Hemosiderin An EPR study of water-insoluble iron in human and rat liver

EPR spectra of the water-insoluble iron fraction, hemosiderin of human and rat liver are described. The homogenate of freshly prepared perfused rat liver shows a non-heme iron signal at g = 4.3 and a high-spin heme-iron signal around g = 6, whereas the washed and sonicated sample of the insoluble iron fraction shows solely a non-heme iron signal at g = 4.3. This indicates that hemosiderin from rat liver does not contain heme iron.Human-liver preparations from post mortem obtained material show in the homogenates as well as in the washed and sonicated samples an intense high-spin heme iron signal at g = 6.0 and a non-heme iron signal at g = 4.3. A comparative experiment, carried out with “aged” rat liver preparations, reveals the same spectra as with the human preparations. It is concluded that the heme present in the insoluble iron fraction is caused by degradation of hemoglobin in the obduction material, and that heme is not a constituent of the insoluble depot iron.     

Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g_|| = 2.24, A_|| = 18.4 mT and g_⊥ = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g_|| = 2.29, A_|| = 16.1 mT and g_⊥ = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu²⁺ with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu²⁺ and three diamagnetic Cu¹⁺ per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per αβγ-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe³⁺ was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu²⁺-EPR signal.     

Electron paramagnetic resonance spectra of mitochondrial and microsomal cytochrome P-450 from the rat adrenal

The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculata mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with g_x values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11β-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spin signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spin parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and high spin ferric g_xy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

The effect of phospholipid depletion on the EPR behavior of cytochrome oxidase

Phospholipids are essential components for electron transport activity of cytochrome oxidase. Recently, we have found that the removal of phospholipids from the oxidase affected the copper and low-spin heme signals, and conceivably other paramagnetic centers as demonstrated by EPR spectroscopy. At 4.2–30 °K, the signal amplitudes and power saturation behaviors were studied at approximately g = 2.0 for the copper signal, and in the neighborhood of g = 3.0 for the low-spin heme signal. After depletion of phospholipids the amplitude of the copper signal decreased 25–30% at 12–30 °K and below 12 °K 40–50% under nonsaturating conditions. The amplitude of the low-spin heme signal decreased 60–70% at 4.2–20 °K. Below 14 °K both signals became more resistant to power saturation, but the copper signal was more readily saturated above this temperature, compared to the oxidase with about 25% lipid. After removal of phospholipids, the spectral features of the copper signal remained essentially the same, but the low-spin heme signal broadened and became very asymmetric to show two signals as revealed by the second harmonic EPR spectra. These findings may explain, at least partially, the wide variations in percentage of EPR detectable copper and heme of cytochrome oxidase reported by different laboratories. Unequivocally, the EPR behavior of cytochrome oxidase is not only affected by the protein moiety, but also by the associated phospholipids of the enzyme.     

EPR study of iron status in human body during intensive physical activity

The iron metabolism was studied in serum blood samples collected from 26 professional sportsmen undergoing intensive physical exercises using EPR combined with hematological and biochemical laboratory tests. Only 23% of EPR spectra (n = 6) were practically normal while in the rest spectra additional abnormal absorption lines were detected. Presumably, the significant portion of new signals may be caused by different cytochromes. Moreover, the anisotropic signals with g ₁ ? 2.02; g ₂ ? 1.94 and g ₃ ? 1.86 registered in some spectra pointed to the sulfur-iron centers. There was nearly linear correlation between the concentration of Fe³⁺ in transferrin (Fe³⁺-Tf) obtained from the EPR spectra and the serum iron concentration measured by absorption photometry both for sportsmen and controls (healthy individuals and patients with different diseases). At equal serum iron concentrations the Fe³⁺-Tf level was higher in sportsmen than that in controls. The Pearson correlation coefficient (r) for Fe³⁺-Tf and serum iron values was equal to 0.89 in sportsmen versus r = 0.97 in controls. Additional new lines in serum EPR spectra of professional sportsmen prove the suitability of EPR assay for scheduled medical exams since routine biochemical and hematological tests are insufficient to discover all abnormalities in iron metabolism under intensive physical exercises.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : V. On the Reduction of Nonheme Iron and the Cytochromes by Nicotinamide Adenine Dinucleotide and Succinate

The sensitivity of nicotinamide adenine dinucleotide (NADH) oxidase and succinoxidase to metal chelators, the generation of an electron paramagnetic resonance (EPR) signal upon addition of these substrates, and the rate of formation of the EPR signal relative to the rate of the cytochrome reduction suggest the participation of nonheme iron proteins in the respiratory process of Escherichia coli. The most inhibitory metal chelator, thenoyltrifluoro acetone, inhibited the reduction of nonheme iron and cytochromes but did not prevent the reoxidation of the reduced forms. The EPR signal, dehydrogenase, and oxidase activities evoked by NADH are considerably greater than the corresponding activities evoked by succinate. Because both substrates can reduce almost all of the cytochromes, a model in which fewer succinate dehydrogenase-nonheme iron protein complexes are linked to a common cytochrome chain than NADH dehydrogenase-nonheme iron protein complexes is considered likely.     

The Formation of g=2.49-Species of Cytochrome P450 in the Rat Liver by PCB126 Oral Administration: Identification of Heme Axial Ligands by EPR Spectroscopy

Rat livers and microsomes were subjected to electron paramagnetic resonance (EPR) measurements at 77 K. The EPR spectra of the livers from the control group, carbon tetrachloride-, 3-methylcholanthrene-, and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126)-treated rats exhibited an EPR spectrum at g=2.40, 2.24, and 1.93, which is characteristic of P450 in a resting state. The liver of the PCB126-treated rats showed an additional distinct EPR spectrum at g=2.49, 2.26, and 1.87 (g=2.49-species). The heme environmental structure of g=2.49-species was identified by crystal field analysis using three EPR g-values of the microsome treated with various chemicals. These results indicated that g=2.49-species is a hemeprotein with cysteine thiolate at the 5th coordination site, and a nitrogenous ligand at the 6th site.     

Correlation of the cytochrome c 2 content of cyanobacterial Photosystem II with the EPR properties of the oxygen-evolving complex

The Mn₄ cluster of PS II advances through a series of oxidation states (S states) that catalyze the breakdown of water to dioxygen in the oxygen-evolving complex. The present study describes the engineering and purification of highly active PS II complexes from mesophilic His-tagged Synechocystis PCC 6803 and purification of PS II core complexes from thermophilic wild-type Synechococcus lividus with high levels of the extrinsic polypeptide, cytochrome c ₅₅₀. The g = 4.1 S₂ state EPR signal, previously not characterized in untreated cyanobacterial PS II, is detected in high yields in these PS II preparations. We present a complete characterization of the g = 4.1 state in cyanobacterial His-tagged Synechocystis PCC 6803 PS II and S. lividus PS II. Also presented are a determination of the stoichiometry of cytochrome c ₅₅₀ bound to His-tagged Synechocystis PCC 6803 PS II and analytical ultracentrifugation results which indicate that cytochrome c ₅₅₀ is a monomer in solution. The temperature-dependent multiline to g = 4.1 EPR signal conversion observed for the S₂ state in cyanobacterial PS II with high cytochrome c ₅₅₀ content is very similar to that previously found for spinach PS II. In spinach PS II, the formation of the S₂ state g = 4.1 EPR signal has been found to correlate with the binding of the extrinsic 17 and 23 kDa polypeptides. The finding of a similar correlation in cyanobacterial PS II with the binding of cytochrome c ₅₅₀ suggests a functional homology between cytochrome c ₅₅₀ and the 17 and 23 kDa extrinsic proteins of spinach PS II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of Nuclear Mutants of Maize Which Lack the Cytochrome f/b-563 Complex

Two high fluorescent, nuclear recessive mutants of maize (Zea mays L.), designated hcf-2 and hcf-6, are described which are missing the chloroplast cytochrome f/b-563 complex. Thylakoids from the mutants show a block in whole chain electron transport activity (H₂O to methyl viologen), while retaining activities associated with photosystem II (H₂O to phenylenediamine) and photosystem I (diaminodurene to methyl viologen). Chemically induced, optical difference spectra indicate a loss of cytochromes f and b-563. Cytochrome b-559 is present in both high and low potential forms. EPR analyses of thylakoid membranes of hcf-6 reveals the lack of a signal (g = 1.90) associated with the Rieske Fe-S center. Additionally, hcf-6 is lacking EPR signals at g = 6 (attributable to the high spin ferric heme of cytochrome b-563) and g = 2.5 (unidentified). The mutant retains signals at g = 2.9 (cytochrome b-559) and at g = 4.3 and 9 (both signals probably arising from a storage form of ferric iron).

Thylakoid polypeptides are examined using polyacrylamide gel electrophoresis. hcf-2 and hcf-6 have identical profiles, showing losses of polypeptides with apparent molecular masses of 33 (cytochrome f), 23 (cytochrome b-563), and 17.5 kilodaltons. The protein associated with the Rieske Fe-S center could not be determined from the gel profiles. Additionally, both mutants show an increase in a band with a molecular mass of 31 kilodaltons.

     

An EPR signal from the "invisible" copper of cytochrome oxidase.

Sulfide is both an inhibitor and a slow reductant of oxidized cytochrome $\text{c}$ oxidase. When the enzyme is exposed to sulfide for short times (one minute or less) and frozen, the resultant electron paramagnetic resonance (EPR) signals show clearly: low spin heme a, low spin heme a₃, the usual “EPR detectable” Cu²⁺ signal (g_⊥ = 2.17, g_⊥ = 2.03), and a new Cu²⁺ signal superimposed on the same region, with (g_⊥ ～ 2.19, g_⊥ = 2.05). This new signal presumably arises because the antiferromagnetic coupling postulated to exist between the iron atom of heme a₃ and this copper is disrupted when heme a₃ is driven to a low spin state by sulfide. The implications of this result with respect to models of the O₂-binding site and redox geometry of oxidase are briefly discussed.     

Renal cytochrome P-450's-electrophoretic and electron paramagnetic resonance studies.

The cytochrome P-450's of the microsomal mixed function oxidase systems from the rabbit renal cortex, outer medulla, inner medulla, and the liver were compared. Sodium dodecyl sulfate-(SDS) gel electrophoresis and electron paramagnetic resonance (EPR) studies detected cytochrome P-450 proteins in the liver, renal cortex, and outer medulla but not the inner medulla of normal animals. Two cytochrome P-450 peptides, which had molecular weights of 54,500 and 58,900 and which comigrated with known hepatic cytochrome P-450's on SDS gels, were identified in the cortex and outer medulla. Treatment of animals with 3-methylcholanthrene (MC) enhanced the 54,500 and 58,900 peptides in the liver and cortex but produced little change in outer medulla. MC treatment induced faint cytochrome P-450 bands in the inner medulla. The EPR studies detected low spin heme iron absorption lines at g = 2.42, 2.26, and 1.92 in liver, cortex, and outer medulla from untreated animals. The amplitude of the low spin absorption lines was increased by ethanol, a reverse type I compound, and reduced by chloroform, a type I compound, in these tissues. MC treatment increased the amplitude of the heme absorption lines in these tissues, and it induced a barely detectable heme spectrum in the inner medulla. Differences in exogenous substrate binding between hepatic and renal microsomes from MC-treated animals were detected by EPR and optical difference spectroscopy. Acetone, 1-butanol, and 2-propanol gave evidence of binding to the hepatic cytochrome P-450's but no evidence of binding to renal cortical microsomes. These results, along with previous enzymatic studies, suggest that the liver and each area of the kidney contain different substrate specificities and pathways for the metabolism of organic compounds.     

Mössbauer spectroscopy and electron paramagnetic resonance studies of iron metabolites inPseudomonas aeruginosa: Fe2+ and Fe3+ ferritin in57ferripyoverdine incubated cells and57ferric citrate fed cells

Pseudomonas aeruginosa samples were studied using Mössbauer spectroscopy and electron paramagnetic resonance (EPR). Samples included whole cells, membranes, and soluble fractions from cells which had been grown with⁵⁷ferric chloride,⁵⁷ferric citrate or incubated with⁵⁷ferripyoverdine. These experiments show for the first time thatP. aeruginosa can accumulate iron in a bacterioferritin when grown under conditions of iron limitation and incubated with its cognate ferrisiderophore, ferripyoverdine. Soluble fraction fromP. aeruginosa cells which were grown iron starved and incubated with⁵⁷ferripyoverdine for 120 min showed the presence of both a ferric and ferrous complex whose Mössbauer spectra matched that of bacterioferritin extracted fromAzotobacter vinelandii and whose EPR spectra showed a characteristic ferritin-like resonance. A second soluble fraction sample from cells which had been grown with⁵⁷ferric citrate also showed the presence of a species with the same EPR and Mössbauer parameters. In addition Western blotting confirmed the presence of bacterioferritin in the soluble fraction of the cells which had been incubated with ferripyoverdine.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and YZ radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S₁ and S₃ states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11–15 signal, were detected in Ca²⁺-depleted PS II. The g=11–15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S₂ in Ca²⁺-depleted PS II. The singlet-like signal was associated with the g=11–15 signal but not with the Y_Z (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y_Z radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y_Z radical was discussed.     

EPR signals and orientation of cytochromes in the spinach chloroplast thylakoid membrane

The cytochromes in spinach chloroplasts were studied using EPR spectroscopy. In addition to the low-spin heme signals previously assigned, cytochrome f (g_z 3.51), high-potential cytochrome b-559 (g_z 3.08) and cytochrome b-559 converted to a low-potential form (g_z 2.94), a high-spin heme signal was induced by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). However, this signal cannot be due to cytochrome b-563 in its native form. The orientation of the cytochromes in the thylakoid membrane was studied in magnetically oriented chloroplasts. Cytochrome b-559 in the native state and in the low-potential form was found to have its heme plane perpendicular to the membrane plane. The orientation was the same for cytochrome b-559 oxidized by low-temperature illumination, which suggests that also the reduced heme is oriented perpendicular to the membrane.     

EPR spectra of Photosystem I and other iron protein components in intact cells of cyanobacteria

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe³⁺ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor ‘X’ of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 ± 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe³⁺, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.     

Thermodynamic resolution of the iron-sulfur centers of the succinic dehydrogenase of Rhodopseudomonas sphaeroides.

A single alkaline wash removes most of the succinic dehydrogenase activity from chromatophores of Rhodopseudomonas sphaeroides. Three iron-sulfur centers are also removed by this washing. Two of these are ferredoxin-like centers with electron paramagnetic resonance signals at g_v = 1.94 and midpoint potentials of +50 and ?250 mV at pH 7. The third is a high-potential iron-sulfur protein type signal centered at g 2.01 and a midpoint potential of +80 mV at pH 7. These centers have very similar properties to those of the well-characterized mammalian succinic dehydrogenase and account for the majority of iron-sulfur centers observed in chromatophores. Because it is so easily removed, it is concluded that succinic dehydrogenase is located on the outer surface of the chromatophore membrane, a conclusion supported by the fact that removal of the enzyme does not interfere with the kinetics of light-induced electron flow, nor does it allow cytochrome c₂ to escape from inside the chromatophore vesicles.     

The origin of split EPR signals in the Ca2+-depleted Photosystem II

A light-driven reaction model for the Ca²⁺-depleted Photosystem (PS) II is proposed to explain the split signal observed in electron paramagnetic resonance (EPR) spectra based on a comparison of EPR assignments with recent x-ray structural data. The split signal has a splitting linewidth of 160 G at around g = 2 and is seen upon illumination of the Ca²⁺-depleted PS II in the S₂ state associated with complete or partial disappearance of the S₂ state multiline signal. Another g=2 broad ESR signal with a 110 G linewidth was produced by 245 K illumination for a short period in the Ca²⁺-depleted PS II in S₁ state. At the same time a normal Y_Z^· radical signal was also efficiently trapped. The g=2 broad signal is attributed to an intermediate S₁X^· state in equilibrium with the trapped Y_Z^· radical. Comparison with x-ray structural data suggests that one of the split signals (doublet signal) is attributable to interaction between His 190 and the Y_Z^· radical, and other signals is attributable to interaction between His 337 and the manganese cluster, providing further clues as to the mechanism of water oxidation in photosynthetic oxygen evolution.     

Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria

Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features.