Directed alteration of the D1 polypeptide of photosystem II: evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680

In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R.J., Barry, B.A., Babcock, G.T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA- occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA- occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.     

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.     

Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II.

Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence-quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition.     

Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.

We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700-chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light-activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild-type cells showed a 4-fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700-apoprotein; greatly reduced whole-chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA-C and PSA-D were not detected in this mutant. ADK9 does assemble near wild-type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6-dichloro-p-benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site-directed mutagenesis of the PS I core.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: identification of the sites of photodamage.

Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P680+ were made of NH2OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D.J., & Cheniae, G.M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K3Fe(CN)6, gave evidence for rapid (t1/2 = 2-3 min) and slow (t1/2 = 3-4) losses of formation of the tyrosyl radicals YZ+ and YD+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 degrees C in subsequent darkness in rather constant abundance (approximately 1/2 spin per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH2OH greater than or equal to NH2NH2 greater than DPC much greater than Mn2+) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P680+, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P680, via pheophytin (Pheo), to the first stable electron acceptor, QA. However, a 4-microsecond decay of P680+, reflecting YZ----P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140 microsecond decay, presumably reflecting QA-/P680+ back-reaction. Photoinhibition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O2 evolution is impaired is Chl/Car greater than YZ greater than YD much greater than P680, Pheo, QA.     

The YF161D1 mutant of Synechocystis 6803 exhibits an EPR signal from a light-induced photosystem II radical.

The currently accepted model for the location of the redox-active tyrosines, D and Z, in photosystem II suggests that they are symmetrically located on the D1 and D2 polypeptides, which are believed to form the heterodimer core of the reaction center. Z, the electron conduit from the manganese catalytic site to the primary chlorophyll donor, has been identified with tyrosine-161 of D1. The YF161D1 mutant of Synechocystis 6803 [Debus, R. J., Barry, B. A., Sithole, I., Babcock, G. T., & McIntosh, L. (1988b) Biochemistry 27, 9071-9074; Metz, J. G., Nixon, P. J., Rogner, M., Brudvig, G. W., & Diner, B. A. (1989) Biochemistry 28, 6960-6969], in which this tyrosine has been changed to a phenylalanine, should have no light-induced EPR (electron paramagnetic resonance) signal from a tyrosine radical. This negative result has indeed been obtained in analysis of one of two independently constructed mutants through the use of a non-oxygen-evolving core preparation (Metz et al., 1989). Here, we present an analysis of a YF161D1 mutant through the use of a photosystem II purification procedure that gives oxygen-evolving particles from wild-type Synechocystis cultures. In our mutant preparation, a light-induced EPR signal from a photosystem II radical is observed under conditions in which, in a wild-type preparation, we can accumulate an EPR signal from Z+. This EPR signal has a different lineshape from that of the Z+ tyrosine radical, and spin quantitation shows that this radical can be produced in up to 60% of the mutant reaction centers. The EPR lineshape of this radical suggests that photosystem II reaction centers of the YF161D1 mutant contain a redox-active amino acid.     

P680(+)* reduction kinetics and redox transition probability of the water oxidizing complex as a function of pH and H/D isotope exchange in spinach thylakoids.

The rise of fluorescence as an indicator for P680(+)* reduction by YZ and the period-four oscillation of oxygen yield induced by a train of saturating flashes were measured in dark-adapted thylakoids as a function of pH in the absence of exogenous electron acceptors. The results reveal that: (i) the average amplitude of the nanosecond kinetics and the average of the maximum fluorescence attained at 100 micros after the flash in the acidic range decrease with decreasing pH; (ii) the oxygen yield exhibits a pronounced period-four oscillation at pH 6.5 and higher damping at both pH 5.0 and pH 8.0; (iii) the probability of misses in the Si-state transitions of the water oxidizing complex is affected characteristically when exchangeable protons are replaced by deuterons [at pH <6.5, the ratio alpha(D)/alpha(H) is larger than 1 whereas at pH >7.0 values of <1 are observed]. The results are discussed within the framework of a combined mechanism for P680(+)* reduction where the nanosecond kinetics reflect an electron transfer coupled with a "rocket-type" proton shift within a hydrogen bridge from YZ to a nearby basic group, X [Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431], and subsequent relaxations within a network of hydrogen bonds. It is concluded that in the acidic region the hydrogen bond between YZ and X (most likely His 190 of polypeptide D1) is interrupted either by direct protonation of X or by conformational changes due to acid-induced Ca2+ release. This gives rise to a decreased P680(+)* reduction by nanosecond kinetics and an increase of dissipative P680(+)* recombination at low pH. A different mechanism is responsible for the almost invariant amplitude of nanosecond kinetics and increase of alpha in the alkaline region.     

Intramolecular translocation of the protein radical formed in the reaction of recombinant sperm whale myoglobin with H2O2.

A sperm whale myoglobin gene containing multiple unique restriction sites has been constructed in pUC 18 by sequential assembly of chemically synthesized oligonucleotide fragments. Expression of the gene in Escherichia coli DH5 alpha cells yields protein that is identical to native sperm whale myoglobin except that it retains the terminal methionine. Site-specific mutagenesis has been used to prepare all the possible tyrosine----phenylalanine mutants of the recombinant myoglobin, including the three single mutants at Tyr-103, -146, and -151, the three double mutants, and the triple mutant. All of the mutant proteins are stable except the Tyr-103 mutant. Introduction of a second mutation (Lys-102----Gln) stabilizes the Tyr-103 mutant. Absorption spectroscopy suggests that the active sites of the mutant proteins are intact. EPR and absorption spectroscopy show that all the proteins, including the triple mutant devoid of tyrosine residues, react with H2O2 to give a ferryl species and a protein radical. The presence of a protein radical in all the mutants suggests that the radical center is readily transferred from one amino acid to another. Cross-linking studies show, however, that protein dimers are only formed when Tyr-151 is present. Tyr-103, shown earlier to be the residue that primarily cross-links to Tyr-151 (Tew, D., and Ortiz de Montellano, P. R. (1988) J. Biol. Chem. 263, 17880-17886), is not essential for cross-linking. Electron transfer from Tyr-151 to the heme, which are 12 A apart, occurs in the absence of the intervening tyrosines at positions 103 and 146. The present studies show that the peroxide-generated myoglobin radical readily exchanges between remote loci, including non-tyrosine residues, but protein cross-linking only occurs when radical density is located on Tyr-151.     

Protein-tyrosyl radical interactions in photosystem II studied by electron spin resonance and electron nuclear double resonance spectroscopy: comparison with ribonucleotide reductase and in vitro tyrosine.

The stable tyrosine radical in photosystem II, YD*, has been studied by ESR and ENDOR spectroscopies to obtain proton hyperfine coupling constants from which the electron spin density distribution can be deduced. Simulations of six previously published ESR spectra of PSII (one at Q band; five at X band, of which two were after specific deuteration and two others were of oriented membranes) can be achieved by using a single set of magnetic parameters that includes anisotropic proton hyperfine tensors, an anisotropic g tensor, and noncoincident axis systems for the g and A tensors. From the spectral simulation of the oriented samples, the orientation of the phenol head group of YD* with respect to the membrane plane has been determined. A similar orientation for YZ*, the redox-active tyrosine in PSII that mediates electron transfer between P680 and the oxygen-evolving complex, is expected. ENDOR spectra of YD* in PSII preparations from spinach and Synechocystis support the set of hyperfine coupling constants but indicate that small differences between the two species exist. Comparison with the results of spectral simulations for tyrosyl radicals in ribonucleotide reductase from prokaryotes or eukaryotes and with in vitro radicals indicates that the spin density distribution remains that of an odd-alternant radical but that interactions with the protein can shift spin density within this basic pattern. The largest changes in spin density occur at the tyrosine phenol oxygen and at the ring carbon para to the oxygen, which indicates that mechanisms exist in the protein environment for fine-tuning the chemical and redox properties of the radical species.     

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.     

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.     

Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344.

The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.     

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680+ in PS II membrane fragments from spinach

The functional connection between redox component Y _z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680⁺ was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680⁺ reduction by Y _z under conditions where the catalytic site of water oxidation stays in the redox state S₁. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca²⁺. The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the Q_B-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680⁺ reduction by Y _z , and (c) Ca²⁺ could play a functional role for the electronic and vibrational coupling between the redox groups Y _z and P680. The electron transport from Y _z to P680⁺ is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - ANT 2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5 dinitrothiophene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2[N-Morpholino]ethanesulfonic acid - PS II photosystem II - Q_A, Q_B primary and secondary plastoquinone acceptor of photosystem II - S _i redox states of the catalytic site of water oxidation - Y _z redox active Tyr-161 of polypeptide D-1     

Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex.

To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.     

Aspartate 170 of the photosystem II reaction center polypeptide D1 is involved in the assembly of the oxygen-evolving manganese cluster.

Eleven site-directed mutations were constructed at aspartate 170 of the D1 polypeptide of the photosystem II (PSII) reaction center of the cyanobacterium Synechocystis sp. PCC 6803. The light-saturated rates of O2 evolution (VO2) measured in whole cells range from close to that of wild-type for Asp170Glu to zero for Asp170Ser and Ala. Those mutant strains that are best able to evolve O2 are also those that show the lowest Km in PSII core complexes for the oxidation of Mn2+ by oxidized Tyr161, the normal oxidant of the Mn cluster responsible for O2 evolution. To a first approximation, the lower the pKa of the residue at position 170, the higher the VO2 and the lower the Km. D1-Asp170 appears to participate in the early steps associated with the assembly of the Mn cluster. It is also the first reported example of an amino acid residue critical to the function and assembly of the oxygen-evolving complex.     

Perturbation of the structure of P680 and the charge distribution on its radical cation in isolated reaction center complexes of photosystem II as revealed by fourier transform infrared spectroscopy

The structure and the electronic properties of P680 and its radical cation in photosystem II (PSII) were studied by means of Fourier transform infrared spectroscopy (FTIR). Light-induced P680+/P680 FTIR difference spectra in the mid- and near-IR regions were measured using PSII membranes from spinach, core complexes from Thermosynechococcus elongatus, and reaction center (RC) complexes (D1-D2-Cytb559) from spinach. The spectral features of the former two preparations were very similar, indicating that the structures of P680 and its radical cation are virtually identical between membranes and cores and between plants and cyanobacteria. In sharp contrast, the spectrum of the RC complexes exhibited significantly different features. A positive doublet at approximately 1724 and approximately 1710 cm-1 due to the 131-keto C=O stretches of P680+ in the membrane and core preparations were changed to a prominent single peak at 1712 cm-1 in the RC complexes. This observation was interpreted to indicate that a positive charge on P680+ was extensively delocalized over the chlorophyll dimer in RC, whereas it was mostly localized on one chlorophyll molecule (70-80%) in intact P680. The significant change in the electronic structure of P680+ in RC was supported by a dramatic change in the characteristics of a broad intervalence band in the near-IR region and relatively large shifts of chlorin ring bands. It is proposed that the extensive charge delocalization in P680+ mainly causes the decrease in the redox potential of P680+/P680 in isolated RC complexes. This potential decrease explains the well-known phenomenon that YZ is not oxidized by P680+ in RC complexes.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P(680), and the first quinone electron acceptor, Q(A), were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S(2)Q(A)(-) and S(2)Q(B)(-) states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S(2)Q(A)(-) and S(2)Q(B)(-) states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q(A)/Q(A)(-) relative to that observed in the presence of DCMU, charge recombination from the S(2)Q(A)(-) state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P(680)(+)Phe(-) radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P(680)(*) from (1)[P(680)(+)Ph(-)] and direct recombination of the (3)[P(680)(+)Ph(-)] and (1)[P(680)(+)Ph(-)] radical states, respectively. An additional non-radiative pathway involves direct recombination of P(680)(+)Q(A)(-). The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of DeltaG(P(680)(*)<-->P(680)(+)Phe(-)) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of DeltaG(P(680)(+)Phe(-)<-->P(680)(+)Q(A)(-)) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Influence of histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the PD1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA1 and psbA2 genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT*) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA3 gene. The O2-evolving activity of His-tagged PSII isolated from WT* was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA1 is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT*. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P680, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to PD1 is not an important determinant of the redox and spectroscopic properties of P680 in T. elongatus.     

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.     

Histidine residue 252 of the Photosystem II D1 polypeptide is involved in a light-induced cross-linking of the polypeptide with the alpha subunit of cytochrome b-559: study of a site-directed mutant of Synechocystis PCC 6803

Properties of the Photosystem II (PSII) complex were examined in the wild-type (control) strain of the cyanobacterium Synechocystis PCC 6803 and its site-directed mutant D1-His252Leu in which the histidine residue 252 of the D1 polypeptide was replaced by leucine. This mutation caused a severe blockage of electron transfer between the PSII electron acceptors Q(A) and Q(B) and largely inhibited PSII oxygen evolving activity. Strong illumination induced formation of a D1-cytochrome b-559 adduct in isolated, detergent-solubilized thylakoid membranes from the control but not the mutant strain. The light-induced generation of the adduct was suppressed after prior modification of thylakoid proteins either with the histidine modifier platinum-terpyridine-chloride or with primary amino group modifiers. Anaerobic conditions and the presence of radical scavengers also inhibited the appearance of the adduct. The data suggest that the D1-cytochrome adduct is the product of a reaction between the oxidized residue His(252) of the D1 polypeptide and the N-terminal amino group of the cytochrome alpha subunit. As the rate of the D1 degradation in the control and mutant strains is similar, formation of the adduct does not seem to represent a required intermediary step in the D1 degradation pathway.