Heme methyl 1H chemical shifts as structural parameters in some low-spin ferriheme proteins

 The different paramagnetic shifts of the four methyl groups in ferriheme proteins have been described as being due to the effect of the axial ligand nodal plane orientation. An equation, heuristically found and theoretically explained, describing the relation between contact and pseudocontact shifts and the position of the axial ligand(s) has been derived for bis-histidine ferriheme proteins and for cyanide-histidine ferriheme proteins. The values of the heuristic parameters contained in the equations were found by fitting the shifts of bovine cytochrome b ₅ and several bis-histidine cytochromes c ₃ and histidine-cyanide systems. The agreement between the observed and the calculated shifts was found to be good. Therefore, by taking advantage of this study, information on the position of the axial ligands, that can be used as a constraint for structure determination, can be obtained from the shifts of the methyl protons. Received: 13 April 1999 / Accepted: 4 June 1999     

The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example

 The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only. Received: 11 July 1995 / Accepted: 30 October 1995     

The effect of axial ligands on the reactivity and stability of the oxoferryl moiety in model complexes of Compound I of heme-dependent enzymes

 The contribution of simple inorganic model complexes to the understanding of related processes in biomolecules is demonstrated by a series of Compound I analogs of heme-dependent enzymes. The oxoiron(IV) porphyrin cation radical state in these synthetic complexes is stable enough to be studied by spectroscopic methods as a function of only one variable, the axial ligand trans to the oxoiron bond. Complementary information from kinetic investigations of the reactivity in epoxidation of olefins enables the separation of the thermodynamic and kinetic effects of the axial ligands. The results clearly indicate that epoxidation by these complexes proceeds by two distinguishable steps, which are affected differently by the axial ligands. The first step is electron transfer from the olefin to the ferryl moiety, probably followed by intramolecular charge rearrangement and product release. It is proposed that part of the enhanced oxygenation activities of cytochrome P-450 monooxygenases and chloroperoxidases is due to a lowering of the energy barrier for the second step via participation of their redox-active cysteinate ligand in charge rearrangement. Received and accepted: 7 May 1996     

The influence of axial ligands on the reduction potential of blue copper proteins

 The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values. We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-S_Met bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials: stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins. The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around the copper site. Received: 7 April 1999 / Accepted: 26 July 1999     

On the role of the axial ligand in heme-based catalysis of the peroxidase and P450 type

 The present commentary focusses on the role of the axial ligand in peroxidase- and P450-type catalysis. Based on molecular orbital calculations and the experimental evidence available, it is argued that the ligand of a heme-containing enzyme may be a factor in setting the relative chance, although not the intrinsic capability, of the enzyme to catalyse a specific type of heme-based reaction chemistry. The ligand can do so by influencing the electrophilicity, i.e. the redox potential of the high-valency iron-oxo complex, and also by influencing the energy barrier for a reaction pathway through delocalization of valence electrons along the axial ligands, thereby, in the case of a cysteinate but not a histidine axial ligand, stabilizing oxygen transfer pathways. Received and accepted: 7 May 1996     

Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G

A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is presented. MSG catalyzes the condensation of glyoxylate with an acetyl group of acetyl-CoA, producing malate, an intermediate in the citric-acid cycle. We show that despite the size of the protein, important structural and dynamic information about the molecule can be obtained on a per-residue basis. 15N-1HN residual dipolar couplings and carbonyl chemical shift changes upon alignment in Pf1 phage establish that there are no significant domain reorientations in the molecule upon ligand binding, in contrast to what was anticipated on the basis of both the X-ray structure of the glyoxylate-bound form of the enzyme and structural studies of a related set of proteins. The chemical shift changes of 1HN, 15N and 13CO nuclei upon binding of pyruvate, a glyoxylate-mimicking inhibitor, and acetyl-CoA have been mapped onto the three-dimensional structure of the molecule. Binding constants of pyruvate, glyoxylate, and acetyl-CoA (in the presence of pyruvate) have been measured, along with the kinetic parameters for glyoxylate and pyruvate binding. The on-rates of pyruvate and glyoxalate binding, approximately 1.2 x 10(6)M(-1)s(-1) and approximately 2.7 x 10(6)M(-1)s(-1), respectively, are significantly lower than what is anticipated from a simple diffusion-controlled process. Some structural implications of the chemical shift perturbations upon binding and the estimated ligand on-rates are discussed.     

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by ¹H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

The effect of heterogeneous structural diffusion on ligand binding to heme proteins

The kinetics of ligand binding to heme proteins studied by flash photolysis display an algebraic time dependence at low temperatures in contrast exponential recombination observed under physiological conditions. This result shows that protein structures should be viewed as a time average of interconverting microstates which are frozen in at low temperatures. We propose a quasi-one-dimensional model of heterogeneous structural diffusion coupled to ligand binding which describes freezing transition as an inherent property of protein fluctuations. The structural hopping rates are derived from a temperature invariant spectrum of activation energies. The model predicts power law kinetics of the form t ^- at long times. The exponent is constant (0.5) at high temperatures but decreases below a critical temperature in the frozen regime. These results are compared to experiments performed with myoglobin and -chains of hemoglobin.     

The inhibitory effect of soybean and soybean isoflavone diets on 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice

Murine contact hypersensitivity (CHS) is one of the most frequently used animal models of human allergic contact dermatitis. We investigated the inhibitory effects of soybean and soy isoflavone (SI) diets on 2,4-dinitrofluorobenzene- (DNFB) induced CHS in mice. The DNFB-induced ear swelling was inhibited in the soy- and SI-treated groups. Histopathological investigations revealed that oral feeding of soybean and SI attenuated ear tissue edema and reduced the number of Gr-1⁺ cell infiltrations into ear tissues. DNA microarray analysis showed that the expression of Ccl24, Xcl1, Ifng, and Ccl17 in the ear tissues was lower in the soy-treated mice than in the positive controls. In addition, CCL24 mRNA and protein expression in the ear tissues were more highly suppressed in the soy- and SI-treated groups. These results suggest that soybean and SI consumption downregulated the gene and protein expression of CCL24, thereby affording protection against CHS in mice.     

Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2–3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-Å resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme–thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme–thiolate coordination can be displaced by exogenous ligands.     

19F NMR study of protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin

 A novel C ₂-symmetric ring-fluorinated hemin, 13,17-bis(2-carboxyethyl)-2,8,12,18-tetramethyl-3,7-difluoroporphyrinatoiron(III), has been synthesized and was incorporated into sperm whale apomyoglobin to investigate protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin (Mb) using ¹⁹F NMR spectroscopy. NMR signals for ¹⁹F atoms introduced as substituents on the present heme in ferrous low-spin and high-spin and ferric low-spin complexes have been observed and their shifts sharply reflect not only the electronic nature of the heme iron, but also in-plane asymmetry of the heme electronic structure. The two-fold symmetric electronic structure of the ring-fluorinated hemin is clearly manifested in the ¹⁹F and ¹H NMR spectra of its dicyano complex. The chemical equivalence of the two fluorine atoms of the heme is removed in the active site of myoglobin and the splitting of the two ¹⁹F NMR signals provides a quantitative probe for characterizing the rhombic perturbation of the heme electronic structure induced by the heme-protein interaction. The in-plane asymmetry of heme electronic structures in carbonmonoxy and deoxy Mbs have been analyzed for the first time on the basis of the shift difference between the two ¹⁹F NMR signals of the heme and is interpreted in terms of iron-ligand binding and/or the orbital ground state of the heme. A potential utility of ¹⁹F NMR, combined with the use of a symmetric fluorinated hemin, in characterizing the heme electronic structure of myoglobin in a variety of iron oxidation, spin, and ligation states, is presented. Received: 23 December 1999 / Accepted: 3 April 2000     

Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

 In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm^–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on ³⁴S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm^–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm^–1 are consistent with square-planar coordination. Addition of exogenous ¹⁵N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with ¹⁵N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with ¹⁵N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone. Received: 29 July 1996 / Accepted: 9 November 1996     

The effect of multiple binding modes on empirical modeling of ligand docking to proteins.

A popular approach to the computational modeling of ligand/receptor interactions is to use an empirical free energy like model with adjustable parameters. Parameters are learned from one set of complexes, then used to predict another set. To improve these empirical methods requires an independent way to study their inherent errors. We introduce a toy model of ligand/receptor binding as a workbench for testing such errors. We study the errors incurred from the two state binding assumption--the assumption that a ligand is either bound in one orientation, or unbound. We find that the two state assumption can cause large errors in free energy predictions, but it does not affect rank order predictions significantly. We show that fitting parameters using data from high affinity ligands can reduce two state errors; so can using more physical models that do not use the two state assumption. We also find that when using two state models to predict free energies, errors are more severe on high affinity ligands than low affinity ligands. And we show that two state errors can be diagnosed by systematically adding new binding modes when predicting free energies: if predictions worsen as the modes are added, then the two state assumption in the fitting step may be at fault.     

The role of growth hormone and amino acids on brain protein synthesis in aged rats given proteins of different quantity and quality

Summary. The purpose of the present study was to determine whether the regulation of brain protein synthesis was mediated through changes in the plasma concentrations of insulin and growth hormone (GH), and whether the concentrations of amino acids in the brain and plasma regulate the brain protein synthesis when the quantity and quality of dietary protein is manipulated. Two experiments were done on three groups of aged rats given diets containing 20% casein, 5% casein or 0% casein (Experiment 1), and 20% casein, 20% gluten, or 20% gelatin (Experiment 2) for 1 d (only one 5-h period) after all rats were fed the 20% casein diet for 10 d (only 5-h feeding per day). The aggregation of brain ribosomes, the concentration in plasma GH, and the branched chain amino acids in the plasma and cerebral cortex declined with a decrease of quantity and quality of dietary protein. The concentration of plasma insulin did not differ among groups. The results suggest that the ingestion of a higher quantity and quality of dietary protein increases the concentrations of GH and several amino acids in aged rats, and that the concentrations of GH and amino acids are at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

Effect of Rho family GTP-binding proteins on Amoeba proteus

Summary.  While there is a number of studies on the effects of Rho GTPases on the actin-based cytoskeleton in higher eukaryotes, studies in protozoans are rather limited. The problem seems to be intriguing since the structure of protozoan cytoskeletons is distinct from most vertebrate cells. By blocking endogenous Rho family proteins of highly motile Amoeba proteus with C3 transferase and antibodies against human RhoA and Rac1, we tried to assess the in vivo role of these proteins in amoebae. In migrating amoebae, both proteins are concentrated in the cortical layer and seem to colocalize with filamentous actin. Endogenous Rac1, but not RhoA, is accumulated in the perinuclear cytoskeleton. Blocking Rac- or Rho-like proteins caused distinct and irreversible changes in the locomotive shape of the examined amoebae and significant inhibition of their migration. Amoebae microinjected with anti-Rac1 antibodies were contracted, shortened, and developed only few wide pseudopodia. More pronounced changes were observed in cells treated with anti-RhoA antibodies. They exhibited an atypical locomotion not leading to their effective displacement. After treatment with 50 μg of C3 transferase per ml, cells rapidly contracted and almost completely rounded up, became refractile with the granules beaten into a dense mass, detached from the surface and died. Ten times lower concentration of the enzyme caused similar changes as the inhibition of endogenous RhoA-like protein. These results indicate that Rho family-based regulation plays a key role in amoebic migration. Received May 2, 2002; accepted August 2, 2002; published online November 29, 2002     

Snail-type zinc finger proteins prevent neurogenesis in Scutoid and transgenic animals of Drosophila

Scutoid is a classical dominant gain-of-function mutation of Drosophila, causing a loss of bristles and roughening of the compound eye. Previous genetic and molecular analyses have shown that Scutoid is associated with a chromosomal transposition resulting in a fusion of no-oceli and snail genes. How this gene fusion event leads to the defects in neurogenesis was not known until now. Here have found that snail is ectopically expressed in the eye-antennal and wing imaginal discs in Scutoid larvae, and that this expression is reduced in Scutoid revertants. We have also shown that the expressivity of Scutoid is enhanced by zeste mutations. snail and escargot encode evolutionarily conserved zinc-finger proteins involved in the development of mesoderm and limbs. Snail and Escargot proteins share a common target DNA sequence with the basic helix-loop-helix (bHLH) type proneural gene products. When expressed in the developing external sense organ precursors of the thorax and the eye, these proteins cause a loss of mechanosensory bristles in the thorax and perturbed the development of the compound eye. Such phenotypes resemble those associated with Scutoid. Furthermore, the effect of ectopic Escargot on bristle development is antagonized by coexpression of the bHLH gene asense. Thus, our results suggest that the Scutoid phenotype is due to an ectopic snail expression under the control of no-oceli enhancer, antagonizing neurogenesis through its inhibitory interaction with bHLH proteins. Received: 8 February 1999 / Accepted: 24 May 1999     

The pattern of acropetal and basipetal cytoplasmic streaming velocities in Chara rhizoids and protonemata, and gravity effect on the pattern as measured by laser-Doppler-velocimetry

 The spatial pattern of acropetal and basipetal cytoplasmic streaming velocities has been studied by laser-Doppler-velocimetry (LDV) in the positively gravitropic (downward growing) rhizoids of Chara globularis Thuill. and for the first time in the negatively gravitropic (upward growing) protonemata. The LDV method proved to be precise and yielded reproducible results even when tiny differences in velocities were measured. In the apical parts of the streaming regions of both cell types, acropetal streaming was faster than basipetal streaming. Starting at the apical reversal point of streaming, the velocity increased basipetally with the distance from that point and became fairly constant close to the basal reversal point; subsequently, the velocity decreased slightly acropetally as the apical reversal point was again approached. There was no change in velocity at the basal reversal point. However, at the apical reversal point there was an abrupt decrease in velocity. The pattern of the ratio of acropetal to basipetal streaming velocity (VR) was a function of the relative distance of the site of measurement from the apical reversal point rather than a function of the absolute distance. Upon inversion of the rhizoids, the VR decreased on average by 3.8% (±0.4%), indicating that the effect of gravity on the streaming velocity was merely physical and without a physiological amplification. Rhizoids that had developed on the slowly rotating horizontal axis of a clinostat, and had never experienced a constant gravity vector, were similar to normally grown rhizoids with respect to VR pattern. In protonemata, the VR pattern was not significantly different from that in rhizoids although the direction of growth was inverse. In rhizoids, oryzalin caused the polar organization of the cell to disappear and nullified the differences in streaming velocities, and cytochalasin D decreased the velocity of basipetal streaming slightly more than that of acropetal streaming. Cyclopiazonic acid, known as an inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum, also reduced the streaming velocities in rhizoids, but had slightly more effect on the acropetal stream. It is possible that the endogenous difference in streaming velocities in both rhizoids and protonemata is caused by differences in the cytoskeletal organization of the opposing streams and/or loading of inhibitors (like Ca²⁺) from the apical/subapical zone into the basipetally streaming endoplasm. Received: 4 October 1999 / Accepted: 4 November 1999     

The effect of neuropeptides from Limulus on its circadian rhythm in retinal sensitivity

Retinal responses of the Limulus lateral eyes to light are greater at night than during the day. A circadian clock in the brain of the horseshoe crab controls these rhythmic changes of light sensitivity. The increase in sensitivity (as measured by the amplitude of the electroretinogram) is mediated at least in part by octopamine that is released from efferent axons terminating in the visual cells. Earlier studies indicate that certain factors in Limulus hemolymph can act in conjunction with octopamine. More recently, five neuropeptides (LP1-LP4 and Lip-HP) had been isolated from acetone extracts of the Limulus central nervous system using HPLC fractionation and radioimmunoassay with antisera against FMRFamide-like peptides for detection. Presently, we have injected into the Limulus lateral eye these five peptides and observed changes in retinal sensitivity. Injection during daytime had no immediate effect on that daytime electroretinogram but decreased the electroretinogram amplitude for the entire subsequent night (12 h). However, upon injection at night, we observed an immediate but only transitory decrease in electroretinogram amplitude for about 1 h without effect on the subsequent daytime electroretinogram. We suggest that the peptides act antagonistically to octopamine and are highly dependent upon the activity state of the efferent nerve terminals. Accepted: 25 August 1996     

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary. The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.