Probing different conformational states of bovine alpha-lactalbumin: fluorescence studies with 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate]

The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bis-ANS) to bovine alpha-lactalbumin (alpha-LA) was investigated. A strong dependence of the Kd value with the bound calcium stoichiometry was found, with Kd values ranging from 6.2 +/- 0.4 to 64.6 +/- 5.9 microM for apo-alpha-LA and 1:1 Ca(II)-alpha-LA, respectively. A 350-fold enhancement of the bis-ANS emission was observed in the protein-bis-ANS complex, along with an approximately 33-nm blue shift. Both appeared to be related to the hydrophobicity of the binding site and were independent of the Ca(II) ion content. From the difference in bis-ANS affinity between apo-alpha-LA and Ca(II)-alpha-LA, we demonstrated that Zn(II) and Al(III) were able to "lock" the protein into a new "apo-like" conformation, which was similar to, but not identical with, the apo conformation. The protein could be interconverted between all three conformations in a Mn(II) titration. The first Mn(II) shifted the apoprotein to the Ca(II) conformation; at higher Mn(II) levels, binding to the second site shifted the protein toward the apo-like conformation. The same behavior was observed with calcium in large excess. The evidence supported a model for the mutually nonexclusive binding of metals both to site I ("calcium site") and to site II ("zinc site") simultaneously. The results suggest that alpha-lactalbumin possesses a hydrophobic surface that becomes somewhat less accessible upon 1:1 calcium binding in the absence of metals that also bind to the zinc site.     

Quantitative evaluation of metal ion binding to PvuII restriction endonuclease

19 F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with K _d values of  ≈ 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with K _d  ≈ 2 mM. Mn(II) paramagnetic broadening in ¹⁹F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease. Received: 30 March 1999 / Accepted: 28 September 1999     

Competitive ion binding to low density lipoproteins: an electron spin resonance study

The electron spin resonance (ESR) technique was used to evaluate binding constants for Ca(II) and Mg(II) in interaction with low density lipoprotein (LDL). The Ca(II) or Mg(II) ions competed with the paramagnetic Mn(II) ions for the same binding sites of two different classes on the LDL surface. For each ion competing with Mn(II), the solutions of eight non-linear competition equations were fit to the experimental titration curves, with two adjustable parameters, the two binding constants. The derived "intrinsic" values (the values corrected for the electrolyte-induced change of the surface potential) for "strong" binding sites for Ca(II) (170 +/- 85 M-1) and Mg(II) (60 +/- 30 M-1) differ significantly from the respective value for Mn(II) (760 M-1). The values for the "weak" binding sites (18 M-1, 15 M-1 and 10 M-1 for Mn(II), Ca(II) and Mg(II), respectively are in the range of the binding constants for these ions in interaction with model membranes.     

Evidence that divalent cations bind to diphtheria toxin: an ESR approach

We have used electron spin resonance (ESR) techniques to study the binding of divalent cations to diphtheria toxin (DT). Addition of DT to Mn(II) solution at a stoichiometry of 2:1 (DT:Mn) induces a 79% loss in the intensity of the ESR spectrum of Mn(II) suggesting a strong binding of Mn(II) to DT. Inclusion of Ca(II) at a ratio of 1:2:1 (Ca:DT:Mn) in the reaction mixture restores the intensity of the Mn(II) signal to 64%. This indicates that Ca(II) and Mn(II) share same binding site(s) in DT. The results presented in this communication suggest that DT is a Ca(II) binding protein.     

Mn(II)-EPR measurements of cation binding by aequorin

Cation binding at 5 degrees C by aequorin, a bioluminescent protein from the jellyfish Aequorea victoria, was examined by means of Mn(II) EPR. The bioluminescence of aequorin is triggered by Ca(II), as well as by trivalent lanthanides, and is inhibited by Mg(II) and Mn(II). Three EF-hand Ca(II)-binding domains have been identified in the aequorin amino acid sequence. In the work reported here, active native aequorin was found to have a single tight binding site for Mn(II) with an association constant of 0.566 microM-1. Ca(II) and La(III) competed for the Mn(II) site with association constants of 1.92 microM-1 and 1.38 microM-1, respectively. The affinity of Ca(II) and La(III) for their two other (presumed) sites on aequorin was an order of magnitude less than their affinity for the Mn(II) site. Mg(II) competed for the Mn(II) site as well but with a much smaller association constant of 0.0109 microM-1. Ca(II)-independent discharged aequorin did not bind Mn(II) to a significant degree. Conjectures on the location of the Mn(II) site in the aequorin amino acid sequence and on the relationship between the binding parameters of the cations and their influence on aequorin activity are given.     

Dynamics of Ca2+-saturated calmodulin D129N mutant studied by multiple molecular dynamics simulations

Fifteen independent 1-nsec MD simulations of fully solvated Ca(2+) saturated calmodulin (CaM) mutant D129N were performed from different initial conditions to provide a sufficient statistical basis to gauge the significance of observed dynamical properties. In all MD simulations the four Ca(2+) ions remained in their binding sites, and retained a single water ligand as observed in the crystal structure. The coordination of Ca(2+) ions in EF-hands I, II, and III was sevenfold. In EF-hand IV, which was perturbed by the mutation of a highly conserved Asp129, an anomalous eightfold Ca(2+) coordination was observed. The Ca(2+) binding loop in EF-hand II was observed to dynamically sample conformations related to the Ca(2+)-free form. Repeated MD simulations implicate two well-defined conformations of Ca(2+) binding loop II, whereas similar effect was not observed for loops I, III, and IV. In 8 out of 15 MD simulations Ca(2+) binding loop II adopted an alternative conformation in which the Thr62 >C=O group was displaced from the Ca(2+) coordination by a water molecule, resulting in the Ca(2+) ion ligated by two water molecules. The alternative conformation of the Ca(2+) binding loop II appears related to the "closed" state involved in conformational exchange previously detected by NMR in the N-terminal domain fragment of CaM and the C-terminal domain fragment of the mutant E140Q. MD simulations suggest that conformations involved in microsecond exchange exist partially preformed on the nanosecond time scale.     

Chemical speciation of L-proline complexes of Ca(II), Zn(II) and Mn(II) in acetonitrile-water mixtures

Abstract

Chemical speciation of binary complexes of Ca(II), Zn(II) and Mn(II) with L-proline is investigated pH-metrically in acetonitrile-water mixtures. The stability constants are calculated using the computer program MINIQUAD75. The best-fit chemical models are selected based on statistical parameters and residual analysis. The models for the binary species contained ML⁺, MLH²⁺and ML₂H⁺ for Ca(II), Zn(II) and Mn(II). The trend in variation of stability constants with change in the dielectric constant of the medium is explained on the basis of structure forming nature of acetonitrile. Distribution of the species with pH at different variations (0.0-60.0% v/v) in acetonitrile-water mixtures is also presented.     

Metal ion binding to alpha-lactalbumin species

A strong cation (calcium) binding site has been demonstrated to exist in several alpha-lactalbumin species; bovine, goat, human, and guinea pig. A metal ion induced conformational change occurs, resulting in a unique (10-14-nm) blue shift and relative quenching of Trp fluorescence for all species. Calcium ion binding to the alpha-lactalbumins yielded dissociation constants (Kdiss consistently in the 10(-10)--10(-12) M range, while Mn(II) binding was in the 20-30 microM range. Independent determinations of these cation binding equilibria were made by ESR measurements of free unliganded Mn(II) in titrations with the bovine species. One strong site (Kdiss = 30.5 microM) was found, which correlated directly with the fluorescence-associated cation binding, plus three weaker sites (Kdiss = 1.1, 5.0, and 5.0 mM, respectively). Several lanthanides as well as Mg(II) were found to displace Mn(II) from the strong site on bovine alpha-lactalbumin (as monitored by ESR) and to cause the identical fluorescence changes as found for Ca(II) and Mn(II) above. The importance of measuring these equilibria by both fluorescence and ESR was borne out by demonstrating the potential errors in estimating dissociation equilibria by the fluorescence method alone. Also, the errors in estimating Kdiss for samples containing partially metal bound apo-alpha-lactalbumin are described as well as rapid, sensitive methods for estimating the extent of metal-free protein and correctly accounting for residual bound metal in equilibrium calculations.     

The basic properties of the electronic structure of the oxygen-evolving complex of photosystem II are not perturbed by Ca2+ removal

Ca(2+) is an integral component of the Mn(4)O(5)Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S(2)' state of the Ca(2+)-depleted cluster from spinach is examined by X- and Q-band EPR and (55)Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca(2+) removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S(2)' state in spinach PS II is very similar to the native S(2) state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca(2+) a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y(Z) and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel (55)Mn(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca(2+)-depleted PS II. Mn(2+) titration, monitored by (55)Mn ENDOR, revealed a specific Mn(2+) binding site with a submicromolar K(D). Ca(2+) titration of Mn(2+)-loaded, Ca(2+)-depleted PS II demonstrated that the site is reversibly made accessible to Mn(2+) by Ca(2+) depletion and reconstitution. Mn(2+) is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn(4)O(5)Ca cluster during photoassembly and/or D1 repair.     

High-resolution proton and laser photochemically induced dynamic nuclear polarization NMR studies of cation binding to bovine alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) is a calcium binding protein that also binds Mn(II), lanthanide ions, A1(III), Zn(II), Co(II). The structural implications of cation binding were studied by high-resolution proton (200 MHz) NMR and photochemically induced dynamic nuclear polarization (CIDNP) spectroscopy. Marked changes were observed in the NMR spectra of the apoprotein upon addition of a stoichiometric amount of calcium to yield Ca(II)-alpha-LA, manifested particularly in ring current shifted aliphatic peaks and in several shifts in the aromatic region, all of which were under slow exchange conditions. The CIDNP results showed that two surface-accessible tyrosine residues, assigned as Tyr-18 and -36, became inaccessible to the solvent upon addition of 1:1 Ca(II) to apo-alpha-lactalbumin, while Tyr-103 and Trp-104 remained completely accessible in both conformers. The proton NMR spectra of apo-alpha-LA and A1(III)-alpha-LA were extremely similar, which was also consistent with intrinsic fluorescence results [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. The paramagnetic cation Mn(II) bound to the strong calcium binding site on apo-alpha-LA but also to the weak secondary Ca(II) binding site(s) on Ca(II)-alpha-LA. It was also found that Co(II) bound to some secondary sites on Ca(II)-alpha-LA that overlapped the weak calcium site. All of the lanthanide shift reagents [Pr(III), Eu(III), Tb(III), Dy(III), Tm(III), Yb(III)] bound under slow exchange conditions; their relative affinities for apo-alpha-lactalbumin from competitive binding experiments were Dy(III), Tb(III), and Pr(III) greater than Ca(II) greater than Yb(III).     

Stoichiometry of manganese and calcium ion binding to concanavalin A

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.     

The high-affinity binding site for manganese on the oxidizing side of Photosystem II

Electron donation to Photosystem II (PS II) by diphenylcarbazide (DPC) is interrupted by the presence of endogenous Mn in PS II particles. Removal of this Mn by Tris treatment greatly stimulates the electron transport with DPC as donor. Binding of low concentration of exogenous Mn(II) to Tris-treated PS II particles inhibits DPC photooxidation competitively with DPC. This phenomenon was used to locate a highly specific Mn(II) binding site on the oxidizing side of Photosystem II with dissociation constant about 0.15 μM. The binding of Mn(II) is electrostatic in nature. Its affinity depends not only on the ionic strength, but also on the anion species of the salt in the medium. The effectiveness in decreasing the affinity follows the order F⁻ > SO²⁻₄ > CH₃COO⁻ > CI⁻ > Br⁻ > NO₃⁻. This observation is interpreted as follows: smaller ions, like F⁻, CH₃COO⁻, and larger ions, like SO²⁻₄, have inhibitory effects on Mn(II) binding, whereas ions with optimal size, like Cl⁻, Br⁻ and NO₃⁻, can stabilize the binding, resembling the anion requirement for reactivation of Cl⁻-depleted chloroplasts. We suggest that the binding site for Mn(II) we observed is the site for the endogenous Mn in the O₂-evolving complex of PS II. This site remains after Tris treatment, which removes all the endogenous Mn as well as the three extrinsic proteins, indicating that it is on the intrinsic component(s) of PS II reaction centers. Furthermore, the Cl⁻ requirement for O₂ evolution may be attributed, at least partly to its stabilizing effect on Mn binding.     

Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis

To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation. Here, we test global kinetic analysis as an approach to this problem. Taking advantage of the various binding and cleavage constants established for PvuII endonuclease, we apply cleavage data obtained under a range of Mg(II) and Ca(II) concentrations to a number of kinetic models which specify A and B sites for both metal ions and various active species. The data are best fit and simulated with models which feature Ca(II) being held more strongly in the B (or secondary) site. This mixed metal enzyme species is the only one which forms appreciably and exhibits a cleavage rate constant similar to that observed when there is only one Mg(II) per active site (approximately 0.01 s^?1). Thus, in the case of PvuII endonuclease, Ca(II) does not stimulate cleavage. However, a simulated increase in activity at moderate Ca(II) concentrations can be rationalized with a cleavage rate constant for the mixed species similar to that when two Mg(II) ions are present in the active site. This provides an important insight into the underlying basis for the Ca(II)-stimulated activity observed for some metallonucleases that is not accessible by any other means.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Carbon-13 NMR studies on the interaction of manganese ion with the tRNA derived modified nucleoside,t6A

Manganese ion (Mn(II)) has been found to interact preferentially with the side chain of the transer RNA anticodon adjacent modified nucleoside, t⁶A, as monitored by carbon-13 nuclear magnetic resonance. Evidence for Mn(II) binding to the adenine ring is also presented. These findings lend credence to the postulate that this unusual nucleoside may be a binding site for magnesium ion in transfer RNA.     

Calorimetric experiments of Mn2+ -binding to alpha-lactalbumin

We measured by batch microcalorimetry the standard enthalpy change delta H degrees of the binding of Mn2+ to apo-bovine alpha-lactalbumin; delta H degrees = -90 +/- k J.mol-1. The binding constants, KMn2+, calculated from the calorimetric and circular dichroism titration curves, are (4.6 +/- 1).10(5) M-1 and (2.1 +/- 0.4).10(5) M-1, respectively. Batch calorimetry confirms the competitive binding Ca2+, Mn2+ and Na+ to the same site. The relatively small enthalpy change for Mn2+ binding compared to Ca2+ binding favours a model of a rigid and almost ideal Ca2+-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an alpha-helix which is directly connected to the complexating loop.     

Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases.

The activities of restriction enzymes are important examples of Mg(II)-dependent hydrolysis of DNA. While a number of crystallographic studies of enzyme-DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal-ion binding in restriction enzymes. Using PvuII restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction-enzyme conformation. The utility of this method is demonstrated with a study of metal-ion binding by PvuII endonuclease. Replacement of 74% (+/- 10%) of the Tyr residues in PvuII endonuclease by 3-fluorotyrosine produces an enzyme with Mg(II)-supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme. Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(II) does not support activity in either enzyme, a result consistent with previous studies. 1H- and 19F-NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short-range spectral broadening and longer range changes in chemical shift. Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II). Coupled with earlier mutagenesis studies that place Ca(II) in the active site [Nastri, H. G., Evans, P.D., Walker, I.H. & Riggs, P.D. (1997) J. Biol. Chem. 272, 25761-25767], these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes.     

Use of Poisons in Determination of Microbial Manganese Binding Rates in Seawater

A method was developed to determine whether microorganisms mediate the precipitation of manganese(II) in the marine environment. Radioactive ⁵⁴Mn(II) was used as a tracer to measure the precipitation (binding and oxidation) of Mn(II) [i.e., the ⁵⁴Mn(II) trapped on 0.2-μm membrane filters] in the presence and absence of biological poisons. A variety of antibiotics, fixatives, and metabolic inhibitors were tested in laboratory control experiments to select poisons that did not interfere in the chemistry of manganese. The poisons were deemed suitable if (i) they did not complex Mn(II) more strongly than the ion-exchange resin Chelex 100, (ii) they did not interfere in the adsorption of ⁵⁴Mn(II) onto synthetic δMnO₂ (manganate), (iii) they did not cause desorption of ⁵⁴Mn(II) which had been preadsorbed onto synthetic manganate, and (iv) they did not solubilize synthetic ⁵⁴manganate. In addition, several known chelators, reducing agents, and buffers normally added to microbiological growth media or used in biochemical assays were tested. Most additions interfered to some extent with manganese chemistry. However, at least one inhibitor, sodium azide, or a mixture of sodium azide, penicillin, and tetracycline was shown to be appropriate for use in field studies of ⁵⁴Mn(II) binding. Formaldehyde could also be used in short incubations (1 to 3 h) but was not suitable for longer time course studies. The method was applied to studies of Mn(II) precipitation in Saanich Inlet, British Columbia, Canada. Bacteria were shown to significantly enhance the rate of Mn(II) removal from solution in the manganese-rich particulate layer which occurs just above the oxygen-hydrogen sulfide interface in the water column.     

Manganese and calcium requirements for reconstitution of oxygen-evolution activity in manganese-depleted photosystem II membranes

The Mn complex of photosystem II and O2-evolution activity are reconstituted in Mn-depleted photosystem II membranes in a light-dependent process called photoactivation. Recovery of O2-evolution activity requires both Mn2+ and Ca2+ in the photoactivation medium. The Mn2+ and Ca2+ dependences of both the effective rate constant and yield of photoactivation have been determined. A comparison of these data with the predictions of mathematical models for photoactivation leads to the conclusion that photoactivation occurs in two stages. The first stage, photoligation of Mn, requires light and depends primarily on Mn2+. The second stage, binding of Ca2+, is required for expression of O2-evolution activity. This two-stage model affords an excellent fit to the data and provides dissociation constants and binding stoichiometries for Ca2+ and Mn2+. We conclude that one Mn2+ ion is bound and photooxidized in the rate-determining step(s) of photoactivation. On the basis of these results and data already in the literature, the molecular details of the elementary steps in photoactivation are discussed and a mechanism of photoactivation is proposed.     

Unique binding site for Mn2+ ion responsible for reducing an oxidized YZ tyrosine in manganese-depleted photosystem II membranes.

Binding of Mn2+ to manganese-depleted photosystem II and electron donation from the bound Mn2+ to an oxidized YZ tyrosine were studied under the same equilibrium conditions. Mn2+ associated with the depleted membranes in a nonsaturating manner when added alone, but only one Mn2+ ion per photosystem II (PS II) was bound to the membranes in the presence of other divalent cations including Ca2+ and Mg2+. Mn2+-dependent electron donation to photosystem II studied by monitoring the decay kinetics of chlorophyll fluorescence and the electron paramagnetic resonance (EPR) signal of an oxidized YZ tyrosine (YZ+) after a single-turnover flash indicated that the binding of only one Mn2+ ion to the manganese-depleted PS II is sufficient for the complete reduction of YZ+ induced by flash excitation. The results indicate that the manganese-depleted membranes have only one unique binding site, which has higher affinity and higher specificity for Mn2+ compared with Mg2+ and Ca2+, and that Mn2+ bound to this unique site can deliver an electron to YZ+ with high efficiency. The dissociation constant for Mn2+ of this site largely depended on pH, suggesting that a single amino acid residue with a pKa value around neutral pH is implicated in the binding of Mn2+. The results are discussed in relation to the photoactivation mechanism that forms the active manganese cluster.