Tryptophan at position 181 of the D2 protein of photosystem II confers quenching of variable fluorescence of chlorophyll: implications for the mechanism of energy-dependent quenching.

The lumenal CD loop region of the D2 protein of photosystem II contains residues that interact with a reaction center chlorophyll and the redox-active Tyr(D). Using combinatorial mutagenesis, photoautotrophic mutants of Synechocystis sp. PCC 6803 have been generated with multiple amino acid changes in this region. The CD loop mutations were transferred into a photosystem I-less Synechocystis strain to facilitate characterization of photosystem II properties in the mutants. Most of the combinatorial photosystem I-less mutants obtained had a high yield of variable fluorescence, F(V). However, in three mutants, which shared a replacement of Phe181 by Trp, the F(V) yield was dramatically reduced although a high rate of oxygen evolution was maintained. A site-directed F181W D2 mutant shared similar properties. Picosecond time-resolved fluorescence measurements revealed that in the combinatorial F181W mutants the fluorescence lifetimes in closed and open photosystem II centers were essentially identical and were similar to the fluorescence lifetime in open centers of the control strain. These results are explained by quenching of variable fluorescence in the mutants by charge separation between Trp181 and excited reaction center chlorophyll. This reaction competes efficiently with fluorescence and nonradiative decay in closed photosystem II centers, where the lifetime of the excitation in the chlorophyll antenna is long. Thermodynamic considerations favor the formation of oxidized tryptophan and reduced chlorophyll in the quenching reaction, presumably followed by charge recombination. A possible role of tryptophan-chlorophyll charge separation in the mechanism of energy-dependent quenching of excitations in photosynthesis is discussed.     

Random chemical mutagenesis of a specific psbDI region coding for a lumenal loop of the D2 protein of photosystem II in Synechocystis sp. PCC 6803

To identify amino acid residues of the D2 protein that are critical for functional photosystem II (PS II), sodium bisulfite was utilized for in vitro random mutagenesis of the psbDI gene from Synechocystis sp. PCC 6803. Sodium bisulfite reacts specifically with cytosine in single-stranded regions of DNA and does not attack double-stranded DNA. Using a hybrid plasmid that was single-stranded in the region to be mutagenized and that was double-stranded elsewhere, mutations were targeted to a specific psbDI region coding for the lumenal A-B loop of the D2 protein. Several mutants were isolated with a total of 15 different amino acid changes in the loop. The majority of these mutations did not result in a loss of photoautotrophic growth or in significantly altered PS II function. However, mutation of Glu-69 to Lys, Ser-79 to Phe, and Ser-88 to Phe were found to influence photosystem II activity; the importance of the latter two residues for proper PS II function was unexpected. Cells carrying the double mutation S79F/S88F in D2 did not grow photoautotrophically and had no functionally active PS II centers. The single mutant S79F was also incapable of photoautrophic growth, but displayed reasonably stable oxygen evolution, while PS II function in the single mutant S88F appeared to be close to normal. Because of the more pronounced phenotype of the S79F/S88F strain as compared to the single mutants, both Ser residues appear to affect stable assembly and function of the PS II complex. The mechanism by which the S79F mutant loses photoautotrophic growth remains to be established. However, these results show the potential of targeted random mutagenesis to identify functionally important residues in selected regions of proteins.     

Photosystem II function and integrity in spite of drastic protein changes in a conserved region of the D2 protein.

D1 and D2 are structurally related proteins forming the core of the photosystem II reaction center. The two proteins have several loop regions including an extended stroma-exposed loop between transmembrane helix D and parallel helix de. This loop (the D-de loop) is phylogenetically conserved in both proteins. The role of the D-de loop in photosystem II was studied in Synechocystis sp. PCC 6803 by constructing a chimeric D2 protein in which the stroma-exposed loop of D1 replaced that of D2. In one of the transgenic lines, a single-base deletion shifted the reading frame of the chimeric gene leading to loss of D2 accumulation and photosystem II assembly. Selection for spontaneous reversion to photoautotrophy yielded several suppressor mutants, five of which were analyzed. In all, further frameshifts in the inserted loop piece restored the original reading frame allowing readthrough to the normal carboxy terminus. However, the sequences in the restored D-de loop varied widely among the mutants. Changes ranged from a deletion of one amino acid residue to an insertion of 31, while the net charge of the D-de loop increased by up to 12 units. Mutant electron transfer rates and photoautotrophic growth were only mildly affected as compared to wild type. Nevertheless, in all mutants, the hydropathy profile of the stroma-exposed D-de loop region maintained its hydrophilic character including turns in similar locations. We conclude that the stroma-exposed, D-de loop of the D2 protein can accommodate drastic composition and size changes without extensive functional consequences in photosystem II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of manganese stabilizing protein to photosystem II: identification of essential N-terminal threonine residues and domains that prevent nonspecific binding

The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, (4)KRLTYD(10)E and (15)TYL(18)E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038-10045]. To better understand the basis of MSP-photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized. Deletion mutants lacking the first 5, 6, 7, and 15 amino acid residues at the N-terminus of the protein were examined for their ability to reconstitute activity in MSP-depleted photosystem II. The results reported here show that truncations of five and six amino acid residues (mutants DeltaR5M and DeltaL6M mutants) have no negative effect on recovery of oxygen evolution activity or on binding of MSP to photosystem II. Deletion of seven residues (mutant DeltaT7M) decreases reconstitution activity to 40% of the control value and reduces functional binding of the mutant protein to photosystem II from two to one copy. Deletion of 15 amino acid residues (mutant DeltaT15M) severely impairs functional binding of MSP, and lowers O(2) evolution activity to less than 20% of the control. DeltaT7M is the only mutant that exhibited neither nonspecific binding to photosystem II nor changes in tertiary structure. These, and previous results, show that the highly conserved Thr7 and Thr15 residues of MSP are required for functional binding of two copies of the eukaryotic protein to photosystem II. Although the N-terminal domains, (1)EGGKR(6)L, (8)YDEIQS(14)K, and (16)YL(18)E of spinach MSP are unnecessary for specific, functional binding interactions, these sequences are necessary to prevent nonspecific binding of the protein to photosystem II.     

Site-directed mutagenesis of the CP 47 protein of photosystem II: alteration of conserved charged residues which lie within lethal deletions of the large extrinsic loop E

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II which functions in both light-harvesting and oxygen evolution. The large extrinsic loop E of this protein has been shown to interact with the oxygen-evolving site. Previously, Vermaas and coworkers have produced a number of deletions within loop E which yielded mutants which were unable to grow photoautotrophically and which could not evolve oxygen at normal rates. During the course of our site-directed mutagenesis program in Synechocystis 6803, we have altered all of the conserved charged residues which were present within six of these deletions. All ten of these mutants were photoautotrophic and evolved oxygen at normal rates. We speculate that the severe phenotypes of the deletion mutants observed by Vermaas and coworkers in due to large structural perturbations in the extrinsic loop E of CP 47.     

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSII electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSII electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, QB of PSII, provide a strong basis of support for structural and functional models of the PSII reaction center.     

In vitro random mutagenesis of the D1 protein of the photosystem II reaction center confers phototolerance on the cyanobacterium Synechocystis sp. PCC 6803.

The D1 protein of the photosystem II reaction center is thought to be the most light-sensitive component of the photosynthetic machinery. To understand the mechanisms underlying the light sensitivity of D1, we performed in vitro random mutagenesis of the psbA gene that codes for D1, transformed the unicellular cyanobacterium Synechocystis sp. PCC 6803 with mutated psbA, and selected phototolerant transformants that did not bleach in high intensity light. A region of psbA2 coding for 178 amino acids of the carboxyl-terminal portion of the peptide was subjected to random mutagenesis by low fidelity polymerase chain reaction amplification or by hydroxylamine treatment. This region contains the binding sites for Q(B), D2 (through Fe), and P680. Eighteen phototolerant mutants with single and multiple amino acid substitutions were selected from a half million transformants exposed to white light at 320 micromol m(-2) s(-1). A strain transformed with non-mutagenized psbA2 became bleached under the same conditions. Site-directed mutagenesis has confirmed that one or more substitutions of amino acids at residues 234, 254, 260, 267, 322, 326, and 328 confers phototolerance. The rate of degradation of D1 protein was not appreciably affected by the mutations. Reduced bleaching of mutant cyanobacterial cells may result from continued buildup of photosynthetic pigment systems caused by changes in redox signals originating from D1.     

Postbinding events mediated by human immunodeficiency virus type 1 are sensitive to modifications in the D4-transmembrane linker region of CD4.

Evidence from both structural and functional studies of the CD4 molecule suggests that several domains, including the transmembrane (TM) domain and the adjoining extracellular region (D4-TM linker), contribute to the post-gp12O-binding events leading to human immunodeficiency virus-mediated membrane fusion. To investigate such a role in syncytium formation and cell-free infectivity, we generated several deletion and substitution mutations in the TM and D4-TM linker regions of the CD4 molecule. We found that while the TM domain of CD4 was dispensable for cell-cell and virus-cell interactions, modifications in the D4-TM linker led to perturbations in both processes. Deletion of the five amino acid residues linking D4 to the TM domain resulted in a delayed and reduced capacity to form syncytia, whereas replacement of the residues with the heterologous sequence from the CD8 molecule restored the kinetic profile to wild-type CD4 levels. On the other hand, both mutants of the CD4 D4-TM linker demonstrated delayed cell-free human immunodeficiency virus type 1 infectivity profiles. The defective fusion capacity may be linked to structural perturbations identified with anti-CD4 monoclonal antibodies in the D1-D2 interface and D3 domain of the deletion mutant yet absent in D1 and D4. While all cells were found to bind comparable levels of gp120, both D4-TM linker mutants appeared to induce a decrease in the V3 loop exposure of bound gp120. This underexposure may explain the delays in cell-free infectivities observed for both of these mutants. Together, these findings confirm a role for regions of the CD4 molecule located outside D1 in post-gp120-binding events and suggest that the D4-TM interface contributes to the conformational changes that direct the fusion process.     

Structural requirements for N-trimethylation of lysine 115 of calmodulin

Calmodulin is trimethylated at lysine 115 by a highly specific methyltransferase that utilizes S-adenosylmethionine as a co-substrate. Lysine 115 is found within a highly conserved six-amino acid loop (LGEKLT) that forms a 90 degrees turn between EF-hand III and EF-hand IV in the carboxyl-terminal lobe. In the present work a mutagenesis approach was used to investigate the structural features of the carboxyl-terminal lobe that lead to the specificity of calmodulin methylation. Three structural regions within the carboxyl-terminal lobe appear to be involved in methyltransferase recognition: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent alpha-helices (helix 6 and helix 7) from EF-hands III and IV. Site-directed mutagenesis of residues in the loop show that three residues, glycine 113, glutamate 114, and leucine 116 are essential for methylation. In addition, subdomain (individual helix or Ca(2+) binding loop) exchange mutants show that the substitutions of either helix 6 (EF-hand III) with helix 2 (EF-hand I) or helix 7 (EF-hand IV) with helix 3 (EF-hand II) compromises methylation. Charge-to-alanine mutations in helix 7 show that substitution of conserved charged residues at positions 118, 120, 122, 126, and 127 reduced lysine 115 methylation rates, suggesting possible electrostatic interactions between this helix and the methyltransferase. Single substitutions in helix 6 did not affect calmodulin methylation, suggesting this region may play a more indirect role in stabilizing the conformation of the methyltransferase recognition sequence.     

A novel protein involved in the functional assembly of the oxygen-evolving complex of photosystem II in Synechocystis sp. PCC 6803

Mutation of Glu69 to Gln in the D2 protein of photosystem II is known to lead to a loss of photoautotrophic growth in Synechocystis sp. PCC 6803. However, second-site mutants (pseudorevertants) with restored photoautotrophic growth but still maintaining the E69Q mutation in D2 are easily obtained. Using a genomic mapping technique involving functional complementation, the secondary mutation was mapped to slr0286 in two independent mutants. The mutations in Slr0286 were R42M or R394H. To study the function of Slr0286, mutants of E69Q and of the wild-type strain were made that lacked slr0286. Deletion of slr0286 did not affect photoautotrophic capacity in wild type but led to a marked decrease in the apparent affinity of Ca(2+) to its binding site at the water-splitting system of photosystem II and to a reduced heat tolerance of the oxygen-evolving system, particularly in E69Q. Moreover, a small increase in the half-time for photoactivation of the oxygen-evolving complex of photosystem II for both wild type and the E69Q mutant was observed in the absence of Slr0286. The accumulation of photosystem II reaction centers, dark stability of the oxygen-evolving apparatus, stability of oxygen evolution, and the kinetics of charge recombination between Q(A)(-) and the donor side were not affected by deletion of slr0286. Slr0286 lacks clear functional motifs, and no homologues are apparent in other organisms, even not in other cyanobacteria. In any case, Slr0286 appears to help the functional assembly and stability of the water-splitting system of photosystem II.     

Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis

Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the alpha3beta1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with alpha3beta1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu(149) and Glu(213)). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151-CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin-tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151-alpha3beta1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151-alpha3beta1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein.     

Three-Dimensional Analysis of CD6 Site-Directed Mutagenesis and Monoclonal Antibody Binding Studies Using the X-ray Structure of Mac-2 Binding Protein and a Molecular Model of the CD6 Ligand Binding Domain

The extracellular region of CD6 consists of three scavenger receptor cysteine-rich (SRCR) domains and binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). Residues important for the CD6-ALCAM interaction have previously been identified by mutagenesis. A total of 22 CD6 residues were classified according to their importance for anti-CD6 monoclonal antibody (mAb) and/or ALCAM binding. The three-dimensional structure of the SRCR domain of Mac-2 binding protein has recently been determined, providing a structural prototype for the SRCR protein superfamily. This has made a thorough three-dimensional analysis of CD6 mutagenesis and mAb binding experiments possible. Mutation of buried residues compromised both mAb and ALCAM binding, consistent with the presence of structural perturbations. However, several residues whose mutation affected both mAb and ALCAM binding or, alternatively, only ligand binding were found to map to the surface in the same region of the domain. This suggests that the CD6 ligand binding site and epitopes of tested mAbs overlap and provides an explanation for the finding that these mAbs effectively block ALCAM binding. An approximate molecular model of CD6 was used to delineate the ALCAM binding site.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089490050263Abbreviations ALCAM activated leukocyte cell adhesion molecule - CD6D3 third (membrane-proxi-mal) extracellular domain of CD6 - IgSF immunoglobulin superfamily - mAb monoclonal antibody - M2BP Mac-2 binding protein - SRCR scavenger receptor cysteine-rich domain - SRCRSF scavenger receptor cysteine-rich protein superfamily     

The conformational change of the protease inhibitor α2-macroglobulin is triggered by the retraction of the cleaved bait region from a central channel

The protease inhibitor α₂-macroglobulin (A2M) is a member of the ancient α₂-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as complement factor C3. A2M and other A2MF proteins undergo an extensive conformational change upon cleavage of their bait region by proteases. However, the mechanism whereby cleavage triggers the change has not yet been determined. We have previously shown that A2M remains functional after completely replacing its bait region with glycine and serine residues. Here, we use this tabula rasa bait region to investigate several hypotheses for the triggering mechanism. When tabula rasa bait regions containing disulfide loops were elongated by reducing the disulfides, we found that A2M remained in its native conformation. In addition, cleavage within a disulfide loop did not trigger the conformational change until after the disulfide was reduced, indicating that the introduction of discontinuity into the bait region is essential to the trigger. Previously, A2MF structures have shown that the C-terminal end of the bait region (a.k.a. the N-terminal region of the truncated α chain) threads through a central channel in native A2MF proteins. Bait region cleavage abolishes this plug-in-channel arrangement, as the bait region retracts from the channel and the channel itself collapses. We found that mutagenesis of conserved plug-in-channel residues disrupted the formation of native A2M. These results provide experimental evidence for a structural hypothesis in which retraction of the bait region from this channel following cleavage and the channel’s subsequent collapse triggers the conformational change of A2M and other A2MF proteins.     

Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors.

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sect. D 52:93-104, 1996), which includes the site of PII modification, was not required for the binding of small-molecule effectors but was necessary for the interaction with all three receptors. Mutations altering residues of this loop or affecting the nearby B loop of PII, which line a cleft between monomers in the trimeric PII, affected the interactions with protein receptors and the binding of small-molecule ligands. Thus, our results support the predictions made from structural studies that the exposed loops of PII and cleft formed at their interface are the sites of regulatory interactions.     

Modulating the redox potential of the stable electron acceptor, Q(B), in mutagenized photosystem II reaction centers

One of the unique features of electron transfer processes in photosystem II (PSII) reaction centers (RC) is the exclusive transfer of electrons down only one of the two parallel cofactor branches. In contrast to the RC core polypeptides (psaA and psaB) of photosystem I (PSI), where electron transfer occurs down both parallel redox-active cofactor branches, there is greater protein-cofactor asymmetry between the PSII RC core polypeptides (D1 and D2). We have focused on the identification of protein-cofactor relationships that determine the branch along which primary charge separation occurs (P(680)(+)/pheophytin(-)(Pheo)). We have previously shown that mutagenesis of the strong hydrogen-bonding residue, D1-E130, to less polar residues (D1-E130Q,H,L) shifted the midpoint potential of the Pheo(D1)/Pheo(D1)(-) couple to more negative values, reducing the quantum yield of primary charge separation. We did not observe, however, electron transfer down the inactive branch in D1-E130 mutants. The protein residue corresponding to D1-E130 on the inactive branch is D2-Q129 which presumably has a reduced hydrogen-bonding interaction with Pheo(D2) relative to the D1-E130 residue with Pheo(D1). Analysis of the recent 2.9 ? cyanobacterial PSII crystal structure indicated, however, that the D2-Q129 residue was too distant from the Pheo(D2) headgroup to serve as a possible hydrogen bond donor and directly impact its midpoint potential as well as potentially determine the directionality of electron transfer. Our objective was to characterize the function of this highly conserved inactive branch residue by replacing it with a nonconservative leucine or a conservative histidine residue. Measurements of Chl fluorescence decay kinetics and thermoluminescence studies indicate that the mutagenesis of D2-Q129 decreases the redox gap between Q(A) and Q(B) due to a lowering of the redox potential of Q(B). The resulting increased yield of S(2)Q(B)(-) charge recombination in the D2-Q129 mutants leads to an increased susceptibility to photoinhibitory light presumably due to (3)P(680)-mediated oxidative damage. The results indicate that the D2-Q129 residue plays a critical role in stabilizing the charge-separated state in PSII and further documents the structural and functional asymmetry between the two cofactor branches in PSII.     

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.     

Site-directed mutagenesis of the CPa-1 protein of photosystem II: alteration of the basic residue pair 384,385R to 384,385G leads to a defect associated with the oxygen-evolving complex.

The psbB gene encodes the intrinsic chlorophyll-a binding protein CPa-1 (CP-47), a component of photosystem II in higher plants, algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into a segment of the psbB gene encoding the large extrinsic loop region of CPa-1 in the cyanobacterium Synechocystis sp. PCC 6803. Altered psbB genes were introduced into a mutant recipient strain (DEL-1) of Synechocystis in which the genomic psbB gene had been partially deleted. Initial target sites for mutagenesis were absolutely conserved basic residue pairs occurring within the large extrinsic loop. One mutation, RR384385GG, produced a strain with impaired photosystem II activity. This strain exhibited growth characteristics comparable to controls. However, at saturating light intensities this mutant strain evolved oxygen at only 50% of the rate of the control strains. Quantum yield measurements at low light intensities indicated that the mutant had 30% fewer fully functional photosystem II centers than do control strains of Synechocystis. Immunological analysis of a number of photosystem II protein components indicated that the mutant accumulates normal quantities of photosystem II proteins and that the ratio of photosystem II to photosystem I proteins is comparable to that found in control strains. Upon exposure to high light intensities the mutant cells exhibited a markedly increased susceptibility to photoinactivation. However, Tris-treated thylakoid membranes from both the mutant and wild-type exhibited comparable rates of photoinactivation. Thylakoid membranes isolated from RR384385GG exhibited only 15% of the H2O to 2,6-dichlorophenolindophenol electron transport rate observed in wild-type strains.(ABSTRACT TRUNCATED AT 250 WORDS)