Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

The effect of water on the Fe(3+)/Fe(2+) reduction potential of heme

Hemeproteins can act as catalysts, oxygen carriers or electron conductors. The ferric/ferrous reduction potential E(m7) of iron in the center of the prosthetic group ranges from negative values for peroxidases to an extreme positive value for cytochrome a(3) with Hb and Mb in the middle [1]. Proteins exercise their influence on E(m7) in several ways: via substituents at the periphery of the chelate structure, via the proximal ligand, and via interaction with the surrounding medium, amino acid side chains, or polar solvents. Work on recombined proteins and 2,4-substituted free hemes documented that the first two effects are additive [2]. For the third effect, models of the dielectric media on a molecular level have been successfully applied [3-5]. E(m7) has also been empirically correlated to the degree of heme exposure to water [6-8]. The apoprotein/porphyrin and water/porphyrin interfaces are complementary since water molecules fill any empty space in the crevice and surround any pertinent part of heme outside the protein boundary. The present work links to this idea by a combination of statistical mechanics simulations and quantum mechanical calculations comparing heme in water with heme in an apolar environment. Our results show that polarization of the porphyrin pi-electron cloud by the field from water dipoles influences E(m7). The dominant effect of this and other determinates of iron electron availability is perturbations of delocalized electron density in the porphyrin chelate, reproduced by a model where the prosthetic group is treated as a disc of uniform electron density. The present work is also of interest since the interfacial energy constitutes the main barrier for heme-protein separation [9-11].     

Interconversion of Mn(2+)-dependent and -independent protein phosphatase 2A from human erythrocytes: role of Zn(2+) and Fe(2+) in protein phosphatase 2A.

Human erythrocyte Mn(2+)-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito et al. (1999) FEBS Lett. 447, 29-33]. The Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn(2+)-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl(2), zinc-metallothionein, or FeCl(2) activated the Mn(2+)-independent PP activity, but pre-incubation with FeCl(3) did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl(2). Pre-incubation of C'A' with 5 microM ZnCl(2) and 15 microM FeCl(2) in the presence of 1 mM ascorbate synergistically stimulated the Mn(2+)-independent PP activity, with concomitant suppression of the Mn(2+)-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn(2+)-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn(2+)-independent CA is a Zn(2+)- and Fe(2+)-metalloenzyme, whose apoenzyme is Mn(2+)-dependent C'A'.     

Effects of Fe(3+)-tumor cell interaction on Ca(2+)-uptake by Ehrlich ascites tumor cells

Fe3+ ions complexed by various ligands induce an increased Ca2+ uptake by Ehrlich carcinoma ascites cells that is proportional to the thermodynamic stability constant of the complex, and the greatest increase is observed with ferric lactate. The absence of ATPase inhibition showed by this ferric complex, suggests that an increased passive diffusion of Ca2+ due to structural modifications of the cell membrane is the most probable cause of this phenomenon.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.     

Localization of Fe(2+) at an RTGR sequence within a DNA duplex explains preferential cleavage by Fe(2+) and H2O2

Nicking of duplex DNA by the iron-mediated Fenton reaction occurs preferentially at a limited number of sequences. Of these, purine-T-G-purine (RTGR) is of particular interest because it is a required element in the upstream regulatory regions of many genes involved in iron and oxidative-stress responses. In order to study the basis of this preferential nicking, NMR studies were undertaken on the RTGR-containing duplex oligonucleotide, d(CGCGATATGACACTAG)/d(CTAGTGTCATATCGCG). One-dimensional and two-dimensional 1H NMR measurements show that Fe(2+) interacts preferentially and reversibly at the ATGA site within the duplex at a rate that is rapid relative to the chemical-shift timescale, while selective paramagnetic NMR line-broadening of the ATGA guanine H8 suggests that Fe(2+) interacts with the guanine N7 moiety. Localization at this site is supported by Fe(2+) titrations of a duplex containing a 7-deazaguanine substitution in place of the guanine in the ATGA sequence. The addition of a 100-fold excess of Mg(2+) over Fe(2+) does not affect the Fe(2+)-dependent broadening. When the ATGA site in the duplex is replaced by ATGT, an RTGR site (GTGA) is created on the opposite strand. Preferential iron localization then takes place at the 3' guanine in GTGA but no longer at the guanine in ATGT, consistent with the lack of preferential cleavage of ATGT sites relative to ATGA sites.     

Near-infrared Yb(3+) vibronic sideband spectroscopy: application to Ca(2+)-binding proteins.

We have used near-infrared (NIR) vibronic fluorescence spectroscopy to study the vibrational structure of ligands associated with model complexes of the lanthanide Yb(3+). This technique exploits the similar binding properties of the lanthanide Yb(3+) to probe Ca(2+)-binding sites in proteins. The (NIR) fluorescence of complexed Yb(3+) exhibits, in addition to main 0-0 (2F5/2----2F7/2) electronic transition of Yb(3+), weak vibronic sidebands which provide infrared-like, local vibrational spectra of the chelates (inner sphere ligands) of Yb(3+). A similar approach has been used for the lanthanide Gd(3+) (MacGregor, R.B., Jr (1989) Arch. Biochem. Biophys. 274, 312-316) which fluoresces in the UV and which is usually complicated by amino-acid residues fluorescing in the same spectral region. In this same spectral region, other complications in studying photosynthetic membranes occur in the form of the excitation wavelength being actinic, promoting photodegradation of the membranes, as well as the reabsorption of Gd(3+) fluorescence. NIR excitation and fluorescence detection of Yb(3+) avoid these problems when studying photosynthetic membranes. A preliminary study has been conducted here on rat muscle parvalbumin.     

Fe(3+)-eta(2)-peroxo species in superoxide reductase from Treponema pallidum. Comparison with Desulfoarculus baarsii

Superoxide reductases (SORs) are superoxide (O2-)-detoxifying enzymes that catalyse the reduction of O2- into hydrogen peroxide. Three different classes of SOR have been reported on the basis of the presence or not of an additional N-terminal domain. They all share a similar active site, with an unusual non-heme Fe atom coordinated by four equatorial histidines and one axial cysteine residues. Crucial catalytic reaction intermediates of SOR are purported to be Fe(3+)-(hydro)peroxo species. Using resonance Raman spectroscopy, we compared the vibrational properties of the Fe3+ active site of two different classes of SOR, from Desulfoarculus baarsii and Treponema pallidum, along with their ferrocyanide and their peroxo complexes. In both species, rapid treatment with H2O2 results in the stabilization of a side-on high spin Fe(3+)-(eta(2)-OO) peroxo species. Comparison of these two peroxo species reveals significant differences in vibrational frequencies and bond strengths of the Fe-O2 (weaker) and O-O (stronger) bonds for the T. pallidum enzyme. Thus, the two peroxo adducts in these two SORs have different stabilities which are also seen to be correlated with differences in the Fe-S coordination strengths as gauged by the Fe-S vibrational frequencies. This was interpreted from structural variations in the two active sites, resulting in differences in the electron donating properties of the trans cysteine ligand. Our results suggest that the structural differences observed in the active site of different classes of SORs should be a determining factor for the rate of release of the iron-peroxo intermediate during enzymatic turnover.     

Antioxidant properties of S-adenosyl-L-methionine in Fe(2+)-initiated oxidations

S-Adenosylmethionine (SAM) is protective against a variety of toxic agents that promote oxidative stress. One mechanism for this protective effect of SAM is increased synthesis of glutathione. We evaluated whether SAM is protective via possible antioxidant-like activities. Aerobic Hepes-buffered solutions of Fe2+ spontaneously oxidize and consume O2 with concomitant production of reactive oxygen species and oxidation of substrates to radical products, e.g., ethanol to hydroxyethyl radical. SAM inhibited this oxidation of ethanol and inhibited aerobic Fe2+ oxidation and consumption of O2. SAM did not regenerate Fe2+ from Fe3+ and was not consumed after incubation with Fe2+. SAM less effectively inhibited aerobic Fe2+ oxidation in the presence of competing chelating agents such as EDTA, citrate, and ADP. The effects of SAM were mimicked by S-adenosylhomocysteine, but not by methionine or methylthioadenosine. SAM did not inhibit Fe2+ oxidation by H2O2 and was a relatively poor inhibitor of the Fenton reaction. Lipid peroxidation initiated by Fe2+ in liposomes was associated with Fe2+ oxidation; these two processes were inhibited by SAM. However, SAM did not show significant peroxyl radical scavenging activity. SAM also inhibited the nonenzymatic lipid peroxidation initiated by Fe2+ + ascorbate in rat liver microsomes. These results suggest that SAM inhibits alcohol and lipid oxidation mainly by Fe2+ chelation and inhibition of Fe2+ autoxidation. This could represent an important mechanism by which SAM exerts cellular protective actions and reduces oxidative stress in biological systems.     

On the use of chiral compounds for probing the DNA handedness: Z to B conversion in poly(dGm5dC) upon binding of Fe(phen)3(2+) and Ru(phen)3(2+)

In order to examine whether chiral metal complexes can be used to discriminate between right- and left-handed DNA conformational states we have studied the enantioselective interactions of Fe(phen)3(2+) and Ru(phen)3(2+) (phen = 1,10-phenanthroline) with poly(dGm5dC) under B- and Z-form conditions. With the inversion-labile Fe(phen)3(2+), enantioselectivity leads to shifts in the diastereomeric binding equilibria. This effect, known as the "Pfeiffer effect" (1-4), is monitored as slowly emerging circular dichroism of the solution, corresponding to a net excess of the favoured enantiomer. With Ru(phen)3(2+), which is stable to intramolecular inversion, the difference in DNA-binding strengths of the enantiomers results in an excess of the less favoured enantiomer in the bulk solution. This excess is detected in the dialysate of the DNA/metal complex solution. With both complexes we find that the delta-enantiomer is favoured when the polynucleotide adopts the B-form, as previously shown, but also when it initially adopts the Z-form conformational state. This observation, together with evidence from UV-circular dichroism and binding data, indicates that the binding of these metal complexes induces a Z- to B-form transition in Z-form poly(dGm5dC). Consequently, neither of the studied chiral DNA-binders can easily be used to discriminate the DNA handedness.     

Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography

Phosphoproteins and phosphoamino acids bind to ferric ions immobilized on iminodiacetate-agarose gel and can be eluted by increasing pH or by introducing phosphate ions to the eluant. Some other metals were found to resemble iron with regard to the interaction with protein-bound phosphate and phosphoamino acids. These observations were utilized to develop purification procedures for phosphoproteins. Hen egg albumin (ovalbumin) was fractionated into three components of varying phosphate contents. Porcine pepsin was purified in a similar manner.     

Uncoupling protein 3 adjusts mitochondrial Ca(2+) uptake to high and low Ca(2+) signals

Uncoupling proteins 2 and 3 (UCP2/3) are essential for mitochondrial Ca(2+) uptake but both proteins exhibit distinct activities in regard to the source and mode of Ca(2+) mobilization. In the present work, structural determinants of their contribution to mitochondrial Ca(2+) uptake were explored. Previous findings indicate the importance of the intermembrane loop 2 (IML2) for the contribution of UCP2/3. Thus, the IML2 of UCP2/3 was substituted by that of UCP1. These chimeras had no activity in mitochondrial uptake of intracellularly released Ca(2+), while they mimicked the wild-type proteins by potentiating mitochondrial sequestration of entering Ca(2+). Alignment of the IML2 sequences revealed that UCP1, UCP2 and UCP3 share a basic amino acid in positions 163, 164 and 167, while only UCP2 and UCP3 contain a second basic residue in positions 168 and 171, respectively. Accordingly, mutants of UCP3 in positions 167 and 171/172 were made. In permeabilized cells, these mutants exhibited distinct Ca(2+) sensitivities in regard to mitochondrial Ca(2+) sequestration. In intact cells, these mutants established different activities in mitochondrial uptake of either intracellularly released (UCP3(R171,E172)) or entering (UCP3(R167)) Ca(2+). Our data demonstrate that distinct sites in the IML2 of UCP3 effect mitochondrial uptake of high and low Ca(2+) signals.     

The structure of CorA: a Mg(2+)-selective channel

The crystal structure of a closed form of the CorA Mg2+ transporter from Thermatoga maritima completes a set of representative structures of transport systems for all of the major biological elements, Mg2+, Ca2+, Na+, K+ and Cl-. The CorA monomer has a C-terminal membrane domain containing two transmembrane segments and a large N-terminal cytoplasmic soluble domain. In the membrane, CorA forms a homopentamer shaped like a funnel. Comparison of the structure of CorA with that of other ion channels and transporters suggests numerous common features, but, as might be predicted from the unique chemistry of the Mg2+ cation, the structure of CorA has several unusual features. Among these are initial binding in the periplasm of a fully hydrated Mg2+ ion; a ring of positive charge external to the ion-conduction pathway at the cytosolic membrane interface; and highly negatively charged helices in the cytosolic domain that appear capable of interacting with the ring of positive charge to facilitate Mg2+ entry. Finally, there are bound Mg2+ ions in the cytosolic domain that are well placed to control the interaction of the ring of positive charge and the negatively charged helices, and thus control Mg2+ entry.     

Oxidation of deoxy myoglobin by [Fe(CN)6]3-.

The ability of myoglobin (Mb) to reversibly bind O2 and other ligands has been well characterized. Mb also participates with a variety of redox metals to form metmyoglobin (metMb). By using an anaerobic stopped-flow device we have measured outer-sphere oxidation by [Fe(CN)6]3 of native sperm whale myoglobin, recombinant wild-type Mb, and a series of mutant Mb proteins in which the distal His-64 was changed to Gly, Phe, Leu or Val. Second-order rate constants for oxidation of mutant proteins are 10-15 times greater than for recombinant or native (kox approximately 10(6) M-1 s-1). We attribute the reduced rate of oxidation of wild-type protein to a higher reorganization energy imposed by the presence of the unique water/His-64/heme interaction, which is absent in the mutant proteins.     

Iron (Fe3+)-mediated redox responses and amelioration of oxidative stress in cadmium (Cd2+) stressed mung bean seedlings: a biochemical and computational analysis

Journal of Plant Biochemistry and Biotechnology - The present investigation aims to evaluate the possible ameliorative role of iron (Fe3+) on cadmium (Cd2+)-induced oxidative stress in mung bean...