Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

Heme ferrous-hydroperoxo complexes: some theoretical considerations

We report density functional calculations on complexes of ferrous hemes with hydroperoxide, where the axial ligand trans to OOH(-) is imidazole, thiolate, or phenoxide. The geometrical parameters and charge distributions within the Fe-O-O-H moiety are identical between the ferrous complexes reported here and their ferric counterparts previously described, even though the latter contain one unpaired electron on iron as opposed to the former, which are diamagnetic. The extra negative charge upon going from a formally ferric state to formally ferrous appears to be distributed essentially on the porphyrin. These findings support recent experimental data showing that the ferrous state of certain hemoproteins can interact with peroxides in a catalytically competent fashion, cleaving the O-O bond heterolytically in a manner reminiscent of the "canonical" ferric-peroxo complexes, and contrary to any expectations based on the Fenton concept commonly invoked in non-heme chemistry.     

Spectral properties of nitric oxide complex of cytochrome c' from Rhodopseudomonas capsulata B100

The spectral properties for NO complexes of ferric and ferrous cytochrome c' from photosynthetic bacterium Rhodopseudomonas capsulata B100 are reported. The electronic absorption, MCD, and EPR spectra have been compared with those of the NO complexes of the other cytochromes c' and horse heart cytochrome c. The NO-ferrous cytochrome c' would be a mixture of NO complexes with six- and five-coordinate nitrosylheme, suggesting that the heme-iron to histidine bond in the ferrous cytochrome c' is more stable than that from chemoheterotrophic bacteria. The reaction product of ferric cytochrome c' with NO exhibited the spectra similar to NO-ferric derivatives of the other hemoproteins, which indicates the formation of NO-ferric cytochrome c'.     

Rapid P450 heme iron reduction by laser photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1. Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (Fe(II)-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for Fe(II)-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast ( approximately 10,000-11,000 s(-1)) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/Fe(II)-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 Fe(II)-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.     

Variation of the oxidation state of verdoheme in the heme oxygenase reaction

Heme oxygenase (HO) converts hemin to biliverdin, CO, and iron applying molecular oxygen and electrons. During successive HO reactions, two intermediates, α-hydroxyhemin and verdoheme, have been generated. Here, oxidation state of the verdoheme-HO complexes is controversial. To clarify this, the heme conversion by soybean and rat HO isoform-1 (GmHO-1 and rHO-1, respectively) was compared both under physiological conditions, with oxygen and NADPH coupled with ferredoxin reductase/ferredoxin for GmHO-1 or with cytochrome P450 reductase for rHO-1, and under a non-physiological condition with hydrogen peroxide. EPR measurements on the hemin-GmHO-1 reaction with oxygen detected a low-spin ferric intermediate, which was undetectable in the rHO-1 reaction, suggesting the verdoheme in the six-coordinate ferric state in GmHO-1. Optical absorption measurements on this reaction indicated that the heme degradation was extremely retarded at verdoheme though this reaction was not inhibited under high-CO concentrations, unlike the rHO-1 reaction. On the contrary, the Gm and rHO-1 reactions with hydrogen peroxide both provided ferric low-spin intermediates though their yields were different. The optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in reaction mixtures and were slowly converted to the ferric biliverdin complex. Consequently, in the physiological oxygen reactions, the verdoheme is found to be stabilized in the ferric state in GmHO-1 probably guided by protein distal residues and in the ferrous state in rHO-1, whereas in the hydrogen peroxide reactions, hydrogen peroxide or hydroxide coordination stabilizes the ferric state of verdoheme in both HOs.     

Spectral properties of cytochrome c' from Rhodopseudomonas capsulata B100 and its CO complex

The spectral properties of cytochrome c' from photosynthetic bacterium Rhodopseudomonas capsulata (= Rhodobacter capsulatus) B100 and its CO complex are reported. The electronic absorption, MCD, and EPR spectra have been compared with those of the other cytochromes c' and horse heart cytochrome c. EPR and electronic spectral results for the ferric cytochrome c' suggest that the ground state of heme-iron(III) at neutral pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state and that at pH 11.0 is in a high-spin state. In the MCD spectrum of the CO-ferrous cytochrome c', the MCD intensity in the Soret band region was much higher than that of CO complexes of hemoproteins with a protoheme. The differences in a stereochemistry of the sixth-coordination position is discussed.     

The green hemoproteins of bovine erythrocytes. II. Spectral, ligand-binding, and electrochemical properties

The two green hemoproteins isolated from bovine erythrocytes (form I and form II) have been characterized as to spectral, electrochemical, and chemical properties. The absorption spectra of the isolated hemoproteins are typical of high spin ferric states. Reduction of the hemoproteins yields high spin ferrohemoproteins. Complexation of the ferrohemoproteins with CO and the ferrihemoproteins with cyanide yields low spin complexes, demonstrating the presence of an exchangeable weak field ligand in both the ferrous and ferric states of the hemoproteins. The differences in position and intensity of the absorption peaks of the visible spectra allow the two forms to be distinguished from one another. The midpoint potential of forms I and II were found to be +0.075 and +0.019 V, respectively, at pH 6.4 and +0.038 and -0.005 V, respectively, at pH 7.0. This is consistent with the gaining of 1 proton/electron during the reduction. The Nernst plot reveals an unusual 0.5-electron transfer, whereas a quantitative titration demonstrates a 1-electron transfer. Form I binds cyanide more tightly than form II (KD of 84 and 252 micrometer, respectively). The observed spectral, electrochemical, and ligand-binding differences between forms I and II can be explained in terms of a greater electron-withdrawing ability of the side chains of the heme of form I relative to the heme of form II.     

Heme oxygenase 1 overexpression increases iron fluxes in caco-2 cells

Heme oxygenase-1 is a microsomal enzyme that, when induced by stress, protects the cells from oxidative injury. Heme oxygenase-1 participates in the cleavage of the heme ring producing biliverdin, CO and ferrous Fe. The released Fe becomes part of intracellular Fe pool and can be stored in ferritin or released by an iron exporter. The mechanism by which heme enters cells is not completely understood, although it had been suggested that it might be internalized by an endocytosis process. In this study, we expressed a full-length Heme oxygenase-1 cDNA in Caco-2 cells and measured intracellular iron content, heme-iron uptake and transport and immunolocalization of heme oxygenase-1 in these cells. We found that heme oxygenasc-1 expressing cells showed increased apical heme iron uptake and transepithelial transport when compared to control cells. These results suggested that heme oxygenase-1 mediates heme iron influx and efflux in intestinal cells.     

Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups.

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. M?ssbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.     

Activity of soybean lipoxygenase in the absence of lipid hydroperoxide

Soybean lipoxygenase was assayed under conditions such that the concentration of the enzyme was in excess of the concentration of the substrate, arachidonic acid. Under these conditions, the concentration of lipid hydroperoxides present as contaminants in the substrate was negligible relative to the enzyme concentration, and the concentration of lipid hydroperoxide product could be determined accurately. The ferric form of the enzyme was observed to be fully active and to catalyze the oxidation of arachidonic acid at a near-diffusion-controlled rate, 1.4 X 10(7) M-1 s-1 at 0 degree C, at concentrations of lipid hydroperoxides as low as 5% of the enzyme concentration. From this, it can be concluded that the higher oxidation states that would be accessible by oxidation of Fe(III) by hydroperoxide are not required for catalysis by soybean lipoxygenase. Surprisingly, the activation of the ferrous form of the enzyme was also observed at insignificantly low lipid hydroperoxide concentrations. This activation presumably involves oxidation of the ferrous to the ferric form of the enzyme and must be more facile than has hitherto been reported. This result may rationalize previous reports that the ferrous and the ferric forms of the enzyme are both active.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

The heme-iron geometry of ferrous nitrosylated heme-serum lipoproteins,hemopexin, and albumin: a comparative EPR study

Serum high and low density lipoproteins, albumin, and hemopexin (HDL, LDL, SA, and HPX, respectively) serve as traps of toxic plasma heme and participate in its complete clearance by transportation to the liver. Moreover, SA-(heme) and HPX-heme have been proposed to facilitate NO scavenging in vivo. Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins (HDL-heme-NO and LDL-heme-NO, respectively) as well as of ferrous nitrosylated heme-rabbit serum hemopexin (HPX-heme-NO) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO). HDL-heme-NO and LDL-heme-NO as well as SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are five-coordinate heme-iron species, characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum. On the other hand, SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO are six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry. The heme-iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-heme-NO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL>HDL>SA>HPX. Altogether, these observations suggest that HPX displays a cleft much more suitable for heme binding than other heme-carriers.     

Carbon monoxide -- a "new" gaseous modulator of gene expression

Carbon monoxide (CO) is an odorless, tasteless and colorless gas which is generated by heme oxygenase enzymes (HOs). HOs degrade heme releasing equimolar amounts of CO, iron and biliverdin, which is subsequently reduced to bilirubin. CO shares many properties with nitric oxide (NO), an established cellular messenger. Both CO and NO are involved in neural transmission and modulation of blood vessel function, including their relaxation and inhibition of platelet aggregation. CO, like NO, binds to heme proteins, although CO binds only ferrous (FeII) heme, whereas NO binds both ferrous and ferric (FeIII). CO enhances the activity of guanylate cyclase although it is less potent than NO. In contrast, CO inhibits other heme proteins, such as catalase or cytochrome p450. The effects of CO on gene expression can be thus varied, depending on the cellular microenvironment and the metabolic pathway being influenced. In this review the regulation of gene expression by HO/CO in the cardiovascular system is discussed. Recent data, derived also from our studies, indicate that HO/CO are significant modulators of inflammatory reactions, influencing the underlying processes such as cell proliferation and production of cytokines and growth factors.     

Kinetic behavior of the monodehydroascorbate radical studied by pulse radiolysis

The reactions of the monodehydroascorbate radical (As.-) with various biological molecules were investigated by pulse radiolysis. As.- reacted with both fully reduced and semiquinone forms of hepatic NADH-cytochrome b5 reductase with second-order rate constants of 4.3 x 10(6) and 3.7 x 10(5) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of As.- with ferrous cytochrome b5 could be detected by pulse radiolysis, whereas the oxidation of cytochrome b5 by As.- was observed by ascorbate-ascorbate oxidase method. This suggests that the rate constant of As.- with the ferrous cytochrome b5 must be several orders in magnitude smaller than that of the disproportionation of As.-. On the other hand, As.- reduced Fe3+EDTA with a second-order rate constant of 4.0 x 10(6) M-1 s-1 but did not reduce ferric hemoproteins such as metmyoglobin, methemoglobin, and cytochrome b5 by either the pulse radiolysis or the ascorbate-ascorbate oxidase method.     

Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein.

A simple and sensitive method for the direct measurement of lipid peroxides in lipoprotein and liposomes is described. The method is based on the principle of the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fe(3+)-xylenol orange complex which can be measured spectrophotometrically at 560 nm. Calibration with standard peroxides, such as hydrogen peroxide, linoleic hydroperoxide, t-butyl hydroperoxide, and cumene hydroperoxide gives a mean apparent extinction coefficient of 4.52 x 10(4) M-1 cm-1 consistent with a chain length of approximately 3 for ferrous ion oxidation by hydroperoxides. Endoperoxides are less reactive or unreactive in the assay. The assay has been validated in the study of lipid peroxidation of low density lipoprotein and phosphatidyl choline liposomes. By pretreatment with enzymes known to metabolize peroxides, we have shown that the assay measures lipid hydroperoxides specifically. Other methods for measuring peroxidation, such as the assessment of conjugated diene, thiobarbituric acid reactive substances and an iodometric assay have been compared with the ferrous oxidation-xylenol orange assay.     

Reduced flavins promote oxidative DNA damage in non-respiring Escherichia coli by delivering electrons to intracellular free iron

When cells are exposed to external H(2)O(2), the H(2)O(2) rapidly diffuses inside and oxidizes ferrous iron, thereby forming hydroxyl radicals that damage DNA. Thus the process of oxidative DNA damage requires only H(2)O(2), free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state. Previous work showed that H(2)O(2) kills Escherichia coli especially rapidly when respiration is inhibited either by cyanide or by genetic defects in respiratory enzymes. In this study we established that these respiratory blocks accelerate the rate of DNA damage. The respiratory blocks did not substantially affect the amounts of intracellular free iron or H(2)O(2), indicating that that they accelerated damage because they increased the availability of the electron donor. The goal of this work was to identify that donor. As expected, the respiratory inhibitors caused a large increase in the amount of intracellular NADH. However, NADH itself was a poor reductant of free iron in vitro. This suggests that in non-respiring cells electrons are transferred from NADH to another carrier that directly reduces the iron. Genetic manipulations of the amounts of intracellular glutathione, NADPH, alpha-ketoacids, ferredoxin, and thioredoxin indicated that none of these was the direct electron donor. However, cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD. The K(m) value of this enzyme for NADH is much higher than the usual intracellular NADH concentration, which explains why its flux increased when NADH levels rose during respiratory inhibition. Flavins that were reduced by purified flavin reductase rapidly transferred electrons to free iron and drove a DNA-damaging Fenton system in vitro. Thus the rate of oxidative DNA damage can be limited by the rate at which electron donors reduce free iron, and reduced flavins become the predominant donors in E. coli when respiration is blocked. It remains unclear whether flavins or other reductants drive Fenton chemistry in respiring cells.     

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased (P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.     

Utilization of the ferrous sulfate (Fricke) dosimeter for evaluating the radioprotective potential of cystamine: experiment and Monte Carlo simulation

Cystamine, an organic disulfide (RSSR), is among the best of the known radiation-protective compounds and has been used to protect normal tissues in clinical radiation therapy. Recently, it has also proved to be beneficial in the treatment of disorders of the central nervous system in animal models. However, the underlying mechanism of its action at the chemical level is not yet well understood. The present study aims at using the ferrous sulfate (Fricke) dosimeter to quantitatively evaluate, both experimentally and theoretically, the radioprotective potential of this compound. The well-known radiolysis of the Fricke dosimeter by (60)Co γ rays or fast electrons, based on the oxidation of ferrous ions to ferric ions by the oxidizing species (?)OH, HO(2)(?), and H(2)O(2) produced in the radiolytic decomposition of water, forms the basis for our method. The presence of cystamine in Fricke dosimeter solutions during irradiation prevents the radiolytic oxidation of Fe(2+) and leads to decreased ferric yields (or G values). The observed decrease in G(Fe(3+)) increases upon increasing the concentration of the disulfide compound over the range 0-0.1 M under both aerated and deaerated conditions. To help assess the basic radiation-protective mechanism of this compound, a full Monte Carlo computer code is developed to simulate in complete detail the radiation-induced chemistry of the studied Fricke/cystamine solutions. Benefiting from the fact that cystamine is reasonably well characterized in terms of radiation chemistry, this computer model proposes reaction mechanisms and incorporates specific reactions describing the radiolysis of cystamine in aerated and deaerated Fricke solutions that lead to the observable quantitative chemical yields. Results clearly indicate that the protective effect of cystamine originates from its radical-capturing ability, which allows this compound to act by competing with the ferrous ions for the various free radicals--especially (?)OH radicals and H(?) atoms--formed during irradiation of the surrounding water. Most interestingly, our simulation modeling also shows that the predominant pathway in the oxidation of cystamine by (?)OH radicals involves an electron-transfer mechanism, yielding RSSR(?+) and OH(-). A very good agreement is found between calculated G(Fe(3+)) values and experiment. This study concludes that Monte Carlo simulations represent a very efficient method for understanding indirect radiation damage at the molecular level.     

The oscillator strengths of hemoproteins. Their relation to the coordination structure and magnetic susceptibility

The oscillator strengths of hemoproteins in the light frequency range of 1.11 X 10(4) to 3.23 X 10(4) cm-1 (wavelength range of 900 to 310 nm) were measured by means of computer-assisted spectrophotometry. The obtained values of oscillator strength per molar heme ranged from about 1.4 to 2.2. By comparing the oscillator strength values of the ferric and ferric cyanide-bound forms of hemoproteins and also the values of low molecular weight ferric heme complexes, it was found that the oscillator strength was lower for those hemoproteins whose heme was coordinated with strong field ligands. It was also found that the hemoproteins showing a smaller pH-dependent change in the carbon monoxide-difference spectrum had lower oscillator strengths. The following linear relation was observed, with various ligand complexes of bovine methemoglobin, horse metmyoglobin, and ferric horseradish peroxidase, between the oscillator strength (f) determined in the present study and the respective magnetic susceptibility (10(6) X chi 20 degrees M) values in the literature: f = A (10(6) X chi 20 degrees M) + B. The values of constants A and B in the equation were estimated for horseradish peroxidase, methemoglobin, and metmyoglobin. On varying the temperature in the range of 0 to 40 degrees C, the oscillator strength of the metmyoglobin-azide complex changed in parallel with the change in the spin state. Taking advantage of the fact that fluoride complexes of many hemoproteins show 10(6) X chi 20 degrees M values close to 14,500 and also that the values of intersection B are around 86.4% of the respective values of the fluoride complexes of ferric horseradish peroxidase, methemoglobin, and metmyoglobin, an empirical equation was evolved for the calculation of an approximate 10(6) X chi 20 degrees M value from the f value of a given complex (fobs) and that of the fluoride complex (fF) of a hemoprotein. The approximate magnetic susceptibilities of various ligand complexes of bovine lactoperoxidase could be thus calculated with the equation. The oscillator strengths of ferrous hemoproteins were also investigated and ligand-dependent regular changes were found.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.