Mössbauer studies of the non-heme iron and cytochrome b559 in a Chlamydomonas reinhardtii PSI- mutant and their interactions with alpha-tocopherol quinone

Spin and valence states of the non-heme iron and the heme iron of cytochrome b559, as well as their interactions with alpha-tocopherol quinone (alpha-TQ) in photosystem II (PSII) thylakoid membranes prepared from the Chlamydomonas reinhardtii PSI- mutant have been studied using M?ssbauer spectroscopy. Both of the iron atoms are in low spin ferrous states. The Debye temperature of the non-heme is 194 K and of the heme iron is 182 K. The treatment of alpha-TQ does not change the spin and the valence states of the non-heme iron but enhances the covalence of its bonds. alpha-TQ oxidizes the heme iron into the high spin Fe3+ state. A possible role of the non-heme iron and alpha-TQ in electron flow through the PSII is discussed.     

Importance of iron location in iron-induced hydroxyl radical production by brain slices

Iron imbalance has been implicated in oxidative injury associated with many brain diseases. The present study investigated the importance of iron location in hydroxyl radical (.OH) generation and the link between .OH production evaluated by the salicylate method and lipid peroxidation monitored by thiobarbituric acid-reactive substances assay. Brain slices were exposed to increasing doses (2, 10 and 50 microM) of Fe(III) that was complexed either to a lipophilic (8-hydroxyquinoline, HQ) or to a hydrophilic (ammoniacal citrate) ligand. Both iron complexes resulted in an increased salicylate hydroxylation and lipid peroxidation, these effects being significantly more potent in presence of Fe(III)-HQ. Salicylate hydroxylation was linearly correlated to the intensity of TBARS formation but the slope of the curve was found to be higher with Fe(III)-HQ. The present results demonstrate that 1) cell-associated reactive iron is more prone than extracellular iron to induce .OH generation, 2) the level of lipid peroxidation depending on the site of .OH production, cannot be used as an index of the level of total .OH formation, 3) the salicylate method is a convenient method to detect .OH formed intracellularly, at least in vitro.     

Biosynthesis of fosfomycin, re-examination and re-confirmation of a unique Fe(II)- and NAD(P)H-dependent epoxidation reaction

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) catalyzes the epoxide ring closure of (S)-HPP to form fosfomycin, a clinically useful antibiotic. Early investigation showed that its activity can be reconstituted with Fe(II), FMN, NADH, and O2 and identified HppE as a new type of mononuclear non-heme iron-dependent oxygenase involving high-valent iron-oxo species in the catalysis. However, a recent study showed that the Zn(II)-reconstituted HppE is active, and HppE exhibits modest affinity for FMN. Thus, a new mechanism is proposed in which the active site-bound Fe2+ or Zn2+ serves as a Lewis acid to activate the 2-OH group of (S)-HPP and the epoxide ring is formed by the attack of the 2-OH group at C-1 coupled with the transfer of the C-1 hydrogen as a hydride ion to the bound FMN. To distinguish between these mechanistic discrepancies, we re-examined the bioautography assay, the basis for the alternative mechanism, and showed that Zn(II) cannot replace Fe(II) in the HppE reaction and NADH is indispensable. Moreover, we demonstrated that the proposed role for FMN as a hydride acceptor is inconsistent with the finding that FMN cannot bind to HppE in the presence of substrate. In addition, using a newly developed HPLC assay, we showed that several non-flavin electron mediators could replace FMN in the HppE-catalyzed epoxidation. Taken together, these results do not support the newly proposed "nucleophilic displacement-hydride transfer" mechanism but are fully consistent with the previously proposed iron-redox mechanism for HppE catalysis, which is unique within the mononuclear non-heme iron enzyme superfamily.     

Iron sources forming nitrosyl complexes in animal tissues

Treatment of perfused mouse liver with nitric oxide does not change the intensities of ESR signals of iron-sulfur proteins characteristic of this tissue. Proceeding from this evidence and also from the ratio between the iron content in these proteins and dinitrosyl iron complexes (complexes 2.03) formed in the liver when it contacts with NO, it is concluded that iron-sulfur proteins are not involved in the formation of complexes 2.03. It seems that only the loosely bound form of non-heme iron-free iron is involved in this process.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr(Z)) and side-path electron donors (Car/Chl(Z)/Cyt(b)(559)) can be oxidized by P(680)(+) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z) EPR signal were independent of the treatment of K(3)Fe(CN)(6), whereas formation and decay of the Car(+)/Chl(Z)(+) EPR signal correlated with the reduction and recovery of the Fe(3+) EPR signal of the non-heme iron in K(3)Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe(3+) reduction, the oxidation of non-heme iron by K(3)Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe(3+) EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr(Z) oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K(3)Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe(3+) state is probably not the intermediate species for the turnover of quinone reduction.     

The source of non-heme iron that binds nitric oxide in cultivated macrophages.

In cultured macrophages (J 774 line) a decrease in iron-sulfur centers (ISC) was not observed after 5 min treatment with nitric oxide (NO) (10(-7) M NO/10(7) cells). The content of these centers was measured by electron spin resonance (ESR) spectroscopy at 16-60 K. However, the appearance of a characteristic ESR signal at g(av) = 2.03 indicated the formation of dinitrosyl iron complex (DNIC) in these cells. These findings suggest that loosely bound non-heme iron (free iron) but not iron from ISC is mainly involved in DNIC formation. ISC might release iron for DNIC formation after their destruction induced by the products of NO oxidation (NO2, N2O3, etc).     

Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei.

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.     

Probing the quinone binding site of photosystem II from Thermosynechococcus elongatus containing either PsbA1 or PsbA3 as the D1 protein through the binding characteristics of herbicides

The main cofactors involved in Photosystem II (PSII) oxygen evolution activity are borne by two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is predominantly encoded by either the psbA(1) or the psbA(3) gene, the expression of which depends on the environmental conditions. In this work, the Q(B) site properties in PsbA1-PSII and PsbA3-PSII were probed through the binding properties of DCMU, a urea-type herbicide, and bromoxynil, a phenolic-type herbicide. This was done by using helium temperature EPR spectroscopy and by monitoring the time-resolved changes of the redox state of Q(A) by absorption spectroscopy in PSII purified from a His(6)-tagged WT strain expressing PsbA1 or from a His(6)-tagged strain in which both the psbA(1) and psbA(2) genes have been deleted and which therefore only express PsbA3. It is shown that, in both PsbA1-PSII and PsbA3-PSII, bromoxynil does not bind to PSII when Q(B) is in its semiquinone state which indicates a much lower affinity for PSII when Q(A) is in its semiquinone state than when it is in its oxidized state. This is consistent with the midpoint potential of Q(A)(-)/Q(A) being more negative in the presence of bromoxynil than in its absence [Krieger-Liszkay and Rutherford, Biochemistry 37 (1998) 17339-17344]. The addition in the dark of DCMU, but not that of bromoxynil, to PSII with a secondary electron acceptor in the Q(B)(-) state induces the oxidation of the non-heme iron in a fraction of PsbA3-PSII but not in PsbA1-PSII. These results are explained as follows: i) bromoxynil has a lower affinity for PSII with the non-heme iron oxidized than DCMU therefore, ii) the midpoint potential of the Fe(II)/Fe(III) couple is lower with DCMU bound than with bromoxynil bound in PsbA3-PSII; and iii) the midpoint potential of the Fe(II)/Fe(III) couple is higher in PsbA1-PSII than in PsbA3-PSII. The observation of DCMU-induced oxidation of the non-heme iron leads us to propose that Q(2), an electron acceptor identified by Joliot and Joliot [FEBS Lett. 134 (1981) 155-158], is the non-heme iron.     

Photoinhibition affects the non-heme iron center in photosystem II.

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.     

Synthesis and characterization of iron(llI) chelating analogues of siderophores on organic solid supports

A series of iron(III)-selective chelating resins have been modeled after the structural features of the naturally occurring siderophore compounds with hydroxamate, catecholate and salicylate iron binding groups. Amberlite IRC-50 was derivatized via an acid chloride intermediate to produce poly(hydroxamic acid) (IRC-50 PHA/N-H and IRC-50 PHA/N- CH₃), poly(catecholate) (IRC-50 PEDA 2,3-DHBAD and IRC-50 PEDA 3,4-DHBA) and poly(salicylate) (IRC-50 PEDA 2-HBAD) chelating resins with enhanced iron(III) binding capacity. The poly(hydroxamic acid) IRC-50 PHA/N-CH₃ was produced in 57% yield, the highest conversion yet reported for such derivatizations. This is the first report of a catechol or salicylate derivatization of Amberlite IRC-50. The highest overall iron(III) binding capacities yet reported for poly(hydroxamic acid) modifications of commercially available polymer supports were obtained for IRC-50 PHA/N-H (1.75 mmol Fe/g dry resin) and IRC-50 PHA/N-CH₃ (1.52 mmol Fe/g dry resin). IRC-50 PHA/N-H was also found to be an effective iron chelator when tested at ambient environmental conditions. Selectivity for iron- (III) was also determined by measuring Fe³⁺ binding capacity in the presence of Ca²⁺. UVVis spectroscopy with photoacoustic detection was used to assign the coordination environment of iron(III) in these chelating resins as bis-(FeL₂) and tris-(FeL₃) chelates. As an alternative synthetic approach, a poly(amidoxime) (PAO) chelating resin was synthesized by polymerization of appropriate monomers. Comparison between the two synthetic approaches showed that PAO bound a greater amount of iron(III) at flow-through column conditions, while IRC-50 PHA/N-H exhibited a greater iron(III) binding capacity at batch equilibration conditions.     

Cysteine-dependent inactivation of hepatic ornithine decarboxylase.

When rat liver homogenate or its postmitochondrial supernatant was incubated with L-cysteine, but not D-cysteine, ornithine decarboxylase (ODC) lost more than half of its catalytic activity within 30 min and, at a slower rate, its immunoreactivity. The inactivation correlated with production of H2S during the incubation. These changes did not occur in liver homogenates from vitamin B6-deficient rats. A heat-stable inactivating factor was found in both dialysed cytosol and washed microsomes obtained from the postmitochondrial supernatant incubated with cysteine. The microsomal inactivating factor was solubilized into Tris/HCl buffer, pH 7.4, containing dithiothreitol. Its absorption spectrum in the visible region resembled that of Fe2+ X dithiothreitol in Tris/HCl buffer. On the other hand FeSO4 inactivated partially purified ODC in a similar manner to the present inactivating factor. During the incubation of postmitochondrial supernatant with cysteine, there was a marked increase in the contents of Fe2+ loosely bound to cytosolic and microsomal macromolecules. Furthermore, the content of such reactive iron in the inactivating factor preparations was enough to account for their inactivating activity. These data suggested that H2S produced from cysteine by some vitamin B6-dependent enzyme(s) converted cytosolic and microsomal iron into a reactive loosely bound form that inactivated ODC.     

Transport of Iron in the Blood-Brain-Cerebrospinal Fluid System

Abstract: Iron is an important constituent in brain and, in certain regions, e.g., the basal nuclei, reaches concentrations equivalent to those in liver. It has a role in electron transfer and is a cofactor for certain enzymes, including those involved in catecholamine and myelin synthesis. Iron in CSF is likely to be representative of that in interstitial fluid of brain. Transferrin in CSF is fully saturated, and the excess iron may be loosely bound as Fe(II). Brain iron is regulated in iron depletion, suggesting a role for the blood-brain barrier (BBB). Iron crosses the luminal membrane of the capillary endothelium by receptor-mediated endocytosis of ferric transferrin. This results in an initial linear uptake of radioactive iron into brain at an average rate relative to serum of about 3.3 × 10^?3 ml·g of brain^?1·h^?1 in the adult rat. This corresponds to about 80 nmol·kg^?1·h^?1. Much higher rates occur in the postnatal rat. These increase during the first 15 days of life and decline thereafter. Within the endothelium, most of the iron is separated from transferrin, presumably by the general mechanism of acidification within the endosome. Iron appears to be absorbed from the vesicular system into cytoplasm and transported across the abluminal plasma membrane into interstitial fluid as one or more species of low molecular weight. There is some evidence that ionic Fe(II) is involved. Certainly Fe(II) ions presented on the luminal side rapidly cross the complete BBB, i.e., luminal and abluminal membranes. Within interstitial fluid, transported iron will bind with any unsaturated transferrin synthesized or transported into the brain-CSF system. Oligodendrocytes are one site of synthesis. From interstitial fluid, ferric transferrin is taken up by neurones and glial cells by the usual receptor-mediated endocytosis. Calculations of the amount of iron leaving the system with the bulk flow of CSF indicate that most iron entering brain across the capillary endothelium finally leaves the system with the bulk outflow of CSF through arachnoid villi and other channels. A system in which influx of iron into brain is by regulated receptor-mediated transport and in which efflux is by bulk flow is ideal for homeostasis of brain iron.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

Forming the phosphate layer in reconstituted horse spleen ferritin and the role of phosphate in promoting core surface redox reactions.

Apo horse spleen ferritin (apo HoSF) was reconstituted to various core sizes (100-3500 Fe3+/HoSF) by depositing Fe(OH)3 within the hollow HoSF interior by air oxidation of Fe2+. Fe2+ and phosphate (Pi) were then added anaerobically at a 1:4 ratio, and both Fe2+ and Pi were incorporated into the HoSF cores. The resulting Pi layer consisted of Fe2+ and Pi at about a 1:3 ratio which is strongly attached to the reconstituted ferritin mineral core surface and is stable even after air oxidation of the bound Fe2+. The total amount of Fe2+ and Pi bound to the iron core surface increases as the core volume increases up to a maximum near 2500 iron atoms, above which the size of the Pi layer decreases with increasing core size. M?ssbauer spectroscopic measurements of the Pi-reconstituted HoSF cores using 57Fe2+ show that 57Fe3+ is the major species present under anaerobic conditions. This result suggests that the incoming 57Fe2+ undergoes an internal redox reaction to form 57Fe3+ during the formation of the Pi layer. Addition of bipyridine removes the 57Fe3+ bound in the Pi layer as [57Fe(bipy)3]2+, showing that the bound 57Fe2+ has not undergone irreversible oxidation. This result is related to previous studies showing that 57Fe2+ bound to native core is reversibly oxidized under anaerobic conditions in native holo bacterial and HoSF ferritins. Attempts to bury the Pi layer of native or reconstituted HoSF by adding 1000 additional iron atoms were not successful, suggesting that after its formation, the Pi layer "floats" on the developing iron mineral core.     

17O-water and cyanide ligation by the active site iron of protocatechuate 3,4-dioxygenase. Evidence for displaceable ligands in the native enzyme and in complexes with inhibitors or transition state analogs

Hyperfine broadening is observable in the EPR spectrum of Brevibacterium fuscum protocatechuate 3,4-dioxygenase after lyophilization and rehydration in 17O-enriched water, demonstrating H2O ligation to the active site iron. Lack of detectable broadening in the sharp features of the spectra of three substrate complexes suggests that H2O is displaced by substrate. Water is bound in the monodentate complex with the competitive inhibitor 3-hydroxybenzoate which binds directly to the iron showing that two iron ligation sites can be occupied by nonprotein ligands. Ketonized substrate analogs which mimic a proposed transition state of the reaction cycle, 2-hydroxyisonicotinic acid N-oxide (2-OHINO) and 6-hydroxynicotinic acid N-oxide (6-OH NNO), have H2O bound in their final, bleached enzyme complexes, suggesting that these complexes are also monodentate. In contrast, a transient, initial complex of 6-OH NNO which is spectrally similar to the substrate complex, apparently does not have H2O bound. Cyanide binding occurs in two steps. The active site Fe3+ of the initial, rapidly formed, violet complex is high spin while that of the second, slowly formed, green complex is low spin; a unique state for mononuclear non-heme iron enzymes. The data suggest that the Fe-CN- and Fe-(CN-)2 complexes form sequentially. CN- binds to enzyme complexes with 2-OH INO and 6-OH NNO in one step to yield high spin Fe3+ species. In contrast, preformed substrate complexes prevent CN- binding. CN- binding eliminates the broadening due to 17O-water in the EPR spectra of both native enzyme and the enzyme-ketonized analog complexes. A model is proposed in which H2O is displaced by bidentate binding of the substrate but can potentially rebind after a subsequent substrate ketonization. The proximity of the vacatable H2O-binding site of the iron to the site of oxygen insertion suggests, however, that this site may serve to stabilize an oxygenated intermediate during the reaction cycle.     

Effect of temperature and chloride on steady-state inhibition of angiotensin I-converting enzyme by enalaprilat and ramiprilat.

Recent work has provided new evidence that ATP is the major constituent of the low-Mr iron pool in the reticulocyte. The interaction of the iron complex of ATP with mitochondria was investigated in the present experiments. When ATP-Fe3+ was incubated with mitochondria, Fe3+, free of ATP, bound with high affinity to Fe3+ receptors on the mitochondria. The binding was saturable and reversible. Iron which was complexed to PPi, nitrilotriacetate, citrate, ADP and GTP also showed saturable binding to mitochondria; Fe3+ complexed to AMP bound non-specifically, as did Fe2+/ascorbate complexed to AMP bound non-specifically, as did Fe2+/ascorbate and Fe2+/dithionite.     

Melatonin exerts a more potent effect than S-adenosyl-l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats

Melatonin and S-adenosyl-l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.     

The Effect of Calcium on Non-heme Iron Uptake,Efflux, and Transport in Intestinal-like Epithelial Cells (Caco-2 Cells)

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of ⁵⁵Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.     

Stimulation and inhibition of iron-dependent lipid peroxidation by desferrioxamine

Peroxidation of rat brain synaptosomes was assessed by the formation of thiobarbituric acid reactive products in either 50 mM potassium phosphate buffer (pH 7.4) or pH adjusted saline. In phosphate, addition of Fe2+ resulted in a dose-related increase in lipid peroxidation. In saline, stimulation of lipid peroxidation by Fe2+ was maximal at 30 uM, and was less at concentrations of 100 uM and above. Whereas desferrioxamine caused a dose-related inhibition of iron-dependent lipid peroxidation in phosphate, it stimulated lipid peroxidation with Fe2+ by as much as 7-fold in saline. The effects of desferrioxamine depended upon the oxidation state of iron, and the concentration of desferrioxamine and lipid. The results suggest that lipid and desferrioxamine compete for available iron. The data are consistent with the hypothesis that either phosphate or desferrioxamine may stimulate iron-dependent lipid peroxidation under certain circumstances by favoring formation of Fe2+/Fe3+ ratios.     

Characterization of the multiple EPR line shapes of iron-semiquinones in photosystem 2.

We have compared the temperature-dependence characteristics of the EPR signals of Qa and Qb iron-semiquinones from both purple bacterial and plant photosystems. The data obtained were analyzed and estimates of the splitting parameters of the non-heme Fe2+ spin sublevels obtained. The study confirms the similarities of the g = 1.8 Qa iron-semiquinone signal (D/k = 15.6 K, E/k = 3.3 K) formed in formate-treated plant photosystem 2 to the signal found in purple bacteria. However, the g = 1.9 Qa iron-semiquinone signal (D/k = 7.1 K, E/k = much less than 1 K), formed in photosystem 2 when bicarbonate remains bound, has a unique temperature behavior. A series of spectral features associated with the iron-semiquinone in bicarbonate-bound photosystem 2 appear as the temperature is lowered, and the analysis of these data requires that some of these features be assigned to the higher spin states. The results are discussed in terms of the requirement for bicarbonate to be a ligand of the non-heme iron.