Site-directed mutations were introduced to replace D1-His198 and D2-His197 of the D1 and D2 polypeptides, respectively, of the photosystem II (PSII) reaction center of Synechocystis PCC 6803. These residues coordinate chlorophylls P A and P B which are homologous to the special pair Bchlorophylls of the bacterial reaction centers that are coordinated respectively by histidines L-173 and M-200 (202). P A and P B together serve as the primary electron donor, P, in purple bacterial reaction centers. In PS II, the site-directed mutations at D1 His198 affect the P + -P-absorbance difference spectrum. The bleaching maximum in the Soret region (in WT at 433 nm) is blue-shifted by as much as 3 nm. In the D1 His198Gln mutant, a similar displacement to the blue is observed for the bleaching maximum in the Q y region (672.5 nm in WT at 80 K), whereas features attributed to a band shift centered at 681 nm are not altered. In the Y Z •-Y Z -difference spectrum, the band shift of a reaction center chlorophyll centered in WT at 433-434 nm is shifted by 2-3 nm to the blue in the D1-His198Gln mutant. The D1-His198Gln mutation has little effect on the optical difference spectrum, 3 P-1 P, of the reaction center triplet formed by P + Pheo -charge recombination (bleaching at 681-684 nm), measured at 5-80 K, but becomes visible as a pronounced shoulder at 669 nm at temperatures g150 K. Measurements of the kinetics of oxidized donor-Q A -charge recombination and of the reduction of P + by redox active tyrosine, Y Z , indicate that the reduction potential of the redox couple P + /P can be appreciably modulated both positively and negatively by ligand replacement at D1-198 but somewhat less so at D2-197. On the basis of these observations and others in the literature, we propose that the monomeric accessory chlorophyll, B A , is a long-wavelength trap located at 684 nm at 5 K. B A * initiates primary charge separation at low temperature, a function that is increasingly shared with P A * in an activated process as the temperature rises. Charge separation from B A * would be potentially very fast and form P A + B A -and/or B A + Pheo -as observed in bacterial reaction centers upon direct excitation of B A ) Proc. Natl. Acad Sci. 96, 2054-2059. The cation, generated upon primary charge separation in PSII, is stabilized at all temperatures primarily on P A , the absorbance spectrum of which is displaced to the blue by the mutations. In WT, the cation is proposed to be shared to a minor extent (∼20%) with P B , the contribution of which can be modulated up or down by mutation. The band shift at 681 nm, observed in the P + -P difference spectrum, is attributed to an electrochromic effect of P A + on neighboring B A . Because of its low-energy singlet and therefore triplet state, the reaction center triplet state is stabilized on B A at e80 K but can be shared with P A at >80 K in a thermally activated process.

The reaction center of photosystem II of oxygenic photosynthesis contains two redox-active tyrosines called Z and D, each of which can act as an electron donor to the oxidized primary electron donor, P680+. These tyrosines are located in homologous positions on the third transmembrane alpha-helix of each of the two homologous polypeptides, D1 and D2, that comprise the reaction center. Tyrosine D of polypeptide D2 has been proposed, upon oxidation, to give up its phenolic proton to a nearby basic amino acid residue, forming a neutral radical. Modeling studies have pointed to His190 (spinach numbering) as a likely candidate for this basic residue. As a test of this hypothesis, we have constructed three site-directed mutations in the D2 polypeptide of the cyanobacterium Synechocystis sp. PCC6803. His189 (the Synechocystis homologue of His190 of spinach) has been replaced by glutamine, aspartate, or leucine. Instead of the normal D. EPR signal (g = 2.0046; line width 16-19 G), PSII core complexes isolated from these three mutants show an altered dark-stable EPR signal with a narrowed line width (11-13 G), and g values of 2.0046, 2.0043, and 2.0042 for the His189Gln, His189Asp, and His189Leu mutants, respectively. Despite the reduced line width, these EPR signals show g values and microwave-power saturation properties similar to the normal D. signal. Furthermore, specific deuteration in one of those mutants at the 3 and 5 positions of the phenol ring of the photosystem II reaction center tyrosines results in a loss of hyperfine structure of the EPR signal, proving that the signal indeed arises from tyrosine.2+ This observation provides support for a model in which an imidazole nitrogen of His189 accepts the phenolic proton of Tyr160 upon oxidation of D, forming a back hydrogen bond to the phenolic oxygen of the neutral tyrosyl radical.

The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ. has been hampered by the simultaneous presence of the much more stable YD., the short lifetime of YZ., and the difficulty in trapping the YZ. radical at low temperature. We present here a method for obtaining an uncontaminated YZ. radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the beta-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ. EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ. radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ., this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ.. We have compared the environments of YZ. and YD. by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ. and YD. differ in line shape and line width. (2) Both YZ. and YD. exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD., indicating the hydrogen bond to be well-defined. In the case of YZ. it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD. possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 A. (4) All of the 1H couplings of YD. are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ. spectrum are broad. The environment surrounding YZ. appears to be more disordered and solvent-accessible.

Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.

We report here the first three-dimensional structure of the D1 C-terminal processing protease (D1P), which is encoded by the ctpA gene. This enzyme removes the C-terminal extension of the D1 polypeptide of photosystem II of oxygenic photosynthesis. Proteolytic processing is necessary to allow the light driven assembly of the tetranuclear manganese cluster, which is responsible for photosynthetic water oxidation. The X-ray structure of the Scenedesmus obliquus enzyme has been determined at 1.8 A resolution using the multiwavelength anomalous dispersion method. The enzyme is monomeric and is composed of three folding domains. The middle domain is topologically homologous to known PDZ motifs and is proposed to be the site at which the substrate C-terminus binds. The remainder of the substrate likely extends across the face of the enzyme, interacting at its scissile bond with the enzyme active site Ser 372 / Lys 397 catalytic dyad, which lies at the center of the protein at the interface of the three domains.

Two mutants of Synechocystis PCC 6803 lacking the psbC gene product CP43 were constructed by site-directed mutagenesis. Analysis of cells and thylakoid membranes of these mutants indicates that PS II reaction centers accumulate to a concentration of about 10% of that of WT cells. PS II core complexes isolated from mutants lacking the CP43 subunit show light-driven electron transfer from the secondary electron donor Z to the primary quinone electron acceptor QA with a quantum yield similar to that of wild type, indicating that CP43 is not required for binding or function of QA. The use of mutants for the removal of CP43 thus avoids the loss of QA function associated with biochemical extraction of CP43 from intact core complexes. Both absorbance and fluorescence emission maxima of the mutant complexes show a blue shift in comparison to the WT PS II core complex, indicating that the absorbance spectrum of CP43 is red-shifted relative to that of the remainder of the core complex. The antenna size of these CP43-less complexes is about 70% of that of WT, indicating that approximately 15 chlorophyll molecules are bound by CP43. The molecular mass of the PS II complex, including the detergent shell, shifts from 310 +/- 15 kDa in WT to 285 +/- 15 kDa in the CP43-less mutants.

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Polypeptide D1 of the photosystem II reaction center of oxygenic photosynthesis is expressed in precursor form (pre-D1), and it must be proteolytically processed at its C terminus to enable assembly of the manganese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtiter plate method is described for assaying this D1 C-terminal processing protease. A protocol is described for the isolation and purification to homogeneity of the enzyme from the green alga, Scenedesmus obliquus. Amino acid sequence information on the purified protease was used to clone the corresponding gene, the translated sequence of which is presented. A comparison of the gene product with homologous proteases points to a region of conserved residues that likely corresponds to the active site of a new class of serine protease. The LF-1 mutant strain of Scenedesmus (isolated by Dr. Norman Bishop) is incapable of processing pre-D1. We show here that the C-terminal processing protease gene in this strain contains a single base deletion that causes a frame shift and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophic and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations provide the first definitive proof that the enzyme isolated is responsible for in vivo proteolytic processing of pre-D1 and that no other protease can compensate for its loss.The photosystem II reaction center contains two homologous polypeptides, D1 and D2, that are responsible for the coordination of the primary photoreactants (1, 2). D1 has also been shown to harbor the redox-active tyrosine, Tyr Z (3, 4), that links the oxidized primary electron donor of photosystem II to the oxygen-evolving manganese cluster and is thought to provide at least some of the coordinating ligands to this complex (5-8). D1 is expressed in precursor form (9 -11), inserted into the thylakoid membrane, and processed at its C terminus (6, 12-15) by a proteolytic enzyme, D1 C-terminal processing protease (Ctp-protease).1 In cyanobacteria, 16 residues are cleaved from precursor D1 (6), 9 in higher plants (15, 16), and 8 in Chlamydomonas 2 with processing occurring in all cases at the carboxyl side of D1-Ala-344. Euglena is the sole oxygenic photosynthetic organism that is believed not to undergo C-terminal processing (1), although, in this case, no C-terminal extension is present. Based on a comparison of 43 psbA genes, the amino acid sequence on the amino side of the processing site is strictly conserved within the 8 residues immediately upstream of the processing site (1). On the carboxyl side of the processing site, no residue is conserved throughout all 43 sequences. While position 345 is normally occupied by either an alanine or a serine, replacement of Ser-345 in Synechocystis 6803 with arginine or alanine (6) or in Chlamydomonas with glycine, cysteine, valine,...