Glutathione enhances fibroblast collagen contraction and protects keratinocytes from apoptosis in hyperglycaemic culture.

BACKGROUND: Cutaneous wound healing is relatively slow in patients with diabetes. OBJECTIVES: To test the hypothesis that this defect in healing of wounds in patients with diabetes results from dysfunction of skin fibroblasts and epidermal keratinocytes and that this dysfunction is related to disrupted intracellular glutathione (GSH) homeostasis. METHODS: We investigated the effects of esterified GSH on the contraction of fibroblasts in a fibroblast-populated collagen lattice and on keratinocyte apoptosis. RESULTS: High glucose medium (hyperglycaemia) reduced the contraction ability of fibroblasts (P < 0.05). The normalization of glucose medium concentrations for hyperglycaemic fibroblasts did not restore the contraction capacity. The percentage of apoptotic keratinocytes was statistically higher in hyperglycaemic cells (P < 0.05). GSH media concentrations ranging from 0.1 to 100 micromol L(-1) restored the ability of hyperglycaemic fibroblasts to contract the gels in a concentration-dependent manner. Primary human keratinocytes grown in hyperglycaemic medium were more susceptible to apoptosis, and treatment with esterified GSH rescued the keratinocytes from apoptosis. CONCLUSIONS: These data suggest that intracellular GSH can normalize skin cell functions disrupted by in vitro cell growth under hyperglycaemic conditions.

g-Strain, ENDOR, and structure of active centers of two-iron ferredoxins.

Collaborative chemical and spectroscopic work from several laboratories resulted in a qualitative structure for the active center in the two-iron ferredoxins, with each iron being in a distorted tetrahedron of sulfur atoms (two acid-labile sulfurs bridging the two iron atoms and the other two from cysteine sulfurs). Subsequent X-ray data from other laboratories confirmed this structure. Detailed EPR spectral syntheses showed that there is a distribution of structures in any given protein (even in a single crystal) resulting in a distribution of the principal values of the g tensor, which may be described by a statistical distribution of still another tensor whose principal axes are, in general, not coincident with the principal-axis frame of the g tensor.

Three-dimensional structure of a purple lipoxygenase.

Polyunsaturated fatty acid metabolism is governed primarily by two enzymes, prostaglandin H synthase and lipoxygenase. The crystal structure of the metastable product-oxidized purple form of soybean lipoxygenase-3 was determined at 2.0 A resolution. The data reveal that the chromophore corresponds to an iron-peroxide complex, a potential intermediate in the catalyzed reaction. A significant alteration of the iron site accompanies the formation of the complex. The structure, the first for a fatty acid-lipoxygenase complex, also reveals an unexpected mode of binding, and identifies amino acid residues that may play significant roles in catalysis, regio- and stereoselectivity.

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1.

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.

Iron extraction from soybean lipoxygenase 3 and reconstitution of catalytic activity from the apoenzyme.

Lipoxygenases contain a unique nonheme iron cofactor with a redox role in the catalyzed reaction. The conditions for the extraction of the metal atom were investigated for one of the soybean lipoxygenase isoenzymes. Removal of the iron by o-phenanthroline was attained in the presence of substrate under anaerobic conditions, but the apoenzyme could not be isolated and reconstituted. The freshly regenerated sodium form of Chelex-100 also removes the iron atom from native soybean lipoxygenase 3, but only in sodium bicarbonate buffer at pH 8.0. The soluble but inactive apoenzyme was reconstituted with ferric ammonium sulfate in Tris--HCl buffer at pH 7.0. Stoichiometric iron in the reconstituted enzyme was established using inductively coupled plasma-atomic emission spectroscopy. The reconstituted enzyme contained 90 +/- 10% of the specific activity of the native enzyme. The native configuration of the reconstituted iron site was confirmed by electron paramagnetic resonance spectroscopy.

The effect of three Korean traditional medicines on the growth rate of cultured human keratinocytes.

The effect of three different Korean Traditional Medicines (KTM) was studied on several functional parameters of adult human cells in culture. The cells were non-transformed strains of normal, skin epidermal cells (keratinocytes) from adult humans. Aqueous extracts of the herbal medicines were tested using two types of cell strains: one type was essential fatty acid deficient (EFAD) cells which grow rapidly in medium that was low in calcium and had no essential fatty acids; the second type was a cell strain grown in medium supplemented with essential fatty acid (EFA-supplemented). These cells had much slower, in vivo skin growth rates, and the fatty acid composition resembled that measured in epidermal biopsy tissue. The KTMs chosen for this study were tae-gang-hual-tang (for treating osteoarthritis), hual-ak-tang (for pain relief) and sip-zeon-tae-bo-tang (for fortifying immune systems). Because high proliferation rates usually correlate with skin inflammation and because many of the chemotactic agents mediating inflammatory response are modified fatty acids, this study focused on cell growth rate and membrane fatty acid composition as signals for the effects of the herbal medicines. By monitoring growth rate, these experiments measured both a stimulatory and a regulatory effect on the growth of keratinocytes. Some toxicity was seen at the highest doses of the KTMs. These effects were modeled mathematically, and the results showed varying effects on growth rate depending on dose and herbal recipe. The fitting parameters were discussed as they relate to biological function. The experimental design was also discussed and alternatives were suggested.

EPR spectrometry of cytochrome P450 2B4: effects of mutations and substrate binding.

The EPR spectra of NH(2)-terminal-truncated P450 cytochrome 2B4 and of several active site mutants that were previously shown to be profoundly altered in catalytic properties were determined. From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximately 40% of the cytochrome is present as the high spin ferric form (g approximately 8, 4, 2). A similar situation was observed in the double mutant E310L T302A, but not in the single mutant E301L. A rhombic high spin signal (g approximately 8, 4, 2) was observed when a substrate such as styrene, benzphetamine, or cyclohexane was added to the truncated cytochrome. Accompanying this change was the appearance of a signal at g = 1.98. Conversely, an axial high spin signal was observed (g approximately 6, 6, 2) when cyclohexanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P450 2B4.

Rearrangement of L-2-hydroxyglutarate to L-threo-3-methylmalate catalyzed by adenosylcobalamin-dependent glutamate mutase.

Adenosylcobalamin-dependent enzymes catalyze a variety of chemically difficult isomerizations in which a nonacidic hydrogen on one carbon is interchanged with an electron-withdrawing group on an adjacent carbon. We describe a new isomerization, that of L-2-hydroxyglutarate to L-threo-3-methylmalate, involving the migration of the carbinol carbon. This reaction is catalyzed by glutamate mutase, but k(cat) = 0.05 s(-)(1) is much lower than that for the natural substrate, L-glutamate (k(cat) = 5.6 s(-)(1)). EPR spectroscopy confirms that the major organic radical that accumulates on the enzyme is the C-4 radical of L-2-hydroxyglutarate. Pre-steady-state kinetic measurements revealed that L-2-hydroxyglutarate-induced homolysis of AdoCbl occurs very rapidly, with a rate constant approaching those measured previously with glutamate and methylaspartate as substrates. These observations are consistent with the rearrangement of the 2-hydroxyglutaryl radical being the rate-determining step in the reaction. The slow rearrangement of the 2-hydroxyglutaryl radical can be attributed to the poor stabilization by the hydroxyl group of the migrating glycolyl moiety of the radical transiently formed on the migrating carbon. In contrast, with the normal substrate the migrating carbon atom bears a nitrogen substituent that better stabilizes the analogous glycyl moiety. These studies point to the importance of the functional groups attached to the migrating carbon in facilitating the carbon skeleton rearrangement.

Electron paramagnetic resonance measurements of the ferrous mononuclear site of phthalate dioxygenase substituted with alternate divalent metal ions: direct evidence for ligation of two histidines in the copper(II)-reconstituted protein.

The metalloenzyme phthalate dioxygenase (PDO) contains two iron-based sites. A Rieske-type [2Fe-2S] cluster serves as an electron-transferring cofactor, and a mononuclear iron site is the putative site of substrate oxygenation. A reductase, which contains FMN and a plant-type [2Fe-2S] ferredoxin domain, transfers electrons from NADH to the Rieske center. Any of the metal ions, Fe(II), Cu(II), Co(II), Mn(II), and Zn(II), can be used to populate the mononuclear site, but only Fe(II) is competent for effecting hydroxylation. Nevertheless, studies of how these metal ions affect both the EPR spectra of the reduced Rieske site and the kinetics of electron transfer in the PDO system indicated that each of these metal ions binds tightly and affects the protein similarly. In this study, EPR spectra were obtained from samples in which iron of the mononuclear site was replaced with Cu(II). The use of (63)Cu(II), in combination with PDO obtained from cultures grown on media enriched in (15)N [using ((15)NH(4))(2)SO(4) as a sole nitrogen source], [delta,epsilon-(15)N]histidine, as well as natural abundance sources of nitrogen, enabled detailed spectral analysis of the superhyperfine structure of the Cu(II) EPR lines. These studies clearly show that two histidines are coordinated to the mononuclear site. Coupled with previous studies [Bertini, I., Luchinat, C., Mincione, G., Parigi, G., Gassner G. T., and Ballou, D. P. (1996) J. Bioinorg. Chem. 1, 468-475] that show the presence of one or two water molecules coordinated to the iron, it is suggested that the mononuclear site is similar to several other mononuclear nonheme iron proteins, including naphthalene dioxygenase, for which crystal structures are available. The lack of observable EPR interaction signals between Cu(II) in the mononuclear site and the reduced Rieske center of PDO suggest that the two sites are at least 12 A apart, which is similar to that found in the naphthalene dioxygenase crystal structure.

The high-potential flavin and heme of nitric oxide synthase are not magnetically linked: implications for electron transfer.

BACKGROUND: The homodimeric nitric oxide synthase (NOS) catalyzes conversion of L-arginine to L-citrulline and nitric oxide. Each subunit contains two flavins and one protoporphyrin IX heme. A key component of the reaction is the transfer of electrons from the flavins to the heme. The NOS gene encodes two domains linked by a short helix containing a calmodulin-recognition sequence. The reductase domain binds the flavin cofactors, while the oxygenase domain binds heme and L-arginine and additionally mediates the dimerization of the NOS subunits. We investigated the origin of the unusual magnetic properties (rapid-spin relaxation) of an air-stable free radical localized to a reductase domain flavin cofactor. RESULTS: We characterized the air-stable flavin in wild-type NOS, both in the presence and absence of calcium and calmodulin, the imidazole-bound heme complex of wild-type NOS, the NOS Cys415-->Ala mutant, and the isolated reductase domain. All preparations of NOS had the same flavin electron-spin relaxation behavior. No half-field transitions or temperature-dependent changes in the linewidth of the radical spin signal were detected. CONCLUSIONS: These data suggest that the observed relaxation enhancement of the NOS flavin radical is caused by the environment provided by the reductase domain. No magnetic interaction between the heme and flavin cofactors was detected, suggesting that the flavin and heme centers are probably separated by more than 15 A.

Kinetic Lability, Structural Diversity, and Oxidation Reactions of New Oligomeric, Anionic Carboxylate-Pyridine Complexes.

The dimeric [M(2)(OAc)(5)(py)(2)&mgr;-(OH(2))]Et(4)N complexes, I (M = Mn, Fe, Co), have been isolated from pyridine solutions of M(II)(OAc)(2).xH(2)O and Et(4)N(OAc).4H(2)O. The X-ray structures of I (M = Mn, Fe, Co) have been determined and show the metal ions asymmetrically bridged by two acetate ligands and a water molecule. One of the metal ions is bound by a pyridine ligand and two monodentate acetate ligands that are hydrogen bonded to the bridging water molecule. The second metal ion is bound to a bidentate acetate ligand and a pyridine ligand. Recrystallization of I from acetonitrile leads to the reorganization of I and isolation of the M(3)(OAc)(8)(Et(4)N)(2) complexes, II (M = Mn, Fe, Co). The X-ray structure of II (M = Mn, Co) has been determined and shows the three metal ions connected by four bridging acetate ligands in a &mgr;(1), &mgr;(2) mode and two acetate ligands in the &mgr;(1),eta(1) mode, with a bidentate acetate ligand on each of the external metal ions completing the distorted octahedral geometry. Air oxidation of I-Fe in propionitrile leads to the formation of the mixed-valence [Fe(3)&mgr;(3)-(O)(OAc)(7)(OH(2))]Et(4)N(III). The X-ray structure of III has been determined and resembles the core of the basic acetate complexes; however, it has five bridging acetate ligands. The Mössbauer spectrum of III shows two quadrupole doublets in a 1:2 ratio with delta(Fe) = 1.29(1) and 0.48 mm/s; DeltaE(q) = 1.89 and 0.71 mm/s. The oxidation of I-Fe by H(2)O(2)/O(2) in pyridine solution in the presence of Cl(-) ligands affords Fe(4)&mgr;(3)-(O)(2)(OAc)(6)(py)(4)Cl(2) (IV). The X-ray structure of IV shows a rhombic {Fe(III)(4)(&mgr;(3)-O)(2)} core previously found in iron and manganese chemistry. The reaction of ferrocenium ion with I-Fe under basic conditions in dichloromethane solution led to the formation of the familiar mixed-valence Fe(3)&mgr;(3)-(O)(OAc)(6)(py)(3) complex (V) with the basic acetate structure. Complexes I-Fe, II-Fe, III, and IV catalyze the reaction of O(2) with adamantane under GiF conditions to give adamantanols and adamantanone. The similarity of the results in comparison to similar studies previously reported for iron/carboxylate complexes are noted and discussed.

Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII.

Coagulation factor VIII (FVIII) is a heterodimer consisting of a light chain of 80 kDa (domains A3-C1-C2) in a metal ion-dependent association with a 220-kDa heavy chain (domains A1-A2-B). The nature of the metal ion-dependent association between the heavy and light chains was investigated using atomic absorption spectroscopy, electron paramagnetic resonance spectroscopy (EPR), and site-directed mutagenesis and expression of the FVIII cDNA. Whereas copper ion was not detected in intact recombinant FVIII, EDTA dissociation of the chains yielded an EPR signal consistent with 1 mol of Cu(I)/mol of active protein, supporting the hypothesis that a single molecule of reduced copper ion is buried within intact FVIII and is released and oxidized upon treatment with EDTA. Cu(I), and not Cu(II), was able to reconstitute FVIII activity from dissociated chains, demonstrating a requirement for Cu(I) in FVIII function. Three potential copper ion binding sites exist within FVIII: one type-2 site and two type-1 sites. The importance of these potential copper ion ligands was tested by studying the effect of site-directed mutants. Of the two histidines that compose the type-2 binding site, the His-1957 --> Ala mutant displayed secretion, light and heavy chain assembly, and activity similar to wild-type FVIII, while mutant His-99 --> Ala was partially defective for secretion and had low levels of heavy and light chain association and activity. In contrast, FVIII having the mutation Cys-310 --> Ser within the type-1 copper binding site in the A1 domain was inactive and partially defective for secretion from the cell, and the heavy and light chains of the secreted protein were not associated. Mutant Cys-2000 --> Ser within the A3 domain displayed secretion, assembly, and activity similar to that for wild-type FVIII. These results support the hypothesis that Cu(I) is buried within the type-1 copper binding site within the A1 domain and is required for FVIII chain association and activity.

Retinoic acid stimulates essential fatty acid-supplemented human keratinocytes in culture.

The effect of all-trans retinoic acid on the proliferation of essential fatty acid (EFA)-deficient and of EFA-supplemented adult human keratinocytes was investigated. EFA-deficient cell strains were supplied with one of four different fatty acid-supplemented media at the P0 to P1 passage. All-trans retinoic acid at 0.5 or 1.0 microM was added to the cultures at the P1 to P2 passage. At passage P3, and 3 and 7 d thereafter, the cell growth rate was determined. The fatty acid content of cultures grown in each medium was measured using gas chromatography. All the EFA media "normalized" the cellular fatty acid composition and drastically decreased the cell number and total DNA and protein of the cultures. All-trans retinoic acid at 1 microM prevented the loss of cell viability and growth usually associated with EFA supplementation but did not affect the control (EFA deficient) or 18:1 fatty acid-supplemented cultures. All-trans retinoic acid at 1 microM altered the fatty acid content of the EFA-supplemented cultures. A statistically significant increase in 14:0, 14:1, 16:1, 18:1, and 20:4 fatty acids occurred, whereas the amounts of 18:0 and 18:2 fatty acids decreased. The largest changes were in 16:1 fatty acid (8-14%) and 18:2 fatty acid (12-5%). All-trans retinoic acid at 0.5 microM also affected both cell growth and fatty acid composition without induction of the CRABP II message. These studies demonstrate that all-trans retinoic acid stimulates the growth of EFA-supplemented keratinocyte cultures while also altering the fatty acid composition of the cells.

Fatty acid alteration and the lateral diffusion of lipids in the plasma membrane of keratinocytes.

The fluorescent probe diI was used to study the lateral mobility of lipids in in vitro strains of living adult human keratinocytes grown in four different media. One medium was essential fatty acid deficient (EFAD) and low in calcium ion, a medium known to yield cells that proliferate rapidly and contain lipid with extremely low levels of essential fatty acids. Two other media were supplemented with essential fatty acids (FAS), media that are known to result in cells that grow more slowly and have normalized fatty acid proportions. A fourth medium consisted of 1 microM all-trans-retinoic acid added to the fatty acid-supplemented medium (FAS-RA), a medium known to produce cells that are highly proliferative, with a growth rate greater than that of the FAS strains and similar to that of the EFAD strains. The keratinocytes grown in these four media were studied using the fluorescence recovery after photobleaching (FRAP) technique to determine the lateral diffusion rate of diI in the plasma membranes. Our results showed a positive correlation between growth rate and diffusion coefficient (D): the diffusion coefficient of diI was higher in the EFAD or FAS-RA cells than in the FAS cells. The measurement of D among the FAS cells fell into two groups. One group was similar to the single group seen in the EFAD cells, but the other group was composed of much lower D values. The other FRAP parameters (mobile fraction and bleach depth) were larger in the "slow" group than in the "fast" group. This trend of negative correlation between these parameters and D was also found within the fast group. These results are interpreted in terms of possible changes in membrane structure or morphology that might be indirectly associated with the fatty acid alterations, including the possible presence of areas in senescing keratinocytes where plasma membranes collapse to form an interacting system of lipid bilayers.

Substitution of D-Trp32 in NPY destabilizes the binding transition state to the Y1 receptor site in SK-N-MC cell membranes.

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1N epsilon 4-proxyl]-NPY, the KD was 8 x 10(-10) M and koff was 2.7 x 10(-4) sec-1 yielding a value for kon of 3.3 x 10(5) sec-1 M-1. The [Ac-Tyr1, N epsilon 4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 x 10(-7) M and koff was 1.7 x 10(-4) sec-1 leading to a value for kon of 1.2 x 10(3) sec-1 M-1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N epsilon 4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.

The iron-sulfur clusters 2 and ubisemiquinone radicals of NADH:ubiquinone oxidoreductase are involved in energy coupling in submitochondrial particles.

The behavior of ubisemiquinone radicals and the iron-sulfur clusters 2 of NADH:ubiquinone oxidoreductase (Complex I) in coupled and uncoupled submitochondrial particles (SMP), oxidizing either NADH or succinate under steady-state conditions, was studied. Multifrequency EPR spectra revealed that the two new g2 lines of the clusters 2, only observed during coupled electron transfer under conditions where energy dissipation is rate-limiting [De Jong, A. M. Ph., Kotlyar, A. B., & Albracht, S. P. J. (1994) Biochim. Biophys. Acta 1186, 163-171], are the result of a spin-spin interaction of 2.8 mT. Investigation of the radical signals present in coupled SMP indicated that more than 90% of the radicals can be ascribed to two types of semiquinones which are bound to Complex I (QI-radicals) or ubiquinol:cytochrome c oxidoreductase (Complex III; QIII-radicals). The presence of QIII-radicals, but not that of QI-radicals, was completely abolished by uncoupler. Part of the QI-radicals weakly interact with the clusters 2 of Complex I. This uncoupler-sensitive interaction can amount to a splitting of the radical EPR signal of at most 1 mT, considerably weaker than the 2.8 mT splitting of the g2 lines of the clusters 2. We propose that the 2.8 mT splitting of these g2 lines results from an energy-induced spin-spin interaction between the two clusters 2 within the TYKY subunit of Complex I. The two clusters 2 show no interaction during electron transfer is uncoupled SMP or in fully-reduced anaerobic-coupled SMP. The results point to a direct role of the Fe-S clusters 2 and the QI-radicals in the mechanism of coupled electron transfer catalyzed by Complex I.

Interaction of Escherichia coli cobalamin-dependent methionine synthase and its physiological partner flavodoxin: binding of flavodoxin leads to axial ligand dissociation from the cobalamin cofactor.

Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the last step in de novo methionine biosynthesis. Conversion of the inactive cob(II)alamin form of the enzyme, formed by the occasional oxidation of cob(I)alamin during turnover, to an active methylcobalamin-containing form requires a reductive methylation of the cofactor in which an electron is supplied by reduced flavodoxin and the methyl group is derived from S-adenosyl-L-methionine. E. coli flavodoxin acts specifically in this activation reaction, and neither E. coli ferredoxin nor flavodoxin from the cyanobacterium Synechococcus will substitute, despite their highly similar midpoint potentials for one-electron transfer. As assessed by EPR spectroscopy, the binding of flavodoxin to cob(II)alamin methionine synthase results in a change in the coordination geometry of the cobalt from five-coordinate to four-coordinate. Histidine 759 of methionine synthase, which replaces the normal lower ligand dimethylbenzimidazole on binding of methylcobalamin to methionine synthase, is dissociated from the cobalt of the cobalamin by the binding of flavodoxin. The association of flavodoxin and methionine synthase depends on ionic strength and pH; the pH dependence corresponds to the uptake of one proton on association. The formation of a complex between flavodoxin and methionine synthase perturbs the midpoint potentials of the flavin and cobalamin cofactors only marginally and without any significant thermodynamic advantage for electron transfer to the cobalamin of methionine synthase. No significant binding was seen between oxidized flavodoxin and methylcobalamin methionine synthase. A model for the interaction of methionine synthase with flavodoxin is proposed in which flavodoxin binding leads to changes in the distribution of methionine synthase conformations.

Oleic acid and linoleic acid are the major determinants of changes in keratinocyte plasma membrane viscosity.

Keratinocytes were grown in medium with no essential fatty acids as well as in media with specially selected fatty acid augmentations. Gas chromatographic determinations of 21 fatty acids in the phospholipids were correlated with plasma membrane viscosity obtained by electron paramagnetic resonance studies (n = 24). Using standard procedures from multivariate analysis, we derived an expression that modeled the viscosity data as a function of four key fatty acid levels: [formula see text] where the fatty acids are given in mole percent of total lipids and are identified as two number sequences: number of carbons followed by number of double bonds. No other fatty acid made a significant contribution to the regression equation. The range of viscosity was very large, varying from 60 to 120 cP over the sample population. The results are interpreted to indicate that polyunsaturated fatty acids are replaced with monounsaturated fatty acids by the keratinocytes and that dihomogamma-linolenic acid (20:3, n-6) plays an important role in membrane viscosity when essential fatty acids are available in the growth medium of these adult human cultured keratinocytes.

Spectral and ligand-binding properties of an unusual hemoprotein, the ferric form of soluble guanylate cyclase.

The soluble form of guanylate cyclase (sGC) is a hemoprotein which serves as the only known receptor for the signaling agent nitric oxide (.NO). The enzyme is a heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin type b heme. .NO increases the Vmax of sGC up to 400-fold by binding to the heme to form a 5-coordinate ferrous nitrosyl complex. The electron paramagnetic resonance spectrum of the ferric form of the enzyme has been obtained. The spectrum displays rhombic symmetry and is indicative of a high-spin heme. Computer simulation of the EPR spectrum yields g values of 6.36, 5.16, and 2.0 with linewidths of 3.3, 4.1, and 3.3 mT, respectively. Using electronic absorption spectroscopy, it was observed that the ferric heme binds cyanide to form a 6-coordinate low-spin complex. The rate constants for association (k(on)) and dissociation (k(off)) of cyanide at 10 degrees C have been determined to be (7.8 +/- 0.3) x 10(-2) M(-1) s(- 1) and (7.2 +/- 0.2) x 10(-5) s(-1), respectively. Unlike the ferrous form of the enzyme, which has a low affinity for ligands that form 6-coordinate complexes due to an unusually fast off-rate, the ferric form of the enzyme appears to have a low affinity for ligands due to a slow on-rate. The ferric heme binds azide with a Kd of 26 +/- 4 mM to form a high-spin complex. The ferric form of the enzyme has a specific activity of approximately 57% that of the nonactivated ferrous form of the enzyme. However, in contrast to the mild activation of the ferrous enzyme by carbon monoxide, the ferric enzyme is not activated by cyanide. These results indicate that there may be a significant structural change in the protein upon the oxidation of the heme iron.

Overproduction and physical characterization of SoxR, a [2Fe-2S] protein that governs an oxidative response regulon in Escherichia coli.

SoxR protein governs the soxRS (superoxide response) regulon of Escherichia coli by becoming a transcriptional activator when the cells are exposed to compounds that mediate univalent redox reactions, many of which produce superoxide as a by-product. SoxR was overproduced and purified to near homogeneity from a strain bearing an expression vector. It could bind specifically to the soxS operator even in the absence of RNA polymerase. The aerobically purified protein, which is readily autooxidized, could activate the transcription of soxS DNA even without exposure to known inducing agents. SoxR is a globular homodimer. It contains one [2Fe-2S] cluster per polypeptide chain, as demonstrated by optical and EPR spectroscopy combined with stoichiometric analysis of iron content, unpaired-electron-spin density, and reduction by dithionite. The protein is active in its oxidized ([2Fe-2S]2+) state. The presence of a prosthetic group capable of univalent redox reactions may help to explain the activation of the regulon in vivo by compounds that can mediate such reactions.