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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The spin-labeled anion N-[4-(2,2,6,6-tetramethylpiperidin-1-oxyl)] oxamate has been synthesized and characterized. In the presence of this compound, a specific iron-transferrin-anion complex is formed, as evidenced by the development of a characteristic red color. No EPR signal was observed for the nitroxyl radical in the protein complex, presumably due to broadening of the signal by the paramagnetic metal ion. Failure to observe a signal implies that the metal to nitroxyl distance is less than or equal to 6 A. This suggests that the anion is directly attached to the metal ion in the protein. The pH dependence of iron dissociation from iron-transferrin-oxalate is also reported. This complex is more stable at low pH than iron-transferrin-carbonate.
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PMID:Oxalate and spin-labeled oxalate as probes of the anion binding site of human transferrin. Metal to anion distance. 2 46

The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.
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PMID:Anion binding by human lactoferrin: results from crystallographic and physicochemical studies. 158 1

Continuous wave (cw) X-band EPR spectra at approximately 90 K were obtained for iron-transferrin-anion complexes with 18 anions. Each anion had a carboxylate group and at least one other polar moiety. As the second polar group was varied from hydroxyl to carbonyl to amine to carboxylate, the EPR spectra changed from a dominant signal at g' approximately 4.3 with a second smaller peak at g' approximately 9 to a broad signal with intensity between g' approximately 5 and 7. Computer simulation indicated that the changes in the EPR spectra were due to changes in the zero field splitting parameter ratio, E/D, from approximately 1/3 for carbonate anion to approximately 0.04 for malonate anion. Observation of iron-13C coupling in the electron spin echo envelope modulation (ESEEM) for iron transferrin [1-13C]pyruvate indicated that the carboxylate group was bound to the iron. It is proposed that all of the anions behave as bidentate ligands, with coordination to the iron through both the carboxylate and proximal groups, and the carboxyl group serves as a bridge between the iron and a positively charged group on the protein.
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PMID:Effect of the synergistic anion on electron paramagnetic resonance spectra of iron-transferrin anion complexes is consistent with bidentate binding of the anion. 165 Nov 23

Human lactotransferrin is able to bind two vanadyl(IV) ions in specific metal-binding sites. The EPR signals of the two vanadyl bound ions, however, appear as one. This result suggests that the environments of the binding sites of human lactotransferrin are similar. The binding activity is promoted to pH 4 using carbonate or bicarbonate as synergistic anion. This unusual stability of the anion-binding site, which is destroyed below pH 6 for other transferrins, can explain in part the great stability of the metallic complexes of human lactotransferrin. However, the different sensitivities of the two metal-binding sites towards protonation permit the preparation of mixed vanadyl(IV), iron(III) complexes with VO2+ bound either on the N-terminal (acid-labile or B site) or on the C-terminal (acid-stable or A site) site. Analysis of the spectra of such mixed complexes shows the presence of a third nonspecific VO2+-binding site termed A'. The nonspecific A' site seems to be located on the outer surface of the protein close to the C-terminal site.
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PMID:Comparative study of the iron-binding properties of human transferrins. II. Electron paramagnetic resonance of mixed metal complexes of human lactotransferrin. 630 30

The anion binding properties of human lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, highlight differences between the two sites, and in the anion binding behaviour when different metals are bound. Carbonate, oxalate and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf although it does for the bovine analogue. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+. The anion sites as determined from the crystal structures of Fe2(CO3)2Lf, Fe2(C2O4)2Lf, Cu2(CO3)2Lf, and Cu2(CO3)(C2O4)Lf have been compared. Both the carbonate and oxalate ions bind in bidentate fashion to the metal, except that the carbonate ion in the N-lobe site of dicupric lactoferrin is monodentate. The hybrid copper lactoferrin complex shows that the oxalate ion binds preferentially in the C-lobe site in a bidentate mode. A series of complexes containing the synergistic anion O,N-chelates with increasing substitution on the N atom (glycinate, iminodiacetate and nitrilotriacetate) have been prepared with iron bovine lactoferrin for comparison with the O,O-chelate oxalate. Overall these observations lead to a generalised model for synergistic anion binding by transferrins and allow comparisons to be made with nonsynergistic anions such as citrate and succinate.
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PMID:Synergism and substitution in the lactoferrins. 776 44

Rates of radiolytic inactivation of bacteria suspended in N2O-saturated solutions were dramatically increased over normal background levels when the media contained chloride or bicarbonate ions. The bacteria could be protected from this enhanced toxicity by the addition of free radical scavengers (ethanol, ascorbate, hydrogen peroxide, mannitol, glucose, EDTA, picolinic acid), indicating that the lethal reactions were extracellular in origin. Prior irradiation of chloride-containing solutions led to formation of hypochlorous acid, which was identified by detection of ring-chlorinated products when reacted with fluorescein. Prolonged irradiation of other solutions did not lead to accumulation of bactericidal agents; however, irradiation of bicarbonate-containing solutions in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) led to formation of the EPR-detectable DMPO.CO3- adduct. The results are interpreted in terms of formation of secondary radicals, among which the carbonate and chlorine radicals are uniquely toxic to bacteria. From rate comparisons of the solution components, it was concluded that the reactions involving chloride ion are unlikely to be expressed in biological environments, but that the CO3- radical could be an important intermediary oxidant in peroxide-inflicted cellular damage, particularly in spatially confined environments such as the leukocyte phagosome.
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PMID:Bactericidal potency of hydroxyl radical in physiological environments. 814 63

The electron paramagnetic resonance linewidth of aquo gadolinium(III) ion changes with the counter-ion identity and concentration in aqueous solutions. The EPR linewidth of 2 mM gadolinium(III) chloride increases from 49.2 to 89.0 mT when carbonate ion is added and decreases to 17.3 mT when nitrite ion is added. These observations suggest association reactions between aquo gadolinium(III) ion and anions that change the electron spin relaxation rates of the aquo ion. The concentration dependence of the gadolinium(III) EPR linewidth is consistent with binding constants for nitrite and nitrate ion with the aquo gadolinium(III) ion of 37 +/- 6 and 2.3 +/- 0.3 L mol-1 respectively. The decreases in the EPR linewidth factors with these association reactions are difficult to understand unless the anion reactions increase the symmetry of the metal center. Although first-coordination reactions may not be ruled out, the decrease in EPR linewidth is more consistent with an outer-sphere association reaction that also reduces the coordination number of the metal center from 9 to 8.
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PMID:Ionic association and electron spin relaxation rates in aquo gadolinium (III) complexes. 866 Dec 68

The binding site of the non-heme iron of photosystem II (PS II) is investigated by light-induced Fourier tranform infrared (FTIR) difference spectroscopy on Tris-washed membranes. The non-heme iron is oxidized (Fe3+) in the dark with ferricyanide and reduced (Fe2+) after light-induced charge separation by electron transfer from the semiquinone anion QA-. EPR experiments and IR modes of ferri- and ferrocyanide show that the electron donor side of PS II is reduced in less than 2 s after a flash and that ferricyanide reoxidizes the non-heme iron with a half-time of approximately 20 s. Recording FTIR spectra before and 2 s after flash illumination thus results in the Fe2+/Fe3+ difference spectrum. This spectrum shows band shifts and intensity changes of IR modes from ligands and neighboring residues of the non-heme iron. The IR modes of bicarbonate are revealed by comparison of Fe2+/Fe3+ spectra obtained on PS II membranes with 12C or 13C isotope labeled bicarbonate in H2O and in 2H2O. The nu as(CO) and nu s(CO) modes of bicarbonate in the Fe2+ state are assigned at 1530 +/- 10 and 1338 cm-1, respectively. The low frequency of the nu as(CO) mode is taken as experimental evidence that bicarbonate is a ligand of the non-heme iron. Furthermore, the small frequency difference (192 cm-1) between the nu as(CO) and nu s(CO) modes as compared to even hydrogen-bonded ionic bicarbonate strongly indicates that bicarbonate is a bidentate ligand of the non-heme iron in PS II. Upon iron oxidation, the bicarbonate modes are largely affected. The nu s(CO) mode is assigned at 1228 cm-1, while the nu as(CO) mode is tentatively assigned at 1658 +/- 20 cm-1. The strong up- and downshifts of the nu as and nu s(CO) modes of bicarbonate upon iron oxidation results in a frequency difference of 430 +/- 20 cm-1 that is not only explained by the increased charge on the iron but indicates that bicarbonate is a monodentate ligand of the oxidized iron. The sensitivity of the nu s(CO) mode of bicarbonate to 1H/2H exchange in both the Fe2+ and Fe3+ states and the presence in the Fe2+ state of a delta (COH) mode at 1258 cm-1 confirm that bicarbonate and not carbonate is the iron ligand and further exhibits hydrogen bond(s) with the protein. The 13C isotope-sensitive modes of bicarbonate are not affected by 15N labeling of the PS II membranes. 15N sensitive signals at 1111/1102 and 1094 cm-1 are assigned to side chain modes from histidine ligands of the iron. The latter signal is proposed to account for a histidine ligand that deprotonates upon iron oxidation. The involvement of protein peptide groups and side chains in the hydrogen-bond network around the iron is also discussed.
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PMID:Bicarbonate binding to the non-heme iron of photosystem II investigated by Fourier transform infrared difference spectroscopy and 13C-labeled bicarbonate. 884 53

Bicarbonate anions have a strong positive influence on the electron and proton transfers in photosystem II (PS II). It has been suggested that bicarbonate binds to the non-heme iron and the QB binding niche of the PS II reaction center. To investigate the potential amino acid binding environment of bicarbonate, an arginine residue (R269) of the D1 protein of PS II of Chlamydomonas reinhardtii was mutated into a glycine; our characterization of the resultant mutant (D1-R269G) shows that both the TyrD+ and QA- Fe2+ EPR signals are substantially reduced and assembly of the tetranuclear Mn is lost (R.S. Hutchison, J. Xiong, R.T. Sayre, Govindjee, Biochim. Biophys. Acta 1277 (1996) 83-92). In order to understand the molecular implications of this mutation on the electron acceptor side of PS II, we used chlorophyll (Chl) a fluorescence as a probe of PS II structure and function, and herbicide binding as a probe for changes in the QB binding niche of PS II. Chl fluorescence measurements with the heterotrophically grown D1-R269G mutant cells (or thylakoids), as compared to that of the wild type, show that: rate of electron transfer from QA to the plastoquinone pool, measured by flash-induced Chl a fluorescence decay kinetics, is reduced by - 17 fold; the minimum Chl a fluorescence yield when all QA- is oxidized, is elevated by 2 fold; the level of stable charge separation as inferred from variable Chl fluorescence is reduced by 44%; binary oscillation pattern of variable Chl a fluorescence obtained after a series of light flashes is absent, indicative of the loss of functioning of the two-electron gate on the PS II acceptor side; 77 K PS II Chl a fluorescence emission bands (F685 and F695) are reduced by 20-30% (assuming no change in the PS I emission band). Thermoluminescence data with thylakoids show the absence of the S2QA- and S2QB- bands in the mutant. Herbicide 14C-terbutryn binding measurements, also with thylakoids, show that the QB niche of the mutant is significantly modified, at least 7-8 fold increased terbutryn dissociation constant is shown (220 nM in the mutant versus 29 nM in the wild type); the PS II sensitivity to bicarbonate-reversible formate inhibition is reduced by 5 fold in the mutant, although the formate/bicarbonate binding site still exists in the mutant. This suggests that D1-R269 must play some role in the binding niche of bicarbonate. On the basis of the above observations, we conclude that the D1-R269G mutation has not only altered the structure and function of PS II (QB niche being abnormal), but may also have a decreased net excitation energy transfer from the PS II core to the reaction center and/or an increased number of inactivated reaction center II. We also discuss a possible scenario for these effects using a recently constructed three dimensional model of the PS II reaction center.
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PMID:Modification of the photosystem II acceptor side function in a D1 mutant (arginine-269-glycine) of Chlamydomonas reinhardti. 939 79

It was previously shown in the photosystem II membrane preparation DT-20 that photoxidation of the oxygen-evolving manganese cluster was blocked by 0.1 mM formate, unless 0.2 mM bicarbonate was present as well [Wincencjusz, H., Allakhverdiev, S. I., Klimov, V. V., and Van Gorkom, H. J. (1996) Biochim. Biophys. Acta 1273, 1-3]. Here it is shown by measurements of EPR signal II that oxidation of the secondary electron donor, YZ, is not inhibited. However, the reduction of is greatly slowed and occurs largely by back reaction with reduced acceptors. Bicarbonate is shown to prevent the loss of fast electron donation to . The release of about one or two free Mn2+ per photosystem II during formate treatment, and the fact that these effects are mimicked by Mn-depletion, suggests that formate may act by replacing a bicarbonate which is essential for Mn binding. Irreversible light-induced rebinding in an EPR-silent form of Mn2+ that was added to Mn-depleted DT-20 was indeed found to depend on the presence of bicarbonate, as did the reconstitution in such material of both the fast electron donation to and the UV absorbance changes characteristic of a functional oxygen-evolving complex. It is concluded that bicarbonate may be an essential ligand of the functional Mn cluster.
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PMID:Bicarbonate may Be required for ligation of manganese in the oxygen-evolving complex of photosystem II. 940 62


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