The role of hydrogen bonds for the multiphasic P680(+)* reduction by YZ in photosystem II with intact oxyen evolution capacity. Analysis of kinetic H/D isotope exchange effects

The mechanism of multiphasic P680(+)* reduction by YZ has been analyzed by studying H/D isotope exchange effects on flash-induced changes of 830 nm absorption, DeltaA830(t), and normalized fluorescence yield, F(t)/F0, in dark-adapted thylakoids and PS II membrane fragments from spinach. It was found that (a) the characteristic period four oscillations of the normalized components of DeltaA830(t) relaxation and of F(t)/F0 rise in the nanosecond and microsecond time domain are significantly modified when exchangeable protons are replaced by deuterons; (b) in marked contrast to the normalized steady-state extent of the microsecond kinetics of 830 nm absorption changes which increases only slightly due to H/D exchange (about 10%) the Si state-dependent pattern exhibits marked effects that are most pronounced after the first, fourth, fifth, and eighth flashes; (c) regardless of data evaluation by different fit procedures the results lead to a consistent conclusion, that is, the relative extent of the back reaction between P680(+)*QA-* becomes enhanced in samples suspended in D2O; and (d) this enhancement is dependent on the Si state of the WOC and attains maximum values in S2 and S3, most likely due to a retardation of the "35 micros kinetics" of P680(+)* reduction. In an extension of our previous suggestion on the functional role of hydrogen bonding of YZ by a basic group X (Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431), a model is proposed for the origin of the multiphasic P680(+)* reduction by YZ. Two types of different processes are involved: (a) electron transfer in the nanosecond time domain is determined by strength and geometry of the hydrogen bond between the O-H group of YZ and acceptor X, and (b) the microsecond kinetics reflect relaxation processes of a hydrogen bond network giving rise to a shift of the equilibrium P680(+)*YZ <==> P680YZ(OX) toward the right side. The implications of this model are discussed.     

Electron-transfer reactions in manganese-depleted photosystem II

We have used flash-detection optical and electron paramagnetic resonance spectroscopy to measure the kinetics and yield per flash of the photooxidation of cytochrome b559 and the yield per flash of the photooxidation of the tyrosine residue YD in Mn-depleted photosystem II (PSII) membranes at room temperature. The initial charge separation forms YZ+ QA-. Following this, cytochrome b559 is oxidized on a time scale of the same order and with the same pH dependence as is observed for the decay of YZ+; under the conditions of our experiments, the decay of YZ+ is determined by the lifetime of YZ+ QA-. In order to explain this observation, we have constructed a model for electron donation in which YZ+ and P680+ are in redox equilibrium and cytochrome b559 and YD are oxidized via P680+. Using our results, together with data from earlier investigations of the kinetics of electron transfer from YZ to P680+ and charge recombination of YZ+ QA-, we have obtained the first global fit for electron donation in Mn-depleted PSII that accounts for the data over the pH range from 5 to 7.5. From these calculations, we have obtained the intrinsic rate constants of all the electron-donation reactions in Mn-depleted PSII. These rate constants allow us to calculate the free energy difference between YZ+ P680 and YZ P680+, which is found to increase by 47 +/- 4 mV/pH from pH 5 to 6 and is observed to increase more slowly per pH unit for pH greater than 6. An important conclusion of our experimental work is that the rates of photooxidation of cytochrome b559 and YD are determined by the lifetime of the oxidizing equivalent on YZ/P680. Extension of our model to oxygen-evolving PSII samples leads to the prediction that the kinetics and yields of electron donation from cytochrome b559 and YD to P680+ will depend on the S2- or S3-state lifetime.     

Evidence for impaired hydrogen-bonding of tyrosine YZ in calcium-depleted photosystem II

Photosystem II (PS II) evolves oxygen from two bound water molecules in a four-stepped reaction that is driven by four quanta of light, each oxidizing the chlorophyll moiety P680 to yield P+680. When starting from its dark equilibrium (mainly state S1), the catalytic center can be clocked through its redox states (S0ellipsisS4) by a series of short flashes of light. The center involves at least a Mn4-cluster and a special tyrosine residue, named YZ, as redox cofactors plus two essential ionic cofactors, Cl- and Ca2+. Centers which have lost Ca2+ do not evolve oxygen. We investigated the stepped progression in dark-adapted PS II core particles after the removal of Ca2+. YZ was oxidized from the first flash on. The difference spectrum of YZ-->YoxZ differed from the one in competent centers, where it has been ascribed to a hydrogen-bonded tyrosinate. The rate of the electron transfer from YZ to P+680 was slowed down by three orders of magnitude and its kinetic isotope effect rose up from 1.1 to 2.5. Proton release into the bulk was now a prerequisite for the electron transfer from YZ to P+680. On the basis of these results and similar effects in Mn-(plus Ca2+-)depleted PS II (M. Haumann et al., Biochemistry, 38 (1999) 1258-1267) we conclude that the presence of Ca2+ in the catalytic center is required to tune the apparent pK of a base cluster, B, to which YZ is linked by hydrogen bonds. The deposition of a proton on B within close proximity of YZ (not its release into the bulk!) is a necessary condition for the reduction in nanoseconds of P+680 and for the functioning of water oxidation. The removal of Ca2+ rises the pK of B, thereby disturbing the hydrogen bonded structure of YZB.     

Regulation of the pH sensitivity of human P2X receptors by N-linked glycosylation

The human (h) P2X(3) receptor and its mutants deficient in one out of four N-glycosylation sites were expressed in HEK293 cells. Concentration-response curves were generated by whole-cell recordings of alpha,beta-methylene ATP (alpha,beta-meATP)-induced currents. A gradual change of external pH from the alkaline 8.0 to the acidic 5.0 successively decreased the maximum current amplitude (E(max)) without affecting the EC(50) value. The replacement of Asn-139 and -170 by Asp (N139D, N170D) abolished the pH sensitivity of the wild-type (WT) hP2X(3) receptor. In the case of N194D, the E(max) was again the highest at the alkaline pH value with no change from 7.4 to 6.5, whereas in the case of N290D, there was an inverse pH sensitivity, with an increase of E(max) in the acidic range. However, this effect appeared to be due to enhanced protonation by the insertion of Asp into the receptor, because replacement of Asn by the neutral Thr resulted in a comparable potency of alpha,beta-meATP at any of the pH values investigated. In accordance with the reported finding that His-206 is involved in the modulation of WT P2X(3) receptors by protons, we showed that the normal change of E(max) by an acidic, but not alkaline pH was abolished after substitution of this His by Ala. However, the double mutant H206A + N290D did not react to acidification or alkalinization with any change in E(max). In conclusion, only fully N-glycosylated P2X(3) receptors recognize external pH with a modified sensitivity towards alpha,beta-meATP.     

Photosystem II of peas: effects of added divalent cations of Mn, Fe, Mg, and Ca on two kinetic components of P(+)(680) reduction in Mn-depleted core particles

The catalytic Mn cluster of the photosynthetic oxygen-evolving system is oxidized via a tyrosine, Y(Z), by a photooxidized chlorophyll a moiety, P(+)(680). The rapid reduction of P(+)(680) by Y(Z) in nanoseconds requires the intactness of an acid/base cluster around Y(Z) with an apparent functional pK of <5. The removal of Mn (together with bound Ca) shifts the pK of the acid/base cluster from the acid into the neutral pH range. At alkaline pH the electron transfer (ET) from Y(Z) to P(+)(680) is still rapid (<1 micros), whereas at acid pH the ET is much slower (10-100 micros) and steered by proton release. In the intermediate pH domain one observes a mix of these kinetic components (see R. Ahlbrink, M. Haumann, D. Cherepanov, O. B?gershausen, A. Mulkidjanian, W. Junge, Biochemistry 37 (1998)). The overall kinetics of P(680)(+) reduction by Y(Z) in Mn-depleted photosystem II (PS II) has been previously shown to be slowed down by divalent cations (added at >10 microM), namely: Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+) (C.W. Hoganson, P.A. Casey, O. Hansson, Biochim. Biophys. Acta 1057 (1991)). Using Mn-depleted PS II core particles from pea as starting material, we re-investigated this phenomenon at nanosecond resolution, aiming at the effect of divalent cations on the particular kinetic components of P(+)(680) reduction. To our surprise we found only the slower, proton steered component retarded by some added cations (namely Co(2+)/Zn(2+)>Fe(2+)>Mn(2+)). Neither the fast component nor the apparent pK of the acid/base cluster around Y(Z) was affected. Apparently, the divalent cations acted (electrostatically) on the proton release channel that connects the oxygen-evolving complex with the bulk water, but not on the ET between Y(Z) and P(+)(680), proper. Contrastingly, Ca(2+) and Mg(2+), when added at >5 mM, accelerated the slow component of P(+)(680) reduction by Y(Z) and shifted the apparent pK of Y(Z) from 7.4 to 6.6 and 6.7, respectively. It was evident that the binding site(s) for added Ca(2+) and Mg(2+) were close to Y(Z) proper. The data obtained are discussed in relation to the nature of the metal-binding sites in photosystem II.     

Photoconsumption of oxygen in photosystem II preparations under impairment of the water-oxidizing complex

Oxygen consumption in photosystem II (PSII) preparations in the light was 2 mol O₂/h per mg Chl at weakly acidic and at neutral pH values. It increased fourfold to fivefold at pH 8.5-9.0. The addition of either artificial electron donors for PSII such as MnCl₂ or diphenylcarbazide, or diuron as an inhibitor of electron transfer from Q_A, the primary bound quinone acceptor, to Q_B, the secondary bound quinone acceptor of PSII, resulted in a decrease in oxygen consumption rate at basic pH to value close to ones measured at pH 6.5. Such additions did not affect oxygen consumption at lower pH values. The induction of variable chlorophyll fluorescence yield in the light differed greatly at pH 6.5 and 8.5. While at pH 6.5 the fluorescence yield, after an initial fast rise almost to F_max, only slightly decreased, at pH 8.5 after such a rise it dropped promptly to a low value. The additions of the artificial electron donors at pH 8.5 resulted in the induction kinetics close to that observed at pH 6.5. These data indicate impairment of electron donation to P680⁺ that could be caused by damage to the water oxidation system at basic pH values. In experiments with PSII preparations treated with Tris to destroy the water-oxidizing complex, photoconsumption of oxygen in the entire pH region was close to the values in untreated preparations at basic pH. In untreated preparations the rate of light-induced oxygen consumption decreased in the presence of catalase, which decomposes H₂O₂, as well as in the presence of electron acceptor potassium ferricyanide. From these data it is suggested that the light-induced oxygen consumption in PSII is caused by two processes, by an interaction of O₂ with organic radicals, which were formed due to oxidation of components of the donor side of this photosystem (proteins, lipids, pigments) by cation-radical P680⁺, as well as by oxygen reduction by still unidentified components of PSII.     

Electron Transfer in the Photosynthetic Membrane: Influence of PH and Surface Potential on the P-680 Reduction Kinetics

The primary electron donor P-680 of the Photosystem-II reaction center was photoxidized by a short flash given after dark adaptation of photosynthetic membranes in which oxygen evolution was inhibited. The P-680⁺ reduction rate was measured under different conditions of pH and salt concentration by following the recovery of the absorption change at 820 nm. As previously reported for Tris-washed chloroplasts (Conjeaud, H., and P. Mathis, 1980, Biochim. Biophys. Acta, 590:353-359) a fast phase of P-680⁺ reduction slows down as the bulk pH decreases. When salt concentration increases, this fast phase becomes faster for pH above 4.5-5 and slower below. A quantitative interpretation is proposed in which the P-680⁺ reduction kinetics by the secondary electron donor Z are controlled by the local pH. This pH, at the membrane level, can be calculated using the Gouy-Chapman theory. A good fit of the results requires to assume that the surface charge density of the inside of the membrane, near the Photosystem-II reaction center, is positive at low pH values and becomes negative as the pH increases, with a local isoelectric point ~4.8. These results lead us to propose a functional scheme in which a pH-dependent proton release is coupled to the electron transfer between secondary and primary donors of Photosystem-II. The H⁺/e ratio varies from 1 at low pH to 0 at high pH, with a real pK ~6.5 for the protonatable species.     

Hydrogen bonding to P700: site-directed mutagenesis of threonine A739 of photosystem I in Chlamydomonas reinhardtii

The primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (P(B)) and chlorophyll a' (P(A)). P(A) is involved in a hydrogen bond network with several surrounding amino acid residues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on the properties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 13(1)-keto group of P(A), was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii using site-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700(+)* - P700) FTIR difference spectra of the mutants lack a negative band at 1634 cm(-1) observed in the wild-type spectrum and instead exhibit a new negative band between 1658 and 1672 cm(-1) depending on the mutation. This band can therefore be assigned to the 13(1)-keto group of P(A) which is upshifted to higher frequencies upon removal of the hydrogen bond. (ii) The main bleaching band in the Q(y)() region of the (P700(+)* - P700) and ((3)P700 - P700) absorption difference spectra is blue shifted for the mutants by approximately 6 nm compared to that of the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700(+)* - P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm (negative CD) of approximately equal area. For each mutant, these two components are blue-shifted to the same extent. The results strongly suggest that a blue shift of the Q(y) absorption band of P(A) is responsible for a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential for the oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy shows that the hfc of the methyl protons at position 12 of the spin-carrying Chl P(B) is decreased due to the removal of the hydrogen bond to P(A). This indicates a redistribution of spin density in P700(+)* compared to that in the wild type. This gives evidence for an electronic coupling between the two halves of the dimer in the wild type and mutants.     

Effect of muscle interstitial pH on P2X and TRPV1 receptor-mediated pressor response.

Activation of purinergic P2X receptors and transient receptor potential vanilloid type 1 (TRPV1) on muscle afferent nerve evokes the pressor response. Because P2X and TRPV1 receptors are sensitive to changes in pH, the aim of this study was to examine the effects of muscle acidification on those receptor-mediated cardiovascular responses. In decerebrate rats, the pH in the hindlimb muscle was adjusted by infusing acidic Ringer solutions into the femoral artery. Dialysate was then collected using microdialysis probes inserted into the muscles, and pH was measured. The interstitial pH was 7.53+/-0.01, 7.22+/-0.02, 6.94+/-0.04, and 6.59+/-0.03 in response to arterial infusion of the Ringer solution at pH 7.4, 6.5, 5.5, and 4.5, respectively. Femoral arterial injection of alpha,beta-methylene-ATP (P2X receptor agonist) in the concentration of 0.25 mM (volume, 0.15-0.25 ml; injection duration, 1 min) at the infused pH of 7.4, 6.5, and 5.5 increased mean arterial pressure (MAP) by 29+/-2, 24+/-3, and 21+/-3 mmHg, respectively (P<0.05, pH 5.5 vs. pH 7.4). When pH levels in the infused solution were 7.4, 6.5, 5.5, and 4.5, capsaicin (1 microg/kg), a TRPV1 agonist, was injected into the artery. This elevated MAP by 29+/-4, 33+/-2, 35+/-3, and 40+/-3 mmHg, respectively (P<0.05, pH 4.5 vs. pH 7.4). Furthermore, blocking acid-sensing ion channel (ASIC) blunted pH effects on TRPV1 response. Our data indicate that 1) muscle acidosis attenuates P2X-mediated pressor response but enhances TRPV1 response; 2) exaggerated TRPV1 response may require lower pH in muscle, and the effect is likely to be mediated via ASIC mechanisms. This study provides evidence that muscle pH may be important in modulating P2X and TRPV1 responsiveness in exercising muscle.     

pH dependent competition between Y(Z) and Y(D) in photosystem II probed by illumination at 5 K

The photosystem II (PSII) reaction center contains two redox active tyrosines, YZ and YD, situated on the D1 and D2 proteins, respectively. By illumination at 5 K, oxidation of YZ in oxygen-evolving PSII can be observed as induction of the Split S1 EPR signal from YZ* in magnetic interaction with the CaMn4 cluster, whereas oxidation of YD can be observed as the formation of the free radical EPR signal from YD*. We have followed the light induced induction at 5 K of the Split S1 signal between pH 4-8.5. The formation of the signal, that is, the oxidation of YZ, is pH independent and efficient between pH 5.5 and 8.5. At low pH, the split signal formation decreases with pKa approximately 4.7-4.9. In samples with chemically pre-reduced YD, the pH dependent competition between YZ and YD was studied. Only YZ was oxidized below pH 7.2, but at pH above 7.2, the oxidation of YD became possible, and the formation of the Split S1 signal diminished. The onset of YD oxidation occurred with pKa approximately 8.0, while the Split S1 signal decreased with pKa approximately 7.9 demonstrating that the two tyrosines compete in this pH interval. The results reflect the formation and breaking of hydrogen bonds between YZ and D1-His190 (HisZ) and YD and D2-His190 (HisD), respectively. The oxidation of respective tyrosine at 5 K demands that the hydrogen bond is well-defined; otherwise, the low-temperature oxidation is not possible. The results are discussed in the framework of recent literature data and with respect to the different oxidation kinetics of YZ and YD.     

Effects of Ca2+ and EGTA on P680*+ reduction kinetics and O2 evolution of Photosystem II

We report for the first time significant changes in the P680*+ reduction kinetics of Photosystem II (PS II) in which the 17 and 23 kDa extrinsic polypeptides are intact, in the presence of Ca(2+) or ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) which were added to vary the Ca(2+) concentration from 5 microM to 30 mM. The decrease in the extent of normal P680*+ reduction decay with lifetimes of 40-370 ns and a corresponding increase in the extent of kinetics with lifetimes of 20-220 micros was interpreted as being due to electron transfer from Y(Z) to P680*+ being replaced by slow forward conduction and by processes including P680*+/Q(A)(-) recombination. The question of whether changes in P680*+ reduction kinetics were caused by loss of Ca(2+) from PS II or by direct interaction of EGTA with PS II was addressed by lowering the free-Ca(2+) concentration of suspensions of PS II core complexes by serial dilution in the absence of EGTA. Despite a significant decrease in the rate of O(2) evolution after this treatment, only small changes in the P680*+ reduction kinetics were observed. Loss of Ca(2+) did not affect P680*+ reduction associated with electron transfer from Y(Z). Since much larger changes in the P680*+ reduction kinetics of intact PS II occurred at comparable free-Ca(2+) concentrations in the presence of EGTA, we conclude that EGTA influenced the P680*+ reduction kinetics by directly interacting with PS II rather than by lowering the free Ca(2+) concentration of the surrounding media. Notwithstanding these effects, we show that useful information about Ca(2+) binding to PS II can be obtained when direct interaction of EGTA is taken into account.     

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dual effect of acid pH on purinergic P2X3 receptors depends on the histidine 206 residue

Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X(3) receptors. In HEK293 cells permanently transfected with human (h) P2X(3) receptors (HEK293-hP2X(3) cells), an acidic pH shifted the concentration-response curve for alpha,beta-methylene ATP (alpha,beta-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of alpha,beta-meATP. Further, a low pH value increased the activation time constant (tau(on)) of the alpha,beta-meATP current; the fast and slow time constants of desensitization (tau(des1), tau(des2)) were at the same time also increased. Finally, acidification accelerated the recovery of P2X(3) receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X(3) receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the alpha,beta-meATP currents. The voltage sensitivity and reversal potential of the currents activated by alpha,beta-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X(3) receptor channels. It is suggested that acidification may have a dual effect on P2X(3) channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues.     

Role of D1-His190 in the proton-coupled oxidation of tyrosine YZ in manganese-depleted photosystem II.

To further characterize the role of D1-His190 in the oxidation of tyrosine Y(Z) in photosystem II, the pH dependence of P(680)(*)()(+) reduction was measured in H190A and Mn-depleted wild-type PSII particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803. Measurements were conducted in the presence and absence of imidazole and other small organic bases. In H190A PSII particles, rapid reduction of P(680)(*)()(+) attributed to electron transfer from Y(Z) increased dramatically above pH 9, with an apparent pK(A) of approximately 10.3. In the presence of ethanolamine and imidazole, this dramatic increase occurred at lower pH values, with the efficiency of Y(Z) oxidation correlating with the solution pK(A) value of the added base. We conclude that the pK(A) of Y(Z) is approximately 10.3 in D1-H190A PSII particles. In Mn-depleted wild-type PSII particles, P(680)(*)()(+) reduction was accelerated by all exogenous bases examined (substituted imidazoles, histidine, Tris, and 1,4-diazabicyclo[2.2.2]octane). We conclude that Y(Z) is solvent accessible in Mn-depleted wild-type PSII particles and that its pK(A) is near that of tyrosine in solution. In Mn-depleted wild-type PSII particles, over 80% of the kinetics of P(680)(*)()(+) reduction after a flash could be described by three kinetic components. The individual rate constants of these components varied slightly with pH, but their relative proportions varied dramatically with pH, showing apparent pK(A) values of 7.5 and 6.25 (6.9 and 5.8 in the presence of Ca(2+) and Mg(2+) ions). An additional pK(A) value (pK(A) < 4.5) may also be present. To describe these data, we propose (1) the pK(A) of His190 is 6.9-7.5, depending on buffer ions, (2) the deprotonation of Y(Z) is facilitated by the transient formation of a either a hydrogen bond or a hydrogen-bonded water bridge between Y(Z) and D1-His190, and (3) when protonated, D1-His190 interacts with nearby residues having pK(A) values near 6 and 4. Because Y(Z) and D1-His190 are located near the Mn cluster, these residues may interact with the Mn cluster in the intact system.     

The EPR spectrum of tyrosine Z* and its decay kinetics in O2-evolving photosystem II preparations

The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.     

Isolation of a photosystem 2 preparation from higher plants with highly enriched oxygen evolution activity

Detergent-treatment of higher plant thylakoids with Triton X-100 at pH 6.3 has been used to purify a PS2 fraction with very high rates of oxygen evolution (1000 μmol.mg chl⁻¹.h⁻¹). A photosynthetic unit size of about 300 chlorophyll (chl) molecules has been determined by optical methods, suggesting an average turnover time for PS2 of about 2 ms. The donor system for P680⁺ is particularly well preserved in the preparation, as judged by P680⁺ reduction kinetics, the detection by EPR of Signal II_LT and the presence of the high potential form of cytochrome b-559 (at a ratio of 1:1 with the reaction centre).     

Influence of Acidic pH on Hydrogen and Acetate Production by an Electrosynthetic Microbiome

Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm² at −765 mV (0.065 mA/cm² sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m³/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m³/day formate, and 3.1 kg/m³/day acetate ( = 4.7 kg CO₂ captured).     

Covalent Modification of Mutant Rat P2X2 Receptors with a Thiol-Reactive Fluorophore Allows Channel Activation by Zinc or Acidic pH without ATP

Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C₅-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.