The release of polypeptides and manganese from oxygen-evolving photosystem II preparations following zinc-treatment

Inhibition of oxygen evolution in photosystem II membrane fragments from pea chloroplasts by washing with Zn₂₊ causes appearance of the EPR signal of Mn(H₂O)₆²⁺. This Mn²⁺ remains associated with the membrane fraction. Release of Mn²⁺ into the medium was correlated with the amount of the 23 kDa protein removed from the membrane. This suggests that this protein may function as a ‘gate’ to an aqueous compartment into which Mn²⁺ is released. Inhibition by Zn²⁺ correlated with the release of 1 Mn²⁺ per reaction centre, out of a total stoichiometry of 4 Mn atoms per reaction centre. By comparing the release of Mn following Zn-treatment of NaCI or CaC1₂ washed membranes, it is concluded that the 33 kDa protein is involved in binding of 2 Mn.     

Manganese enhances the phosphorylation of membrane-associated proteins isolated from barley roots

Microsomal membranes isolated from barley roots (Hordeum vulgare L. cv. CM72) contained endogenous protein phosphorylation activities that were greatly enhanced by Mn²⁺. Mg²⁺ions also stimulated protein phosphorylation, but to a lesser extent than Mn²⁺. Ca²⁺ enhanced Mg²⁺, but not Mn²⁺-dependent phosphorylation. It is proposed that this strong enhancement by Mn²⁺ may be due to a greater affinity of Mn²⁺ than either Ca²⁺ or Mg²⁺ for both the Ca²⁺ and Mg²⁺ binding sites of certain kinases. Some Mn²⁺ stimulated kinase activity was eliminated from the membrane by washing with 0.2 mol/L KCl. The KCl extract contained histone and casein kinase activities, and 4 major phosphoproteins that were phosphorylated on serine and threonine residues. Phosphorylation of a 52 kDa polypeptide corresponded with the characteristics of the histone kinase activity and may represent the autophosphorylation of a CDPK-type kinase. Phosphorylation of a 36 kDa polypeptide was Ca²⁺ stimulated and may represent the autophosphorylation of a different type of unknown kinase. Polypeptides of 18 and 15 kDa had characteristics that suggest they were autophosphorylating subunits of a membrane bound nucleotide di-phosphokinase.     

Heat inactivation of oxygen evolution in Photosystem II particles and its acceleration by chloride depletion and exogenous manganese

Heat inactivation of oxygen evolution by isolated Photosystem II particles was accelerated by Cl⁻ depletion and exogenous Mn²⁺. Weak red light also accelerated heat inactivation. Heat treatment released the 33, 24 and 18 kDa proteins and Mn from the Photosystem II particles. The protein release was stimulated by Cl⁻ depletion and exogenous Mn²⁺, and the Mn release was also stimulated by Cl⁻ depletion. A 50% loss of Mn corresponded to full inactivation of oxygen evolution, whereas no direct correlation seemed to exist between the loss of any one protein and inactivation of oxygen evolution. Removal of the 24 and 18 kDa proteins from photosystem II particles only slightly decreased the heat stability of oxygen evolution.     

Superior Therapeutic Index of Calmangafodipir in Comparison to Mangafodipir as a Chemotherapy Adjunct

Mangafodipir is a magnetic resonance imaging contrast agent with manganese superoxide dismutase (MnSOD) mimetic activity. The MnSOD mimetic activity protects healthy cells against oxidative stress-induced detrimental effects, e.g., myelosuppressive effects of chemotherapy drugs. The contrast property depends on in vivo dissociation of Mn²⁺ from mangafodipir—about 80% dissociates after injection. The SOD mimetic activity, however, depends on the intact Mn complex. Complexed Mn²⁺ is readily excreted in the urine, whereas dissociated Mn²⁺ is excreted slowly via the biliary route. Mn is an essential but also a potentially neurotoxic metal. For more frequent therapeutic use, neurotoxicity due to Mn accumulation in the brain may represent a serious problem. Replacement of 4/[5] of Mn²⁺ in mangafodipir with Ca²⁺ (resulting in calmangafodipir) stabilizes it from releasing Mn²⁺ after administration, which roughly doubles renal excretion of Mn. A considerable part of Mn²⁺ release from mangafodipir is governed by the presence of a limited amount of plasma zinc (Zn²⁺). Zn²⁺ has roughly 10³ and 10⁹ times higher affinity than Mn²⁺ and Ca²⁺, respectively, for fodipir. Replacement of 80% of Mn²⁺ with Ca²⁺ is enough for binding a considerable amount of the readily available plasma Zn²⁺, resulting in considerably less Mn²⁺ release and retention in the brain and other organs. At equivalent Mn²⁺ doses, calmangafodipir was significantly more efficacious than mangafodipir to protect BALB/c mice against myelosuppressive effects of the chemotherapy drug oxaliplatin. Calmangafodipir did not interfere negatively with the antitumor activity of oxaliplatin in CT2[6] tumor-bearing syngenic BALB/c mice, contrary calmangafodipir increased the antitumor activity.     

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

The effect of calcium on the oxidation of acetaldehyde by rat liver mitochondria.

Isolated liver mitochondria oxidized acetaldehyde in the following order: State 4< state 3< valinomycin. Ca²⁺, in concentrations greater than 0.10 mM, inhibited the oxidation of acetaldehyde by isolated liver mitochondria under all conditions. Valinomycin-stimulated oxidation of acetaldehyde was more sensitive to inhibition by Ca²⁺ than were the state 3 or 4 rates of acetaldehyde oxidation. Acetaldehyde could support an energy-dependent uptake of Ca²⁺ at rates about 20 percent that found with succinate. Ruthenium red, an inhibitor of Ca²⁺ translocation, almost completely prevented the inhibition by Ca²⁺, under all conditions. The addition of externally added NAD⁺ or NADH provided complete relief against the inhibitions by Ca²⁺ of the state 4 and 3 rates of acetaldehyde oxidation. Although some relief was also observed with the valinomycin-stimulated system, significant inhibition persisted. Cations such as Zn²⁺, Cu²⁺, or Hg²⁺ also inhibited acetaldehyde oxidation, whereas Mg²⁺ and Mn²⁺ were without effect. These cations also blocked glutamate oxidation and presumably inhibit acetaldehyde oxidation by preventing reoxidation of NADH. The greater sensitivity of the ionophore-stimulated oxidation of acetaldehyde to inhibition by Ca²⁺ may reflect release of intramitochondria K⁺, which is known to occur in the presence of Ca²⁺, suggesting that acetaldehyde oxidation is influenced by the cation environment within the mitochondria.     

Divalent cation stimulation of substrate oxidation by corn mitochondria

The oxidation of reduced nicotinamide adenine dinucleotide, malate-pyruvate, and succinate by corn mitochondria in buffered 0.2 m KCl was determined as a function of divalent cations. Ni²⁺, Mg²⁺, Co²⁺, Ca²⁺, Mn²⁺, Sr²⁺, and Ba²⁺ stimulated reduced nicotinamide adenine dinucleotide oxidation in the absence of inorganic phosphate, with Ca²⁺ and Sr²⁺ having the greatest effect. Malate-pyruvate and succinate oxidation was stimulated by Ca²⁺, Ba²⁺, and Sr²⁺, but only in the presence of inorganic phosphate. Ca²⁺, Sr²⁺, and Ba²⁺ produced a simulated state 4 to state 3 transition with all three substrates, but only with malate-pyruvate and succinate was there a return to state 4. The order of divalent cation effectiveness suggests that the rate of water substitution from the cation inner coordination hydration sphere may be a rate-limiting step in certain mitochondrial reactions involving electron transport and phosphorylation.     

Manganese toxicity towards Saccharomyces cerevisiae : Dependence on intracellular and extracellular magnesium concentrations

Inhibition of the growth of Saccharomyces cerevisiae was evident at concentrations of 0.5 mM Mn²⁺ or higher, but a tolerance to lower Mn²⁺ concentrations was observed. The inhibitory effects of 2.0 mM Mn²⁺ were eliminated by supplementing the medium with excess Mg²⁺ (10 mM), whereas addition of excess Ca²⁺ and K⁺ had negligible effect on Mn²⁺ toxicity. Growth inhibition by Mn²⁺, in the absence of a Mg²⁺ supplement, was attributed to Mn²⁺ accumulation to toxic intracellular levels. Mn levels in S. cerevisiae grown in Mg²⁺-supplemented medium were severalfold lower than those of cells growing in unsupplemented medium. Mn²⁺ toxicity was also influenced by intracellular Mg, as Mn²⁺ toxicity was found to be more closely correlated with the cellular Mg:Mn ratio than with cellular Mn levels alone. Cells with low intracellular levels of Mg were more susceptible to Mn²⁺ toxicity than cells with high cellular Mg, even when sequestered Mn²⁺ levels were similar. A critical Mg:Mn ratio of 2.0 was identified below which Mn²⁺ toxicity became acute. The results demonstrate the importance of intracellular and extracellular competitive interactions in determining the toxicity of Mn²⁺. Received: 18 June 1997 / Received last revision: 10 January 1998 / Accepted: 24 January 1998     

Manganese oxidation by microbial consortia from sand filters

The role of microbial consortia on the removal of manganese (Mn) was examined on sand from three different Belgian rapid sand filters for the treatment of ground water. Microorganisms closely associated with deposits of Fe and amorphous Mn precipitates were observed by SEM and EDAX techniques on sand from the filters able to remove Mn efficiently. Bacterial counts were performed. Of the CFU enumerated on PYM-medium, 25–33% displayed Mn-oxidizing activity.Batch cultures were set up by inoculating a Mn-containing, low organic medium with sand from one of the filters. Microbial growth resulted in the formation of Mn-removing bacterial flocs and a pH increase. Suppression of microbial growth by addition of azide, kanamycin, or by autoclaving reduced removal of Mn²⁺ from 0.5 mM/day to 0.05–0.11 mM/day. Buffering the pH of the medium at 7.5 (0.1 mM Hepes) decelerated the Mn removal but did not halt it, whereas microelectrode measurements revealed a clear pH drop of about 0.7 units inside bacterial flocs. In the absence of Mn²⁺, the pH drop was only 0.4 units. The auto-catalytic removal of Mn by the Mn oxide coated filter sand was not sufficient to explain the Mn removal observed. Inactivated cells were not capable of a pronounced autocatalytic Mn removal. Experiments with enrichment cultures indicated that the Mn-removing capacity of the microbial sand filter consortia was not constitutive but was promoted by preadaptation and the presence of a substratum. These results clearly link Mn oxidation in rapid sand filters to microbial processes. Offprint requests to: W. Verstraete.     

Modification of the thermoluminescence properties of Ca2+ depleted Photosystem II membranes by the 23 kDa extrinsic polypeptide and by oligocarboxylic acids

Photosystem II membranes were isolated from chloroplasts of pokeweed (Phytolacca americana) and rendered deficient in Ca²⁺, an inorganic cofactor of photosynthetic water oxidation. The thermoluminescence properties of such membranes were found to depend on the Ca²⁺-depleting method used. This feature was analyzed with respect to the thermoluminescence emission that accompanied the recombination reaction between the reduced acceptor Q_A ^– and the oxidant of the S₂ state. It was determined that the differences observed among various preparations of Ca²⁺-depleted membranes were attributable to the presence or absence of the extrinsic 23 kDa polypeptide on the membranes. The binding of this polypeptide to Ca²⁺-depleted membranes devoid of the 17 and 23 kDa extrinsic polypeptides caused the thermoluminescence to be emitted at a higher temperature due to a further stabilization of an already abnormally stable S₂ state. Addition of the chelators EDTA or EGTA and of citrate brought about a similar response. The conditions required for the upshift of the emission temperature of thermoluminescence strongly resembled those identified by Boussac et al. (FEBS Lett. 277 (1990) 69–74) as responsible for modifying the EPR multiline signal from the S₂ state of Ca²⁺-depleted PS II membranes. Consistent with the authors' interpretation of the reason for this modification, we conclude that the elevated emission temperature of the thermoluminescence emission reflects an abnormal ligand environment of the Mn-center in PS II that may be created by a direct ligation of the added agents to Mn. Evidence is also presented that the return to a normal S₂ after an addition of Ca²⁺ occurs via yet another condition of S₂ which, in terms of its thermoluminescence properties, resembles that of Ca²⁺-depleted membranes before addition of modifying agents, but is not identical to it.     

The 16 and 23 kDa extrinsic polypeptides and the associated Ca2+ and Cl- modify atrazine interaction with the photosystem II core complex

The oxygen evolving complex of photosystem II (PS II) contains three extrinsic polypeptides of approximate molecular weights 16, 23 and 33 kDa. These polypeptides are associated with the roles of Cl^-, Ca²⁺ and Mn²⁺ in oxygen evolution. We have shown that selective removal of 16 and 23 kDa polypeptides from the above complex by NaCl washing of PS II enriched membrane fragments renders the PS II core complex more susceptible to the herbicide atrazine. On the other hand, when both native and depleted preparations were resupplied with exogenous Ca²⁺ and Cl^-, we obtained a reduction of atrazine inhibition which was much stronger in the depleted preparations than in the native ones. It is concluded that removal of 16 and 23 kDa polypeptides in general, and disorganization of associated Ca²⁺ and Cl^- in particular, enhances atrazine penetration to its sites of action in the vicinity of the PS II complex. The above could be interpreted if we assume a reduced plastoquinone affinity at the Q_B (secondary plastoquinone electron acceptor) pocket of D1 polypeptide following transmembranous modifications caused by the depletion of these polypeptides.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - MES 2-(N-morpholino)ethanesulfonic acid - PMSF phenylmethylsul-phonyfluoride - PS II photosystem II - PAGE polyacrilamide gel electrophoresis     

Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Effect of Mn2+ and Ca2+ on O2 evolution and on the variable fluorescence yield associated with Photosystem II in preparations of Anacystis nidulans

Extraction with EDTA of lyophilized and lysozyme treated preparations of the blue-green algae Anacystis nidulans resulted in loss of the capacity for photoevolution of O₂. Reactivation was achieved by the addition of both cations: Mn²⁺ and Ca²⁺ (or to a smaller extent by Mn²⁺ and Sr²⁺). The dual requirement for Mn²⁺ and Ca²⁺ could be demonstrated when the O₂ evolution under short saturating light flashes and the variable chlorophyll fluorescence associated with the reduction of the primary acceptor of Photosystem II was examined. The fluorescence experiments in addition showed that incorporation of the cations was a light dependent step, since the fluorescence rise only started after a lag period.     

Some effects of Ca2+, Mg2+, and Mn2+ on the ultrastructure,light-scattering properties,and malic enzyme activity of adrenal cortex mitochondria

The ultrastructure and 90 ° light-scattering capacity of adrenal cortex mitochondria have been examined under conditions which lead to an activation of malic enzyme activity in these mitochondria. After isolation, the mitochondria display an aggregate ultrastructure which does not resemble the vesicular (orthodox) form normally seen in vivo. Under conditions of malic enzyme activation (presence of malate, NADP⁺, Mg²⁺ and 1 mm Ca²⁺), the ultrastructure reverts to a vesicular form as seen in vivo. Of these required components, only Ca²⁺ affects the ultrastructure. The ultrastructural transformation from the aggregate to the orthodox form is always accompanied by a decrease in the 90 ° light-scattering capacity. When produced by Ca²⁺, transformation requires energy-dependent Ca²⁺ uptake if an oxidizable substrate is present. In the absence of substrate, the transformation occurs as an apparent energy-independent effect. Mn²⁺ can substitute for Ca²⁺ only in the presence of substrate. In de-energized mitochondria, Mn²⁺ prevents the effects of Ca²⁺. The activation of malic enzyme is always preceded by a decrease in light scattering and transformation to the orthodox ultrastructure; however, the presence of the orthodox form is not a sufficient condition since subsequent chelation of free Ca²⁺ fails to reverse either the decrease in light scattering or ultrastructural transformation but does reverse the enzyme activation. In addition, levels of Mn²⁺ which effectively depress light-scattering capacity and produce the orthodox form, fail to activate malic enzyme significantly. The data are discussed as they relate to Ca²⁺-induced damage in mitochondria.     

Requirement of divalent cations for photoactivation of the laten water-oxidation system in intact chloroplasts from flashed leaves

The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn²⁺ (10 μM). (b) A high concentration of Mn²⁺ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca²⁺ (5 mM). (c) The Ca²⁺-dependent restoration was inhibited by a high concentration of Mg²⁺ or Sr²⁺, but the inhibition was restored by the coexistence of Ca²⁺. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca²⁺ inhibits photoactivation in competition with Mn²⁺. (ii) High concentration of Mn²⁺ inhibits photoactivation in competition with Ca²⁺. (iii) High concentration of Mg²⁺ affects photoactivation by inhibiting Ca²⁺-dependent restoration in competition with Ca²⁺. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn²⁺ and Ca²⁺, and that photoactivation takes place in the center having both Mn²⁺ and Ca²⁺ on their respective binding sites.     

Differing responses of the two forms of photosystem II carbonic anhydrase to chloride, cations, and pH

The effects of Cl⁻, Mn²⁺, Ca²⁺, and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl⁻, and strongly inhibited above this concentration. This enzyme is activated by Mn²⁺ and stimulated somewhat by Ca²⁺. The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl⁻ up to 0.4 M. Ca²⁺ appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn₄ complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: II. A binuclear manganese-containing 34 kilodalton protein,a probable component of the water dehydrogenase enzyme

Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O₂-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O₂ evolution activity from functional membranes, such as incubation in 1.5 mM NH₂OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two ⁵⁵Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn₂(III,III) site or an antiferromagnetically coupled mixed-valence Mn₂(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH₂OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn₂(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn₂(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O₂ evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O₂-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O₂ evolving complex.     

An electron paramagnetic resonance study of Mn2+ uptake by the chick chorioallantoic membrane

Mn²⁺ uptake in the chick chorioallantoic membrane, an embryonic epithelial tissue which transports Ca²⁺ in vivo was studied using electron paramagnetic resonance (EPR). Mn²⁺ was used as a paramagnetic analog for Ca²⁺, since there is evidence that Mn²⁺ is accumulated by the Ca²⁺ transport mechanism.After 1.5 h of uptake the EPR spectrum of the Mn²⁺ in the membrane indicated that 89 % of the Mn²⁺ was in a spin-exchange form, indicating close packing of Mn²⁺. The Mn²⁺ spacing was estimated from the line width to be about 4.7 Å. The remaining Mn²⁺ was very likely Mn²⁺ hexahydrate.At pH 7.4 the spin-exchange spectrum tended to broaden when uptake was inhibited, while at pH 5.0 the spin-exchange spectrum was completely abolished in the presence of inhibitors.The EPR spectrum of Mn²⁺ in the chorioallantoic membrane had a broader line width than that of Mn²⁺ in isolated mitochondria, suggesting that in this tissue mitochondria are not directly involved in divalent cation transport. These EPR studies support the concept that divalent cations are sequestered in high concentrations from the rest of the cell contents during transcellular active transport.