Iron bound to the high-affinity Mn-binding site of the oxygen-evolving complex shifts the pK of a component controlling electron transport via Y(Z)

Flash-probe fluorescence spectroscopy was used to compare the pH dependence of charge recombination between Y(Z)(*) and Q(a)(-) in Mn-depleted, photosystem II membranes [PSII(-Mn)] and in membranes with the high-affinity (HA(Z)) Mn-binding site blocked by iron [PSII(-Mn,+Fe); Semin, B. K., Ghirardi, M. L., and Seibert, M. (2002) Biochemistry 41, 5854-5864]. The apparent half-time for fluorescence decay (t(1/2)) in PSII(-Mn) increased from 9 ms at pH 4.4 to 75 ms at pH 9.0 [with an apparent pK (pK(app)) of 7.1]. The actual fluorescence decay kinetics can be fit to one exponential component at pH <6.0 (t(1/2) = 9.5 ms), but it requires an additional component at pH >6.0 (t(1/2) = 385 ms). Similar measurements with PSII(-Mn,+Fe) membranes show that iron binding has little effect on the maximum and minimum t(1/2) values measured at alkaline and acidic pHs but that it does shift the pK(app) from 7.1 to 6.1 toward the more acidic pK(app) value typical of intact membranes. Light-induced Fe(II) blocking of the PSII(-Mn) membrane is accompanied by a decrease in buffer Fe(II) concentration. This decrease was not the result of Fe(II) binding, but rather of its oxidation at two sites, the HA(Z) site and the low-affinity site. M?ssbauer spectroscopy at 80 K on PSII(-Mn,+Fe) samples, prepared under conditions providing the maximal blocking effect but minimizing the amount of nonspecifically bound iron cations, supports this conclusion since this method detected only Fe(III) cations bound to the membranes. Correlation of the kinetics of Fe(II) oxidation with the blocking parameters showed that blocking occurs after four to five Fe(II) cations were oxidized at the HA(Z) site. In summary, the blocking of the HA(Z) Mn-binding site by iron in PSII(-Mn) membranes not only prevents the access of exogenous donors to Y(Z) but also partially restores the properties of the hydrogen bond net found in intact PS(II), which in turn controls the rate of electron transport to Y(Z).     

The carboxyl modifier 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) inhibits half of the high-affinity Mn-binding site in photosystem II membrane fragments

The diphenylcarbazide(DPC)/Mn2+ assay [Hsu, B.-D., Lee, J.-Y., & Pan, R.-L. (1987) Biochim. Biophys. Acta 890, 89-96] was used to assess the amount of the high-affinity Mn-binding site in manganese-depleted photosystem II (PS II) membrane fragments from spinach and Scenedesmus obliquus. The assay mechanism at high DPC concentration was shown to involve noncompetitive inhibition of only half of the control level of DPC donation to PS II by micromolar concentrations of Mn at pH 6.5 (i.e., one of two DPC donation sites is inhibited). At low DPC concentration both DPC and Mn2+ donate to PS II additively. Treatment with the carboxyl amino acid modifier 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) inhibited half of the high-affinity Mn-binding site in spinach and Scenedesmus WT PS II membranes and all of the available site in Scenedesmus LF-1 mutant PS II membranes. A similar EDC concentration dependence was observed in all cases. Addition of 2 mM MnCl2 to the 10 mM EDC modification buffer provided complete protection for the Mn-binding site from modification. This protection was specific for Mn2+; six other divalent cations were ineffective. We conclude that EDC modifies that half of the high-affinity Mn-binding site that is insensitive to the histidine modifier diethyl pyrocarbonate (DEPC) [Seibert, M., Tamura, N., & Inoue, Y. (1989) Biochim. Biophys. Acta 974, 185-191] and directly affects ligands that bind Mn. The effects of EDC and DEPC that influence the high-affinity site are mutually exclusive and are specific to the lumenal side of the PS II membrane. Removal of the two more loosely bound of the four functional Mn from PS II membranes uncovers that part of the high-affinity site associated with carboxyl but not histidyl residues. We suggest that carboxyl residues on reaction center proteins are associated with half of the high-affinity Mn-binding site in PS II and are involved along with histidine residues in binding Mn functional in the O2-evolving process.     

Iron-blocking the high-affinity Mn-binding site in photosystem II facilitates identification of the type of hydrogen bond participating in proton-coupled electron transport via YZ

Incubation of Mn-depleted PSII membranes [PSII(-Mn)] with Fe(II) is accompanied by the blocking of Y(Z)(*) at the high-affinity Mn-binding site to exogenous electron donors [Semin et al. (2002) Biochemistry 41, 5854-5864] and a shift of the pK(app) of the hydrogen bond partner for Y(Z) (base B) from 7.1 to 6.1 [Semin, B. K., and Seibert, M. (2004) Biochemistry 43, 6772-6782]. Here we calculate activation energies (E(a)) for Y(Z)(*) reduction in PSII(-Mn) and Fe-blocked PSII(-Mn) samples [PSII(-Mn, +Fe)] from temperature dependencies of the rate constants of the fast and slow components of the flash-probe fluorescence decay kinetics. At pH < pK(app) (e.g., 5.5), the decays are fit with one (fast) component in both types of samples, and E(a) is equal to 42.2 +/- 2.9 kJ/mol in PSII(-Mn) and 46.4 +/- 3.3 kJ/mol in PSII(-Mn, +Fe) membranes. At pH > pK(app), the decay kinetics exhibit an additional slow component in PSII(-Mn, +Fe) membranes (E(a) = 36.1 +/- 7.5 kJ/mol), which is much lower than the E(a) of the corresponding component observed for Y(Z)(*) reduction in PSII(-Mn) samples (48.1 +/- 1.7 kJ/mol). We suggest that the above difference results from the formation of a strong low barrier hydrogen bond (LBHB) between Y(Z) and base B in PSII(-Mn, +Fe) samples. To confirm this, Fe-blocking was performed in D(2)O to insert D(+), which has an energetic barrier distinct from H(+), into the LBHB. Measurement of the pH effects on the rates of Y(Z)(*) reduction in PSII(-Mn, +Fe) samples blocked in D(2)O shows a shift of the pK(app) from 6.1 to 7.6, and an increase in the E(a) of the slow component. This approach was also used to measure the stability of the Y(Z)(*) EPR signal at various temperatures in both kinds of membranes. In PSII(-Mn) membranes, the freeze-trapped Y(Z)(*) radical is stable below 190 K, but half of the Y(Z)(*) EPR signal disappears after a 1-min incubation when the sample is warmed to 253 K. In PSII(-Mn, +Fe) samples, the trapped Y(Z)(*) radical is unstable at a much lower temperature (77 K). However, the insertion of D(+) into the hydrogen bond between Y(Z) and base B during the blocking process increases the temperature stability of the Y(Z)(*) EPR signal at 77 K. Again, these results indicate that Fe-blocking involves Y(Z) in the formation of a LBHB, which in turn is consistent with the suggested existence of a LBHB between Y(Z) and base B in intact PSII membranes [Zhang, C., and Styring, S. (2003) Biochemistry 42, 8066-8076].     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Ca2+ effects on Fe(II) interactions with Mn-binding sites in Mn-depleted oxygen-evolving complexes of photosystem II and on Fe replacement of Mn in Mn-containing,Ca-depleted complexes

Fe(II) cations bind with high efficiency and specificity at the high-affinity (HA), Mn-binding site (termed the “blocking effect” since Fe blocks further electron donation to the site) of the oxygen-evolving complex (OEC) in Mn-depleted, photosystem II (PSII) membrane fragments (Semin et al. in Biochemistry 41:5854, 2002). Furthermore, Fe(II) cations can substitute for 1 or 2Mn cations (pH dependent) in Ca-depleted PSII membranes (Semin et al. in Journal of Bioenergetics and Biomembranes 48:227, 2016; Semin et al. in Journal of Photochemistry and Photobiology B 178:192, 2018). In the current study, we examined the effect of Ca²⁺ cations on the interaction of Fe(II) ions with Mn-depleted [PSII(-Mn)] and Ca-depleted [PSII(-Ca)] photosystem II membranes. We found that Ca²⁺ cations (about 50 mM) inhibit the light-dependent oxidation of Fe(II) (5 µM) by about 25% in PSII(-Mn) membranes, whereas inhibition of the blocking process is greater at about 40%. Blocking of the HA site by Fe cations also decreases the rate of charge recombination between Q_A^? and Y_Z^?+ from t_1/2?=?30 ms to 46 ms. However, Ca²⁺ does not affect the rate during the blocking process. An Fe(II) cation (20 µM) replaces 1Mn cation in the Mn₄CaO₅ catalytic cluster of PSII(-Ca) membranes at pH 5.7 but 2 Mn cations at pH 6.5. In the presence of Ca²⁺ (10 mM) during the substitution process, Fe(II) is not able to extract Mn at pH 5.7 and extracts only 1Mn at pH 6.5 (instead of two without Ca²⁺). Measurements of fluorescence induction kinetics support these observations. Inhibition of Mn substitution with Fe(II) cations in the OEC only occurs with Ca²⁺ and Sr²⁺ cations, which are also able to restore oxygen evolution in PSII(-Ca) samples. Nonactive cations like La³⁺, Ni²⁺, Cd²⁺, and Mg²⁺ have no influence on the replacement of Mn with Fe. These results show that the location and/or ligand composition of one Mn cation in the Mn₄CaO₅ cluster is strongly affected by calcium depletion or rebinding and that bound calcium affects the redox potential of the extractable Mn4 cation in the OEC, making it resistant to reduction.

     

Characteristic features of the interaction between Fe(II) cations and the donor side of the manganese-depleted photosystem II

Ferrous iron cations Fe(II) can effectively bind to the donor side of the manganese-depleted photosystem II (PSII(-Mn)) and in this way block electron transfer from diphenylcarbazide (DPC) to the major donor for P680, Y_Z. The present study was focused on the characteristic features of this process. The oxidation and subsequent binding of Fe(II) cations to PSII(-Mn) may proceed in the absence of an artificial electron acceptor, and therefore we investigated the role of O₂ as a putative endogenous acceptor. Oxygen was shown to participate in the blockade of Y_Z by Fe cations, apparently as a structural element of Fe cluster formed at the donor side of PSII(-Mn). The kinetic study of blocking Y_Z by Fe(II) as dependent on light intensity demonstrated that the quantum efficiency of Fe cations binding to the donor side of PSII(-Mn) considerably exceeded that of Mn cations. We also compared the possibilities of extracting the native Mn cluster and reconstructed Fe cations from PSII and an alternative electron transport from DPC to P680⁺ under the conditions of the Y_Z blockade by Fe cations. Neither an alternative donor for P680, Y_D , nor cytochrome b ₅₅₉ participated in the latter process. As a whole, our evidence shows that many features of binding Fe cation to the donor side of PSII(-Mn) are in common with photoassembling the Mn cluster.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 12–20.Original Russian Text Copyright © 2005 by Lovyagina, Davletshina, Kultysheva, Timofeev, Ivanov, Semin.     

Accessibility of tyrosine Y(.)(Z) to exogenous reductants and Mn(2+) in various Photosystem II preparations

The reduction of tyrosine Y(.)(Z) by benzidine and exogenous Mn(2+) was studied by kinetic EPR experiments in various Photosystem II (PSII) preparations. Using lanthanide treated PSII membranes it was demonstrated that neither the extrinsic polypeptides (17, 23 and 33 kDa) nor the Mn complex block the accessibility of Y(.)(Z) to exogenous reductants, such as benzidine. In addition, it was shown that in the presence of the native Mn complex exogenous Mn(2+) does not reduce Y(.)(Z).     

A possible role of redox-active histidine in the photoligation of manganese into a photosynthetic O2-evolving enzyme.

Our previous experiments with a histidine modifier suggest that in Mn-depleted photosystem (PS) II a histidine residue is photooxidized and charge recombination between the oxidized histidine and QA-emits the thermoluminescence AT-band [Ono, T., & Inoue, Y. (1991) FEBS Lett. 278, 183-186]. By use of the AT-band as an index for histidine oxidation, and EPR signals IIf and IIs as indexes for tyrosine oxidation, we studied the role of this putative redox-active histidine in the photoactivation of the O2-evolving enzyme in NH2OH-treated PSII. The following results have been obtained. (i) Strong-light photoinhibition of NH2OH-treated PSII quickly impaired both capabilities of photoactivation and AT-band emission with almost the same half-inhibition time of 1-2 s, while signal IIf was well retained and signal IIs was not affected at all after complete loss of photoactivation capability. (ii) The capability of exogenous Mn2+ photooxidation was relatively sensitive to strong-light photoinhibition, but DPC was relatively sensitive to strong-light photoinhibition, but DPC photooxidation was highly resistant. (iii) Weak-light photoinhibition simultaneously impaired the capabilities of photoactivation, AT-band emission, and signal IIf with the same half-inhibition time of 1 min, leaving signal IIs unaffected. (iv) It was inferred that the putative redox-active histidine is essential for the photooxidation of coordinated Mn2+, the probable initial step of photoactivation, and its photodamage results in the loss of the capabilities of photoactivation and AT-band emission. Based on these, a scheme of electron transfer on the donor side of PSII involving histidine oxidation via Z+ is proposed.     

Fluorescence Induction Kinetics in Membrane Preparations of Photosystem II with Heterogeneous Metal Clusters (Mn/Fe) in the Oxygen-Evolving Complex

Transport of electrons in spinach photosystem II (PSII) whose oxygen-evolving complex (OEC) contains heterogeneous metal clusters 2Mn2Fe and 3Mn1Fe was studied by measuring the fluorescence induction kinetics (FIK). PSII(2Mn,2Fe) and PSII(3Mn,1Fe) preparations were produced using Cadepleted PSII membranes (PSII(–Ca)). It was found that FIK in PSII(2Mn,2Fe) membranes is similar in form to FIK in PSII(–Ca) samples, but the fluorescence yield is lower in PSII(2Mn,2Fe). The results demonstrate that, just as in PSII(–Ca) preparations, there is electron transfer from the metal cluster in the OEC to the primary plastoquinone electron acceptor Q_A. They also show that partial substitution of Mn cations with Fe has no effect on the electron transport on the acceptor side of PSII. Thus, these data demonstrate the possibility of water oxidation either by the heterogeneous metal cluster or just by the manganese dimer. We established that FIK in PSII(3Mn,1Fe) preparations are similar in form to FIK in PSII(2Mn,2Fe) membranes but PSII(3Mn,1Fe) is characterized by a slightly higher maximal fluorescence yield, F_max. The electron transfer rate in PSII(3Mn,1Fe) preparations significantly (by a factor of two) increases in the presence of Ca²⁺, whereas Ca²⁺ has hardly any effect on the electron transport in PSII(2Mn,2Fe) membranes. In Mndepleted PSII membranes, FIK reaches its maximum (the so-called peak K), after which the fluorescence yield starts to decrease as the result of two factors: the oxidation of reduced primary plastoquinone Q _A ^? and the absence of electron influx from the donor side of PSII. The replacement of Mn cations by Fe in PSII(?Mn) preparations leads to fluorescence saturation and disappearance of the K peak. This is possibly due to the deceleration of the charge recombination process that takes place between reduced primary electron acceptor Q _A ^? and oxidized tyrosine Y _Z ^+. which is an electron carrier between the OEC and the primary electron donor P680.     

Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.     

4-Nitrocatechol as a probe of a Mn(II)-dependent extradiol-cleaving catechol dioxygenase (MndD): comparison with relevant Fe(II) and Mn(II) model complexes

Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) is an extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis that has 82% sequence identity to and cleaves the same substrate (3,4-dihydroxyphenylacetic acid) as Fe(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HPCD) from Brevibacterium fuscum. We have observed that MndD binds the chromophoric 4-nitrocatechol (4-NCH(2)) substrate as a dianion and cleaves it extremely slowly, in contrast to the Fe(II)-dependent enzymes which bind 4-NCH(2) mostly as a monoanion and cleave 4-NCH(2) 4-5 orders of magnitude faster. These results suggest that the monoanionic binding state of 4-NC is essential for extradiol cleavage. In order to address the differences in 4-NCH(2) binding to these enzymes, we synthesized and characterized the first mononuclear monoanionic and dianionic Mn(II)-(4-NC) model complexes as well as their Fe(II)-(4-NC) analogs. The structures of [(6-Me(2)-bpmcn)Fe(II)(4-NCH)](+), [(6-Me(3)-TPA)Mn(II)(DBCH)](+), and [(6-Me(2)-bpmcn)Mn(II)(4-NCH)](+) reveal that the monoanionic catecholate is bound in an asymmetric fashion (Delta r(metal-O(catecholate))=0.25-0.35 A), as found in the crystal structures of the E(.)S complexes of extradiol-cleaving catechol dioxygenases. Acid-base titrations of [(L)M(II)(4-NCH)](+) complexes in aprotic solvents show that the p K(a) of the second catecholate proton of 4-NCH bound to the metal center is half a p K(a) unit higher for the Mn(II) complexes than for the Fe(II) complexes. These results are in line with the Lewis acidities of the two divalent metal ions but are the opposite of the trend observed for 4-NCH(2) binding to the Mn(II)- and Fe(II)-catechol dioxygenases. These results suggest that the MndD active site decreases the second p K(a) of the bound 4-NCH(2) relative to the HPCD active site.     

A functional role for tyrosine-D in assembly of the inorganic core of the water oxidase complex of photosystem II and the kinetics of water oxidation.

The role of D2-Tyr160 (Y(D)), a photooxidizable residue in the D2 reaction center polypeptide of photosystem II (PSII), was investigated in both wild type and a mutant strain (D2-Tyr160Phe) in which phenylalanine replaces Y(D) in the cyanobacterium Synechocystis sp. (strain PCC 6803). Y(D) is the symmetry-related tyrosine that is homologous to the essential photoactive Tyr161(Y(Z)) of the D1 polypeptide of PSII. We compared the flash-induced yield of O(2) in intact, functional PSII centers from both wild-type and mutant PSII core complexes. The yield of O(2) in the intact holo-enzyme was found to be identical in the mutant and wild-type PSII cores using long (saturating) pulses or continuous illumination, but was observed to be appreciably reduced in the mutant using short (nonsaturating) light pulses (<50 ms). We also compared the rates of the first two kinetically resolved steps of photoactivation. Photoactivation is the assembly process for binding of the inorganic cofactors to the apo-water oxidation/PSII complex (apo-WOC-PSII) and their light-induced photooxidation to form the functional Mn(4)Ca(1)Cl(x)() core required for O(2) evolution. We show that the D2-Tyr160Phe mutant cores can assemble a functional WOC from the free inorganic cofactors, but at a much slower rate and with reduced quantum efficiency vs wild-type PSII cores. Both of these observations imply that the presence of Y(D)(*) leads to a more efficient photooxidation of the Mn cluster relative to deactivation (reductive processes). One possible explanation for this behavior is that the phenolic proton on Y(D) is retained within the reaction center following Y(D) oxidation. The positive charge, likely shared by D2-His189 and other residues, raises the reduction potential of P(680)(+)/P(680), thereby increasing the driving force for the oxidation of Mn(4)Y(Z). There is, therefore, a competitive advantage to organisms that retain the Y(D) residue, possibly explaining its retention in all sequences of psbD (encoding the D2 polypeptide) known to date. We also find that the sequence of metal binding steps during assembly of apo-WOC-PSII centers in cyanobacteria cores differs from that in higher plants. This is seen by a reduced calcium affinity at its effector site and reduced competition for binding to the Mn(II) site, resulting in acceleration of the initial lagtime by Ca(2+), in contrast to retardation in spinach. Ca(2+) binding to its effector site promotes the stability of the photointermediates (IM1 and above) by suppressing unproductive decay.     

Mechanism of irreversible inhibition of O2 evolution in photosystem II by Tris(hydroxymethyl)aminomethane.

The dark reaction of tris(hydroxymethyl)aminomethane (Tris) with the O2-evolving center of photosystem II (PSII) in the S1 state causes irreversible inhibition of O2 evolution. Similar inhibition is observed for several other amines: NH3, CH3NH2, (CH3)2NH, ethanolamine, and 2-amino-2-ethyl-1,3-propanediol. In PSII membranes, both depleted of the 17- and 23-kDa polypeptides and undepleted, the rate of reaction of Tris depends inversely upon the Cl- concentration. However, the rate of reaction of Tris is about 2-fold greater with PSII membranes depleted of the 17- and 23-kDa polypeptides than with undepleted PSII membranes. We have used low-temperature electron paramagnetic resonance (EPR) spectroscopy to study the effect of Tris on the oxidation state of the Mn complex in the O2-evolving center, to monitor the electron-donation reactions in Tris-treated samples, and to observe any loss of the Mn complex (forming Mn2+ ions) after Tris treatment. We find that Tris treatment causes loss of electron-donation ability from the Mn complex at the same rate as inhibition of O2 evolution and that Mn2+ ions are released. We conclude that Tris reduces the Mn complex to labile Mn2+ ions, without generating any kinetically stable, partially reduced intermediates, and that the reaction occurs at the Cl(-)-sensitive site previously characterized in studies of the reversible inhibition of O2 evolution by amines.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

An electron spin-echo envelope modulation study [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708] and a recent Fourier transform infrared study [Noguchi, T., Inoue, Y., and Tang, X.-S. (1999) Biochemistry 38, 10187-10195], both conducted with [(15)N]histidine-labeled photosystem II particles, show that at least one histidine residue coordinates the O(2)-evolving Mn cluster in photosystem II. Evidence obtained from site-directed mutagenesis studies suggests that one of these residues may be His332 of the D1 polypeptide. The mutation D1-H332E is of particular interest because cells of the cyanobacterium Synechocystis sp. PCC 6803 that contain this mutation evolve no O(2) but appear to assemble Mn clusters in nearly all photosystem II reaction centers [Chu, H.-A., Nguyen, A. P. , and Debus, R. J. (1995) Biochemistry 34, 5859-5882]. Photosystem II particles isolated from the Synechocystis D1-H332E mutant are characterized in this study. Intact D1-H332E photosystem II particles exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the S(2) state multiline EPR signal observed in wild-type PSII particles. However, the quantum yield for oxidizing the S(1) state Mn cluster is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*), and the temperature threshold for forming the S(2) state is approximately 100 K higher than in wild-type PSII preparations. Furthermore, the D1-H332E PSII particles are unable to advance beyond the Y(Z)(*)S(2) state, as shown by the accumulation of a narrow "split" EPR signal under multiple turnover conditions. In Mn-depleted photosystem II particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in D1-H332E is accelerated in comparison to wild-type, showing that the mutation alters the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-His332 being located near the Mn-Y(Z) complex and perhaps ligating Mn.     

Beta-carotene redox reactions in photosystem II: electron transfer pathway

A carotenoid (Car), a chlorophyll (Chl(Z)), and cytochrome b(559) (Cyt b(559)) are able to donate electrons with a low quantum yield to the photooxidized chlorophyll, P680(+), when photosystem II (PSII) is illuminated at low temperatures. Three pathways for electron transfer from Cyt b(559) to P680(+) are considered: (a) the "linear pathway" in which Cyt b(559) donates via Chl(Z) to Car, (b) the "branched pathway" in which Cyt b(559) donates via Car and where Chl(Z) is also able to donate to Car, and (c) the "parallel pathway" where Cyt b(559) donates to P680 without intermediate electron carriers and electron donation from Chl(Z) and Car occurs by a competing pathway. Experiments were performed using EPR and spectrophotometry in an attempt to distinguish among these pathways, and the following observations were made. (1) Using PSII with an intact Mn cluster in which Cyt b(559) was preoxidized, Car oxidation was dominant upon illumination at < or =20 K, while electron donation from Chl dominated at >120 K. (2) When Cyt b(559) was prereduced, its light-induced oxidation occurred at < or =20 K in what appeared to be all of the centers and without the formation of a detectable Car(+) intermediate. The small and variable quantity of Car(+) photoinduced in these experiments can be attributed to the residual centers in which Cyt b(559) remained oxidized prior to illumination. (3) The relative rates for irreversible electron donation from Cyt b(559) and Car were determined indirectly at 20 K by monitoring the flash-induced loss of charge separation (i.e., the accumulation of Cyt b(559)(+)Q(A)(-) or Car(+)Q(A)(-)). Similar yields per flash were observed (13% for Cyt b(559) and 8% for Car), indicating similar donation rates. The slightly lower yield with Car as a donor is attributed at least in part to slow charge recombination occurring from the Car(+)Q(A)(-) radical pair in a fraction of centers. (4) Light-induced oxidation of Cyt b(559) and Car at 20 K was monitored directly by EPR, and the rates were found to be indistinguishable. The parallel pathway predicts that when both Cyt b(559) and Car are prereduced, the relative amounts of Cyt b(559)(+) and Car(+) produced upon illumination at 20 K should depend directly on their relative electron donation rates. The measured similarity in the donation rates thus predicts comparable yields of oxidation for both donors. However, what is observed experimentally is that Cyt b(559) oxidation occurs almost exclusively, and this argues strongly against the parallel pathway. The lack of Car(+) as a detectable intermediate is attributed to rapid electron transfer from Cyt b(559) to Car(+). The trapping of Car(+) at low temperature when Cyt b(559) is preoxidized but its absence when Cyt b(559) is prereduced is taken as an argument against the simple linear pathway. Overall, the data reported here and previously favor the branched pathway over the linear pathway, while the parallel pathway is thought to be unlikely. Structural considerations provide further arguments in favor of the branched model.     

pH Dependence of the Efficiency of Binding of Iron Cations to the Donor Side of Photosystem II

Light-induced interaction of Fe(II) cations with the donor side of Mn-depleted photosystem II (PS II(–Mn)) results in the binding of iron cations and blocking of the high-affinity (HA_Z) Mn-binding site. The pH dependence of the blocking was measured using the diphenylcarbazide/2,6-dichlorophenolindophenol test. The curve of the pH dependence is bell-shaped with pK ₁ = 5.8 and pK ₂ = 8.0. The pH dependence of the O₂-evolution mediated by PS II membranes is also bellshaped (pK ₂ = 7.6). The pH dependence of the process of electron donation from exogenous donors in PS II(–Mn) was studied to determine the location of the alkaline pH sensitive site of the electron transport chain. The data of the study showed that the decrease in the iron cation binding efficiency at pH > 7.0 during blocking was determined by the donor side of the PS II(–Mn). Mössbauer spectroscopy revealed that incubation of PS II(–Mn) membranes in a buffer solution containing ⁵⁷Fe(II) + ⁵⁷Fe(III) was accompanied by binding only Fe(III) cations. The pH dependence of the nonspecific Fe(III) cation binding is also described by the same bell-shaped curve with pK ₂ = 8.1. The treatment of the PS II(–Mn) membranes with the histidine modifier diethylpyrocarbonate resulted in an increase in the iron binding strength at alkaline pH. It is suggested that blocking efficiency at alkaline pH is determined by competition between OH^– and histidine ligand for Fe(III). Because the high-affinity Mn-binding site contains no histidine residue, this fact can be regarded as evidence that histidine is located at another (other than high-affinity) Fe(III) binding site. In other words, this means that the blockage of the high-affinity Mn-binding site is determined by at least two iron cations. We assume that inactivation of oxygen-evolving complex and inhibition of photoactivation in the alkaline pH region are also determined by competition between OH^– and a histidine residue involved in coordination of manganese cation outside the high-affinity site.     

Amino acid residues that modulate the properties of tyrosine Y(Z) and the manganese cluster in the water oxidizing complex of photosystem II

The catalytic site for photosynthetic water oxidation is embedded in a protein matrix consisting of nearly 30 different polypeptides. Residues from several of these polypeptides modulate the properties of the tetrameric Mn cluster and the redox-active tyrosine residue, Y(Z), that are located at the catalytic site. However, most or all of the residues that interact directly with Y(Z) and the Mn cluster appear to be contributed by the D1 polypeptide. This review summarizes our knowledge of the environments of Y(Z) and the Mn cluster as obtained from the introduction of site-directed, deletion, and other mutations into the photosystem II polypeptides of the cyanobacterium Synechocystis sp. PCC 6803 and the green alga Chlamydomonas reinhardtii.     

A new mechanism-based inhibitor of photosynthetic water oxidation: acetone hydrazone. 1. Equilibrium reactions

The process of photosynthetic water oxidation has been investigated by using a new type of water oxidation inhibitor, the alkyl hydrazones. Acetone hydrazone (AceH), (CH3)2CNNH2, inhibits water oxidation by a mechanism that is analogous to that of NH2OH. This involves binding to the water-oxidizing complex (WOC), followed by photoreversible reduction of manganese (loss of the S1----S2 reaction). At higher AceH concentrations the S1 state is reduced in the dark and Mn is released, albeit to a lesser extent than with NH2OH. Following extraction of Mn, AceH is able to donate electrons rapidly to the reaction center tyrosine radical Z+ (161Tyr-D1 protein), more slowly to a reaction center radical C+, and not at all to the dark-stable tyrosine radical D+ (160Tyr-D2 protein) which must be sequestered in an inaccessible site. Manganese, Z+, and C+ thus appear to be located in a common protein domain, with Mn being the first accessible donor, followed by Z+ and then C+. Photooxidation of Cyt b-559 is suppressed by AceH, indicating either reduction or competition for donation to P680+. Unexpectedly, Cl- was found not to interfere or compete with AceH for binding to the WOC in the S1 state, in contrast to the reported rate of binding of N,N-dimethylhydroxylamine, (CH3)2NOH [Beck, W., & Brudvig, G. (1988) J. Am. Chem. Soc. 110, 1517-1523]. We interpret the latter behavior as due to ionic screening of the thylakoid membrane, rather than a specific Cl- site involved in water oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)