162-fold) by heat treatment, ammonium sulfate precipitation, and chromatofocusing chromatography. The superoxide dismutase produced during aerobic growth in the same medium, but without manganese and supplemented with high purity iron, was similarly purified (220-fold). The molecular masses of each holoenzyme were approximately 43,000 with a subunit mass of 20,700, indicating that the enzymes were dimers of two equally sized subunits. The superoxide dismutase from manganese-grown cells was a manganese enzyme (MnSOD) containing 1.2 atoms of manganese and 0.25 atoms of iron/subunit. The superoxide dismutase from iron-grown cells was an iron enzyme (FeSOD) containing 0.07 atoms of manganese and 0.78 atoms of iron/subunit. The amino acid compositions of the MnSOD and the FeSOD were virtually identical, and their amino-terminal sequences were identical through the first 22 amino acids. Dialysis of the FeSOD with o-phenanthroline and sodium ascorbate generated aposuperoxide dismutase with 94% loss of activity; subsequent dialysis of apoenzyme with either manganese sulfate or ferrous sulfate reconstituted activity (recoveries of 37 and 30%, respectively). Electrophoretic determination of cytoplasmic radioiron distribution indicated that (during aerobic growth) manganese prevented insertion of iron into superoxide dismutase, although the iron levels of at least two other cytoplasmic fractions were not altered by manganese. Therefore, S. mutans used the same aposuperoxide dismutase to form either FeSOD or MnSOD, depending upon which metal was available in the culture medium. Such "cambialistic" enzymes (those capable of making a cofactor substitution) may represent a previously unrecognized family of superoxide dismutases.