Structure and Function in Photosystem II


Oxygenic photosynthesis occurs in cyanobacteria and green plants, and requires photosystem (PS) II to catalyze the photo-oxidation of water. Most previous molecular engineering work on PS II has been done with the cyanobacterium Synechocystis sp. PCC 6803, because of the well developed molecular genetics technology. However, our recent development of a streamlined system for manipulation of the psbA gene encoding the D1 peptide of PS II of Chlamydomonas reinhardtii makes this an attractive alternative. In contrast to cyanobacteria, this unicellular alga has a chloroplast with all the main characteristics of higher plants, including the same PS II, light harvesting apparatus, and oxygen evolving complex. Cells deficient in photosynthesis can be readily grown on acetate. Biophysical methods based on fluorescence and spectrophotometry are easier to use than with cyanobacteria because PS II is expressed at higher level, and has a chlorophyll (Chl) light harvesting apparatus, allowing techniques developed using chloroplasts to be applied directly to intact cells. We are using molecular engineering and biophysical assay to explore the relation between structure and function in PS II in C. reinhardtii.

Structure of Photosystem II

Biochemical and biophysical evidence has shown that PS II spans the chloroplast membrane, with a water oxidizing site on the lumenal side, a plastoquinone reductase site on the stromal side, and a set of chlorophyll and pheophytin chromophores catalyzing the photochemical reaction which separates charge between these sites. The atomic structure of PS II is not known. However the weak sequence homology of PS II and bacterial reaction centers, along with much biochemical evidence, has been taken to indicated common features in the basic structure and mechanism. In our first models for the core of PS II (1-3) we used the solved structure of a bacterial reaction center (4) as a template, using residue substitution, and manual construction based on structural prediction to accommodate differences (such as extra loops) apparent from homologous alignment. Other models (5, 6) have incorporated inter-helical loops constructed by use of template matching from known structures, energy minimization and simulated annealing. More recent models of photosystem II have included a putative Mn-cluster and bicarbonate group. Others labs have concentrated on modeling the binding pockets for primary and secondary plastoquinone acceptors (QA and QB respectively) (7). There are substantial differences in structure between models in regions of mechanistic interest, reflecting the lack of experimental constraints.

Click here for a Chime tutorial on a model of the Photosytem II core from Jonathan Nugent and colleagues.

Nature of the prosthetic groups

Biochemical evidence suggests an overall structure and complement of chromophores similar to that of the bacterial reaction centers (see 8-10 for recent reviews). On the acceptor side, the basic structure, with binding sites for QA and QB spaced by an Fe ligated by 4 His, seems well established, as does the probability that E130 in D1 provides a ligand to pheophytin. The histidine ligands to the ancillary BChl in the bacterial centers are not conserved in D1 or D2, leaving the Chl ligands uncertain. The lumenal side is even more problematical, since it is here that the major differences from the bacterial reaction center are found. The nature of P680 (the primary donor Chl) is still controversial. The dimeric special pair expected from bacterial centers has not been unambiguously demonstrated, although H198 of D1, and the equivalent in D2, are conserved as putative ligands, and the near IR band of P680+ is diagnostic. Several suggestions have been made that P680 might be monomeric (8-10), and more equivalent to the ancillary Chls of the bacterial reaction center. Important roles for Tyr-161 (D1) as donor to P680+, and for Tyr-160 (D2) as an ancillary donor, have been established (11-15). A Mn-cluster catalyzes O2-evolution (see below).

(PS II chromophores).

Mechanism of plastoquinone reduction

Kinetic measurements and studies of herbicide resistance lesions have suggested that the two-electron gate (see 16 for review of earlier work, 23-28) has the same general mechanism and basic structure in PS II and photosynthetic bacteria. In green plants, the kinetics of the individual partial reactions are well resolved by the fluorescence methods introduced by this lab (29-33). We have studied the mechanism in higher plants and in C. reinhardtii, and have characterized mutations in both which give rise to herbicide resistance and modified function (31-33). The atomic structure has been resolved in bacterial reaction centers (4, 91,92), and the mechanism of inhibition suggested from kinetic studies has been confirmed by the observation that herbicides can replace the quinone substrate at the QB-site. The atomic structures provide no dynamic information, but much work has attempted to relate structure to function through studies of mutant strains (see 28 for recent review). With higher plants and C. reinhardtii, similar kinetic work has been mainly with mutants selected for herbicide resistance (24-26, 29-34).

Mechanism of oxygen evolution

Advances in the past decade have come from the development of methods for biochemical dissection and biophysical characterization of the oxygen evolving complex (see 10 for comprehensive review, 35-54). Three extrinsic oxygen evolution enhancing proteins (OEEP) have been identified, and their role in lowering the concentrations of Ca2+ and Cl- required for an active complex demonstrated, but although they are required for efficient reaction under physiological conditions, oxygen evolution can occur rapidly without them (35-40). The Mn-cluster is therefore assumed to require only ligands from the intrinsic proteins (D1, D2, CP43, CP47, two subunits of cytochrome b-559, and possibly the product of psbI) (10), and the entire electron transfer mechanism is demonstrated to reside in this complex.

The structure of the donor-side can be probed by a variety of spectroscopic methods (reviewed in 8-10, 41). The Mn cluster and its ligand environment have been extensively characterized through the EPR multiline, g=4.1 (S2-state) and g=4.8 (S1) signals (reviewed in 9, 41, see also 46-51). An anomalous EPR-signal observed when the normal transition to the S3-state is perturbed by Ca2+ or Cl- depletion has been attributed to an organic radical tentatively assigned to an oxidized histidine side-chain (64-67). The structure of the Mn-cluster has been probed by EXAFS, and distances to neighboring Mn and [O, N] atoms derived (52-54). Shifts in the K-edge X-ray of Mn have been demonstrated for two of the major transitions (S0S1S2), and interpreted as showing oxidation of Mn (42-44), but oxidation on the S2S3 transition is still controversial (43, 45). The S0-state is probably Mn2+:Mn3+:Mn4+:Mn4+, progressing by 1-electron oxidations to Mn4+:Mn4+:Mn4+:Mn4+ or Mn4+:Mn4+:Mn4+:His+ in S3 (9, 10).

The spectroscopic methods reviewed above provide no kinetic information, and require a commitment of time, biochemical material and equipment which make the cost of detailed analysis of numerous mutant strains exorbitant. Fortunately, much detailed information can be obtained by much simpler and less expensive methods based on fluorescence and spectrophotometry. Kinetic parameters for the S-state transitions (55-62) have been characterized by measurement of absorbance changes in the near UV, and by kinetic methods based on fluorescence (63). Thermodynamic characteristics have been probed with respect to the acceptor-side by measurement of back-reaction kinetics (73), delayed fluorescence (70) and thermoluminescence glow-curves (74). EPR and UV-spectrophotometry, have been used to follow redox changes of the secondary donor (YZ, or Z), identified as Y-161 of D1 (10-15).

The protolytic reactions are also easily measured photometrically (68). In chloroplasts, the 1, 0, 1, 2 pattern for release of the protons from water (in transitions S0-S1-S2-S3-S0) (68-70) has been shown to reflect non-integer stoichiometries for the different transitions, which vary with pH (71, 72). The pK values suggest that dissociable groups of proteins are the immediate source of H+. This is in accord with the electron transfers indicated by redox transitions of the Mn cluster; extensive evidence suggests that the S2 and S3 states carry an excess positive-charge, so the sub-stoichiometric H+-release must involve coulombic effects (71).

Use of molecular engineering to study the structure/function interface

Most work to date has been with the systems developed using the cyanobacterium Synechocystis sp. PCC 6803 for mutagenesis of D1, D2 and CP43 subunits (reviewed in 10, 24, 26).

Donor side. Potential ligands to the Mn-cluster have been explored by extensive mutagenesis of both D1 and D2 (10, 16, 19-21, 67, 75). Coordination of a 4-Mn cluster would be expected to involve 24 sites, of which 10 would be filled by di-µ-oxo bridges, leaving 14 for other groups. Surprisingly few residues have been identified as of critical importance in oxygen evolution, and much speculation has centered around the question of the missing ligands. In other proteins, in addition to polar side-chains, many metal ligands are provided by back-bone carbonyl >C=O groups, and by water bridges, and it would be difficult to identify these by specific residue change. In addition, Cl- may be a ligand in PS II. Exploration of polar side-chains has focused on inter-helical loops on the lumenal side of the proteins. In D1, residue changes giving altered O2-evolution or Mn-binding in loop ab (D59, D61, E65), loop cd (D170, E189, H190), and the C-terminal span (H332, E333, H337, D342, and the terminal -COO- of A344) identify potential ligands. Residue Y161 provides the secondary donor (Z), D170 is necessary for binding of Mn with high affinity in the assembly of the active OEC, but probably does not provide a ligand to the Mn-cluster (127, 144, 145), H198 is a possible ligand to the putative [Chl]2, and H190 is essential for O2-evolution, a possible candidate for the putative redox-active histidine and/or a potential Mn-ligand. H195 as non-essential. These residues are in spans where the structure is unfortunately ill-defined. In D2, only E70 in loop ab looks like a good candidate.

Molecular Engineering of the D1 protein in C. reinhardtii. The past few years have seen major advances in molecular genetics and biochemical protocols which make C. reinhardtii an attractive model system for a combined biophysical and molecular genetic analysis of green-plant photosynthesis (32, 76-90). Efficient chloroplast transformation in C. reinhardtii using a particle gun has opened the way for specific mutagenesis of the chloroplast genome, and molecular engineering of the chloroplast-encoded proteins involved in photosynthesis (81). Two chloroplast genes encode the core proteins of PS II; the psbA gene for D1 (equivalent to the L subunit of bacterial reaction centers) and psbD for D2 (equivalent to the M subunit). The psbA gene in C. reinhardtii has previously been cloned and sequenced (82), and was shown to be the allele on which several herbicide resistant mutations were mapped (83). One copy of the psbA gene is present in each of the inverted-repeats of the chloroplast genome, of which ~40 copies are estimated to be in a gamete cell (84). Unlike those in higher plants, the psbA gene in C. reinhardtii is interrupted by four large introns. Although the polyploid chloroplast genome and the presence of introns have made it difficult to manipulate the psbA gene, several groups have succeeded in introducing site-directed mutations in C. reinhardtii (76-79, 85-87, 127), by transforming wild-type strains using a plasmid-borne mutagenized fragment, with or without a separate plasmid carrying the 16S rRNA gene conferring spectinomycin resistance. These protocols have several drawbacks, which relate to the presence of introns, the need for co-transformation with a separate plasmid to introduce selectable markers, and the lack of tailored features to allow application of cassette mutagenesis protocols. Johanningmeier and Heiss (88) have reported construction of a plasmid containing cDNA of the psbA gene, which was shown to transform a psbA deletion strain, but with poor efficiency. We have constructed an intron-free psbA by PCR-splicing which shows a high efficiency of expression (90, and see below); the poor efficiency seen in (88) could be attributed to the truncated 5'-untranslated region in their construct. Mutations at two positions, H190 and H195, on the donor side in PS II of C. reinhardtii have been constructed by Sayre et al. (76-79), and this lab has collaborated with Dr. Sayre in their characterization (77, 78). As in Synechocystis 6803, H195 as non-essential, and H190 is essential for O2-evolution. Changes to H, T, P and N at D170, thought to be involved in assembly of the Mn complex, have been constructed by Erickson (87), and characterized in some detail (127). Because of the availability of fluorescence methods, kinetic studies of the two-electron gate are more advanced in green plants than bacterial systems, even though we do not have a defined atomic structure. Our previous work has been on kinetic characterization of mutants selected for herbicide resistance (29-33). We demonstrated using fluorescence techniques that we could characterize the changes in rate constants, pK values and binding constants which defined the affects of mutagenesis at the mechanistic level, and this allowed us to explore the structure-function interface. An extensive literature on C. reinhardtii mutants selected for herbicide resistance has been recently reviewed (24-28), and the report of a recent symposium on the topic provides a range of papers covering recent developments (34).

Current Research

Construction of an intron-free psbA gene, with aadA gene in tandem, and unique restriction sites. In order to overcome the difficulties in manipulating the native gene discussed above, we have constructed an intron-free psbA gene incorporating useful restriction sites and the aadA gene as a spectinomycin resistance (specr) cassette in tandem, by using PCR splicing (89, 90). We used this approach because the nucleotide sequence of the psbA gene had already been determined (82) and the clone with the native sequence was available, and because the final construct could be made with long and intact 5'- and 3'-flanking regions. Details of the construction of plasmids pBA155, pBA157 and pBA158 are given in Minagawa and Crofts (90). The plasmids all transformed the ac-u- psbA deletion strain of C. reinhardtii, with expression of psbA and aadA showing ~100% linkage, to give specr and pho+ colonies in high yield.

Mutagenesis of psbA gene and molecular engineering of the D1 protein. Site-directed mutations have been constructed in pBA155, 157 or 158 by a two-step PCR method (89, 90). Fragments carrying mutations were used to replace the wild-type segments as cassettes. Where indicated (Table I), the recombinant plasmids were then shot into a psbA deletion mutant. Bombarded cells were screened for specr and pho+ phenotypes. All the specr transformants are expected to contain the mutagenized psbA gene. Since the recipient deletion strain does not contain the wild-type locus, we do not have to worry about contamination by a wild-type copy of the psbA gene.

Mutagenesis of the psbD gene. The psbD gene encodes D2, the second core protein of PS II. There have been no previous reports of site-directed mutagenesis of this gene in C. reinhardtii, although several groups have introduced mutations in Synechocystis 6803. To construct a plasmid suitable for molecular engineering, a HindIII-PstI fragment containing the psbD gene has been subcloned in a phagemid vector, pGEM3fZ(+). Using single-stranded templates of the resultant construct, pBD102, we have introduced site-directed mutations using the 5-methyl cytosine method, as summarized in Table I. In order to use this system for transformation, we need to introduce a selectable marker, and have concentrated on finding alternatives to aadA (conferring a specr phenotype), as described below. In parallel work, Hutchinson and Sayre have constructed a plasmid with the aadA gene inserted at the 5'-end of psbD, which they have made available to us.

Selectable genetic markers for expression in chloroplasts. In order to extend future work to more ambitious projects using parallel mutagenesis in more than one gene, we need an alternative selection protocol, and have looked for new selectable genetic markers which can be used and expressed in C. reinhardtii. We have used the pBA157 plasmid to introduce the following potential marker genes in place of the aadA gene: gfp (green fluorescence protein), luxAB (luciferase), tetQ (tetracycline resistance), and ermF (erythromycin resistance). Expression of the genes could be readily tested by selection of pho+ colonies after biolistic transformation of the ac-u- psbA deletion strain, followed by appropriate screening. The most promising results have been obtained with ermF, where transformation has given pho+ strains showing resistance to erythromycin, and possibly with expression of tetQ.

Preliminary characterization of strains with mutations in D1. We have demonstrated a number of effects not previously seen in cyanobacterial systems. Table I summarizes our progress in characterization of mutant strains (Donor-side residue changes). Points of interest are: (i) Y161F looks very like the similar cyanobacterial mutant, with a low fluorescence after a single flash, showing no rapid donor to P680+. We have shown that oxidation of Chl and carotenoid provides a source of electrons for slow reduction of P680+. (ii) P162, and conserved aromatics at F180, or F182, are not essential, but F186L has a strongly perturbed donor side, leading to severely reduced growth. We have generated several revertants to this mutation but haven't had a chance to characterize or sequence them. (iii) Mutation of E189 to Q or L showed minor effects but, surprisingly, E189D failed to grow, and showed a blocked donor side after 1 flash, indicating a defect in the S1 or S2 transition. This indicates that charge and polarity are not important, but length of side chain is, suggesting that E189 has no ligand function, but stabilizes local structure by extending to a polar phase which aspartate cannot reach. (iv) N191 mutants showed more subtle effects on the donor side, suggesting stabilization of oxidized products by polarity, but no liganding; (v) S264G in the QB-site showed atraziner and properties similar to the same mutation in higher plants. Electron transfer to QB showed a normal rate constant, but diminished stability of QB-; electron transfer to QB- was normal. (vi) H252 clearly has a critical function; it cannot be replaced by glutamine, which shows a similar size and polarity, suggesting that the protolytic function might be important. The mutation gave rise to a severely blocked electron transport (~100 x slower), and a pho- phenotype, although the QB-site was still functional, as judged by further inhibition on addition of DCMU.

(H252 and S264 in the Qb-pocket)

						TABLE I
Residues under investigation in D1
Residue=>to  Oligo.	Seq.   Plasmid	In 	Pho+	Fluor.rise   Back 	   2-elec. gate
                                     Chlamy.                     reaction
Y161	F       +	+	pBA155	 +	-     very small  presumed rapid    not detect.
P162	L	+	+ 	pBA155	 +	++	NY	      NY	       NY
F180	L	+	+	pBA155	 +	+++	normal	    normal	    normal
F182	L	+	+	pBA155	 +	+++	normal	    normal	    normal
F182	Y	+	+	pBA155	 +	+++	normal	    normal	    normal
F186	L	+	+	pBA155	 +	+	NY	      NY	       NY
E189	Q	+	+	pBA155	 + 	+++	slow?   rapid+norm.phases   normal
E189	L	+	+	pBA158	 +	+++	normal  rapid+norm.phases    ambig
E189	D	+	+	pBA158	 +	-    F1. normal    normal	      ambig
						    F2. truncated -	               -
N191	A	+	+	pBA158	 +	+++	normal	    slow            normal
N191	L	+	+	pBA155	 +	+++	normal	    slow            normal
N191	D	+	+	pBA155	 +	+++	normal	   slowed           normal
H252	Q	+	+	pBA155	 +	-	normal	    normal	  inhibited
S264	G	+	+	pBA155	 +	++	normal	    normal	  perturbed
R257	K,E,M	+	+	pBA158	 +	+++	NY	      NY	       NY 
R257	Q	+	+	pBA158	 +	-	NY	      NY	       NY
Random mutagenesis	NY	pBA158	 +	NA	NY	      NY	       NY
Selectable marker genes co-transformed with pBA155
Marker gene	Plasmid     In Chlamy.	Pho+	Expression 
ErmF		pBA157	+	+++		Yes (ermr)
TetQ		pBA157	+	+++		Weak tetr?
Gfp		pBA157	+	+++		Not detectable
LuxAB		pBA157	NY	NY		NY
Residues under investigation in D2
Residue =>to	Oligo.	Seq.	Plasmid	In Chlamy.	
Y160	F	+	+	pBD102	NY
Y160	L	+	+	pBD102	NY
P161	A	+	NY	pBD102	NY
F179	L	+	NY	pBD102	NY
F181	L	+	NY	pBD102	NY
F185	L	+	NY	pBD102	NY
F188	Y	+	NY	pBD102	NY
N190	D	+	NY	pBD102	NY
Olig, oligonucleotide synthesized; 
Seq, cassette sequenced; 
Plasmid, plasmid used as transformation vehicle; 
Chlamy, C. reinhardtii transformed, with growth on TAP medium; 
Pho+, photosynthetic growth (wild-type rates +++, 
	lower rates indicated by fewer +, - is pho-); 
Fluor. rise, variable fluorescence rise in the sub-micro second time scale; 
Back Reaction, Charge recombination measured by decay of variable fluorescence 
	in the presence of DCMU; 
2-elec. gate, kinetics of QA- oxidation after 1 or 2 flashes, showing binary pattern 
	(normal) or normal kinetics after 1 flash but no binary pattern (ambig); 
NY, not yet completed.


  1. Robinson, H. and Crofts, A. (1987) A hypothetical tertiary structure for the QB-binding pocket of the photosystem II reaction center protein D1. Biophys. J. , 51, 125a.
  2. Crofts, A.R., Robinson, H.H., Andrews, K., Van Doren, S. and Berry, E. (1987) Catalytic sites for reduction and oxidation of quinones. In Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, B. and Ernster, L., eds.), pp. 617-624. Plenum Publ. Corp., New York, NY.
  3. Bowyer, J., Hilton, M., Whitelegge, J., Jewess, P., Camilleri, P., Crofts, A., and Robinson, H. (1989) Molecular modelling studies on the binding of phenylurea inhibitors to the D1 protein of photosystem II. Z. Naturforsch 45c, 379-387.
  4. Michel, H., Epp, O., and Deisenhofer, J. (1986) Pigment-protein interactions in the photosynthetic reaction center from Rhodopseudomonas viridis. EMBO J 5, 2445-2452.
  5. Ruffle, S. V., Donnelly, D., Blundell, T. L., and Nugent, J. H. A. (1992) A three-dimensional model of the photosystem II reaction centre of Pisum sativum. Photosynth Res 34, 287-300.
  6. Svensson, B., Etchebest, C., Tuffery, P., Smith, J., and Styring, S. (1992) A structural model derived using molecular mechanics for the core of the photosystem II reaction centre including the redox components. In Research in Photosynthesis (Murata, N. ed.) Vol. II , pp. 147-150, Kluwer Academic Publ., Dordrecht, Netherlands.
  7. Draber, W., Tietjen, K., Kluth, J. F., and Trebst, A. (1991) Herbicides in photosynthesis research. Angew. Chem. Int .Ed. Engl. 30, 1621-1633.
  8. Andersson, B. and Styring, S. (1991) Photosystem II molecular organization function and acclimation. In Current Topics In Bioenergetics Vol. 16 (Lee, C. P. ed.)., pp. 1-82. Academic Press, Inc., San Diego, CA.
  9. Rutherford, A. W., Zimmermann, J. L., and Boussac, A. (1992) Oxygen evolution. In Topics In Photosynthesis Vol. 11: The Photosystems: Structure, Function and Molecular Biology (Barber, J., eds.), pp. 179-229, Elsevier Science Publishers B. V., Amsterdam, Netherlands.
  10. Debus, R. J. (1992) The manganese and calcium ions of photosynthetic oxygen evolution. Biochim Biophys Acta 1102, 269-352.
  11. Debus, R.J., Barry, B, A., Babcock, G.T. and McIntosh, L. (1988) Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system. Proc. Natl. Acad. Sci. USA 85, 427-430.
  12. Vermaas, W. F. J., Rutherford, A. W., and Hansson, O. (1988) Site-directed mutagenesis in photosystem II of the cyanobacterium Synechocystis sp PCC 6803 donor D is a tyrosine residue in the D2 protein. Proc. Natl. Acad. Sci. U.S.A 85, 8477-8481.
  13. Metz, J. G., Nixon, P., Rogner, M., Brudvig, G. W., and Diner, B. A. (1989) 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. Biochemistry 28, 6960-6969.
  14. Hoganson, C. W. and Babcock, G. T. (1988) Electron-transfer events near the reaction center in oxygen-evolving photosystem II preparations. Biochemistry 27, 5848-5855.
  15. Gerken, S., Brettel, K., Schlodder, E., and Witt, H. T. (1988) Optical characterization of the immediate electron donor to chlorophyll alphaII in oxygen-evolving photosystem II complexes tyrosine as possible electron carrier between chlorophyll alphaII and the water-oxidizing manganese complex. FEBS Lett 237, 69-75.
  16. Boerner, R.J., Nguyen, A.P., Barry, B.A. and Debus, R.J. (1992) 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. Biochemistry 31, 6660-6672.
  17. Vermaas, W. (1993) Molecular-biological approaches to analyze photosystem II structure and function. Ann. Rev. Plant Physiol. and Plant Mol. Biol. 44, 457-481.
  18. Tommos, C., Davidsson, L, Svensson, B., Madsen, C., Vermaas, W. and Styring, S. (1993) Modified EPR spectra of the tyrosine (D) radical in photosystem II in site-directed mutants of Synechocystis SP PCC. 6803 - Identification of side chains in the immediate vicinity of tyrosine (D) on the D2 protein. Biochemistry 32, 5436-5441.
  19. Nixon, P.J., Trost, J.T. and Diner, B.A. (1992) 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. Biochemistry 31, 10859-10871.
  20. Nixon, P.J. and Diner, B.A. (1992) Aspartate-170 of the photosystem-II reaction center polypeptide D1 is involved in the assembly of the oxygen-evolving manganese cluster. Biochemistry 31, 942-948.
  21. Diner, B.A. and Nixon, P.J. (1992) The rate of reduction of oxidized redox-active tyrosine, Z+, by exogenous Mn2+ is slowed in a site-directed mutant, at aspartate 170 of polypeptide D1 of photosystem II, inactive for photosynthetic oxygen evolution. Biochim. Biophys. Acta 1101, 134-138.
  22. Crofts, A. R. and Wraight, C. A. (1983) The electrochemical domain of photosynthesis. Biochim Biophys Acta 726, 149-186.
  23. Crofts, A. R., Robinson, H. H. and Snozzi, M. (1984). Reactions of Quinones at Catalytic Sites; a Diffusional Role in H-transfer. In Advances in Photosynthesis Research (Sybesma, C., ed.), Vol. I, pp. 461-468, Martinus Nijhoff/Dr W. Junk Publishers, The Hague, Netherlands.
  24. Bowyer, J. R., Camilleri, P., and Vermaas, W. F. J. (1991) Photosystem II and its interaction with herbicides. In Topics in photosynthesis Vol. 10: Herbicides (Baker, N. R. and Percival, M. P. eds.) pp. 27-85, Elsevier Science Publishers B. V., Amsterdam, Netherlands.
  25. Diner, B. A., Petrouleas, V., and Wendoloski, J. J. (1991) The iron quinone electron-acceptor complex of photosystem II. Physiol Plant 81, 423-436.
  26. Oettmeier, W. (1992) Herbicides of Photosystem II. In Topics in photosynthesis Vol. 11: The Photosystems: Structure, Function and Molecular Biology (Barber, J. ed.) pp. 349-408, Elsevier Science Publishers B. V., Amsterdam, Netherlands.
  27. Sinning, I. (1992) Herbicide binding in the bacterial photosynthetic reaction center. Trends Biochem Sci 17, 150-154.
  28. Shinkarev, V. and Wraight, C.A. (1993) Electron and proton transfer in the acceptor quinone complex of reaction centers of photosynthetic bacteria. In The Photosynthetic Reaction Center (Deisenhofer, J., ed.), Vol. 1, pp. 193-255. Academic Press, Inc. New York.
  29. Bowes, J. M., Crofts, A. R., and Arntzen, C. J. (1980). Redox reactions on the reducing side of photosystem II in chloroplasts with altered herbicide binding properties. Arch. Biochem. Biophys., 200, 303-308.
  30. Taoka, S. and Crofts, A. R. (1987) Competition of Inhibitors with the secondary quinone in dark-adapted thylakoid membranes. In Progress in Photosynthesis Research (Biggins, J., eds.), Vol. II, pp. 425-428, Martinus Nijgoff Publishers, Dordrecht, Netherlands.
  31. Taoka, S. (1989) Thesis for Ph.D. degree, University of Illinois at Urbana-Champaign
  32. Crofts, A.R., Baroli, I., Kramer, D. and Taoka, S. (1993) Kinetics of electron transfer between QA and QB in wild-type and herbicide-resistant mutants of C. reinhardtii, Z. Naturforsch. 48c, 259-266.
  33. Taoka, S., Dahms, V. and Crofts, A.R., (1994) Kinetics of electron flow in the two-electron gate of a herbicide resistant Amaranthus hybridus with serine 264 changed to glycine. to be submitted.
  34. See papers in Z. Naturforsch. (1993) 48c, 259-266.
  35. Kuwabara, T., Miyao, M., Murata, T., and Murata, N. (1985) The function of 33-kilodalton protein in the photosynthetic oxygen-evolution system studied by reconstitution experiments. Biochim Biophys Acta 806, 283-289.
  36. Bricker, T. M. (1992) Oxygen evolution in the absence of the 33-kilodalton manganese-stabilizing protein. Biochemistry 31, 4623-4628.
  37. Ono, T. -A. and Inoue, Y. (1985) S-state turnover in the oxygen-evolving system of calcium chloride-washed photosystem II particles depleted of 3 peripheral proteins as measured by thermoluminescence removal of 33-kilodalton protein inhibits S3 to S4 transition. Biochim Biophys Acta 806, 331-340.
  38. Franzen, L. G., Hansson, O., and Andreasson, L. E. (1985) The roles of the extrinsic subunits in photosystem II as revealed by EPR. Biochim Biophys Acta 808, 171-179.
  39. Burnap, R. L. and Sherman, L. A. (1991) Deletion mutagenesis in Synechocystis sp PCC6803 indicates that the manganese-stabilizing protein of photosystem II is not essential for oxygen evolution. Biochemistry 30, 440-446.
  40. Philbrick, J. B., Diner, B. A., and Zilinskas, B. A. (1991) Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II. J Biol Chem 266, 13370-13376.
  41. Brudvig, G. W., Beck, W. F., and De, P. J. C. (1989) Mechanism of photosynthetic water oxidation. Ann. Rev. of Biophys. and Biophysical Chemistry 18, 25-46.
  42. Mcdermott, A. E., Yachandra, V. K., Guiles, R. D., Cole, J. L., Dexheimer, S. L., Britt, R. D., Sauer, K., and Klein, M. P. (1988) Characterization of the manganese oxygen-evolving complex and the iron-quinone acceptor complex in photosystem II from a thermophilic cyanobacterium by EPR and X-ray absorption spectroscopy. Biochemistry 27, 4021-4031.
  43. Guiles, R. D., Zimmermann, J. L., Mcdermott, A. E., Yachandra, V. K., Cole, J. L., Dexheimer, S. L., Britt, R. D., Wieghardt, K., Bossek, U., and Et, A. L. (1990) The S3 state of photosystem II differences between the structure of the manganese complex in the S2 and S3 states determined by X-ray absorption spectroscopy. Biochemistry 29, 471-485.
  44. Riggs, P. J., Mei, R., Yocum, C. F., and Penner, H. J. E. (1992) Reduced derivatives of the manganese cluster in the photosynthetic oxygen-evolving complex. J Am Chem Soc 114, 10650-10651.
  45. Ono, T. A., Noguchi, T., Inoue, Y., Kusunoki, M., Matsushita, T., and Oyanagi, H. (1992) X-ray detection of the period-four cycling of the manganese cluster in photosynthetic water oxidizing enzyme. Science 258, 1335-1337.
  46. Dismukes, G. C. and Siderer, Y. (1980) EPR spectroscopic observations of a manganese center associated with water oxidation in spinach chloroplasts. FEBS Lett 121, 78-80.
  47. Brudvig, G. W., Casey, J. L., and Sauer, K. (1983) The effect of temperature on the formation and decay of the multiline EPR signal species associated with photosynthetic oxygen evolution. Biochim. Biophys. Acta 723, 366-371.
  48. Hansson, O., Aasa, R., and Vanngard, T. (1987) The origin of the multiline and g equals 4.1 EPR signals from the oxygen-evolving system of photosystem II. Biophys J 51, 825-832.
  49. Beck, W. F., De, P. J. C., and Brudvig, G. W. (1986) Ammonia binds to the manganese site of the oxygen-evolving complex of photosystem II in the S2 state. J Am Chem Soc 108, 4018-4022.
  50. Boussac, A., Zimmermann, J.L., and Rutherford, A.W. (1990) Factors influencing the formation of modified S2 EPR signal and the S3 EPR signal in calcium-depleted photosystem II. FEBS Lett 277, 69-74.
  51. Dexheimer, S. L. and Klein, M. P. (1992) Detection of a paramagnetic intermediate in the S1 state of photosynthetic oxygen-evolving complex. J Am Chem Soc 114, 2821-2826.
  52. Sauer, K., Yachandra, V.K., Britt, R.D. and Klein, M.P. (1992) In Manganese Redox Enzymes (Pecoraro, V.L., ed.), pp. 141-175, VCH, New York, NY.
  53. Penner-Hahn, J.E., Fronko, R.M., Pecoraro, V.L., Yocum, C.F., Betts, S.D. and Bowlby, N.R. (1990) Structural characterization of the manganese sites in the photosynthetic oxygen-evolving complex using X-ray absorption spectroscopy. J. Am. Chem. Soc. 112, 2549-2557.
  54. Prince, R. C., Cramer, S. P., and George, G. N. (1990) The Manganese Cluster of the Water-splitting Enzyme. In Current Research in Photosynthesis (Baltscheffsky, M., eds.), Vol. I, pp. 685-692, Kluwer Academic Publishers, Dordrecht, Netherlands.
  55. Velthuys, B. R. (1981) Spectrophotometric studies of the S-state transitions of photosystem II and of the interactions of its charged donor chain with lipid soluble anions. In Proceedings of the 5th Intl. Cong. Photosynthesis (Akoyunoglou, G., eds.), Vol. II, pp. 75-86, Balaban International Science Services, Philadelphia, PA.
  56. Dekker, J. P. and Van, G. H. J. (1987) Electron transfer in the water-oxidizing complex of photosystem II. J Bioenerg Biomembr 19, 125-142.
  57. Saygin, O. and Witt, H. T. (1987) Optical characterization of intermediates in the water-splitting enzyme system of photosynthesis. Possible states and configurations of manganese and water. Biochim Biophys Acta 893, 452-469.
  58. Lavergne, J. (1987) Optical-difference spectra of the S-state transitions in the photosynthetic oxygen-evolving complex. Biochim Biophys Acta 894, 91-107.
  59. Dekker, J.P. (1992) In Manganese Redox Enzymes (Pecoraro, V.L., ed.), pp. 85-103, VCH, New York, NY.
  60. Lavergne, J. (1991) Improved UV visible spectra of the S transitions in the photosynthetic oxygen-evolving system. Biochim Biophys Acta 1060, 175-188.
  61. van Leeuwen, P. J., Heimann, C., Dekker, J. P., Gast, P., and van Gorkom, H. J. (1992) Redox changes of the oxygen evolving complex in photosystem II core particles as studied by UV spectroscopy. In Research in Photosynthesis (Murata, N., eds.), Vol. II, pp. 325-328, Kluwer Academic Publishers, Dordrecht, Netherlands.
  62. van Leeuwen, P. J., Heimann, C., Kleinherenbrink, F. A. M., and van Gorkom, H. J. (1992) Kinetics of electron transport on the donor side of spinach photosystem II core particles. In Research in Photosynthesis (Murata, N., eds.), Vol. II, pp. 341-344, Kluwer Academic Publishers, Dordrecht, Netherlands.
  63. Itoh, S., Yerkes, C.T., Koike, H., Robinson, H.H. and Crofts, A.R. (1984). Effect of chloride depletion on electron donation from the water oxidizing complex of photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated Pea chloroplasts. Biochim. Biophys. Acta, 766, 612-622.
  64. van Vliet, P., Boussac, A. and Rutherford, A.W. (1994) Chloride-depletion effects in the calcium-deficient oxygen-evolving complex of photosystem II. Biochemistry 33, 12998-13004.
  65. Tso, J., Sivaraja, M., and Dismukes, G. C. (1991) Calcium limits substrate accessibility or reactivity at the manganese cluster in photosynthetic water oxidation. Biochemistry 30, 4734-4739.
  66. Hallahan, B. J., Nugent, J. H. A., Warden, J. T., and Evans, M. C. W. (1992) Investigation of the origin of the S3 EPR signal from the oxygen-evolving complex of photosystem 2. The role of tyrosine Z. Biochemistry 31, 4562-4573.
  67. Tang, X.S., Diner, B.A., Larsen, B.S., Gilchrist, M.L., Lorigan, G.A. and Britt, R.D. (1994) Identification of Histidine at the catalytic site of the photosynthetic oxygen-evolving complex. Proc. Natl. Acad. Sci. USA 91, 704-708.
  68. Saphon, S. and Crofts, A. R. (1977). Protolytic reactions in photosystem II. A new model for the release of protons accompanying the photooxidation of water. Z. Naturforsch. 32c, 617-626.
  69. Fowler, C. F. (1977) Proton evolution from photosystem II stoichiometry and mechanistic considerations. Biochim Biophys Acta 462, 414-421.
  70. Bowes, J.M. and Crofts, A.R. (1981). The role of pH and membrane potential in the reactions of photosystem II as measured by effects on delayed fluorescence. Biochim. Biophys. Acta., 637, 464-472.
  71. Rappaport, F. and Lavergne, J. (1991) Proton release during successive oxidation steps of the photosynthetic water oxidation process stoichiometries and pH dependence. Biochemistry 30, 10004-10012.
  72. Jahns, P. and Junge, W. (1992) Proton release during four steps of photosynthetic water oxidation induction of 1 1 1 1 pattern due to lack of chlorophyll a/b binding proteins. Biochemistry 31, 7398-7403.
  73. Yerkes, C. T., Babcock, G. T. and Crofts, A. R. (1983). A Tris-induced change in the midpoint potential of Z, the donor to photosystem II, as determined by the kinetics of the backreaction. FEBS Lett., 158, 359-363.
  74. Rutherford, A. W., Crofts, A. R., and Inoue, Y. (1982). Thermoluminescence as a probe of photosystem II photochemistry; the origin of the flash induced glow peaks. Biochim. Biophys. Acta 682, 457-465.
  75. Tang, X.S., Chisholm, D.A., Dismukes, G.C., Brudvig, G.W. and Diner, B.A. (1993) Spectroscopic evidence from site-directed mutants of Synechocystis PCC 6803 in favor of a close interaction between Histidine-189 and redox-active tyrosine-160, both of polypeptide D2 of the photosystem II reaction center. Biochemistry 32, 13742-13748.
  76. Roffey, R. A., Golbeck, J. H., Hille, C. R., and Sayre, R. T. (1991) Photosynthetic electron transport in genetically altered photosystem II reaction centers of chloroplasts. Proc. Natl. Acad. Sci. USA 88, 9122-9126.
  77. Kramer, D. M., Roffey, R.A., Govindjee, and Sayre. R.T. (1994) The AT thermoluminescence band from Chlamydomonas reinhardtii and the effects of mutagenesis of histidine residues on the donor side of the photosystem II D1 polypeptide. Biochim. Biophys. Acta 1185, 228-237
  78. Roffey, R.A., Kramer, D. M., Govindjee, and Sayre. R.T. (1994) Lumenal-side histidine mutations in the D1 protein of photosystem II affect donor side electron transfer in Chlamydomonas reinhardtii. Biochim. Biophys. Acta 1185, 257-270.
  79. Roffey, R. A., van Wijk, K. J., Sayre, R. T., and Styring, S. Spectroscopic characterization of tyrosine-Z in histidine 190 mutants of the D1 protein in photosystem II (PSII) in Chlamydomonas reinhardtii. J. Biol. Chem. 269, 5115-5121.
  80. Rochaix, J.-D. (1987) Molecular genetics of chloroplasts and mitochondria in the unicellular green alga Chlamydomonas. FEMS Microbiol. Rev. 46, 13-34.
  81. Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., Randolph-Anderson, B. L., Robertson, D., Klein, T. M., Shark, K. B., and Sanford, J. C. (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 1534-1538.
  82. Erickson, J. M., Rahire, M., and Rochaix, J. D. (1984) Chlamydomonas reinhardii gene for the 32000-molecular-weight protein of photosystem II contains 4 large introns and is located entirely within the chloroplast inverted repeat. EMBO J 3, 2753-2762.
  83. Erickson, J. M., Rahire, M., Rochaix, J. D., and Mets, L. (1985) Herbicide resistance and cross-resistance changes at 3 distinct sites in the herbicide-binding protein. Science 228, 204-207.
  84. Gillham, N. W. (1978) Organelle Heredity. pp. 347-380, Raven Press, New York, NY.
  85. Heiss, S. and Johanningmeier, U. (1992) Analysis of a herbicide resistant mutant obtained by transformation of the Chlamydomonas chloroplast. Photosynthesis Res. 34, 311-317.
  86. Lers, A., Heifetz, P. B., Boynton, J. E., Gillham, N. W., and Osmond, C. B. (1992) The carboxyl-terminal extension of the D1 protein of photosystem II is not required for optimal photosynthetic performance under CO2-and light-saturated growth conditions. J. Biol. Chem. 267, 17494-17497.
  87. Erickson, J. M., Whitelegge, J., Koo, D., and Boyd, K. (1992) Site-directed mutagenesis of the photosystem II D1 gene and transformation of the Chlamydomonas chloroplast genome. In Research in Photosynthesis (Murata, N., eds.), Vol. III, pp. 421-424, Kluwer Academic Publishers, Dordrecht, Netherlands.
  88. Johanningmeier, U. and Heiss, S. (1993) Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene. Plant Mol Biol 22, 91-99.
  89. Minagawa, J. and Crofts, A.R. (1994) Site-directed mutagenesis of the photosystem II reaction center: Construction of PCR-spliced psbA gene in a plasmid conferring spectinomycin resistance, and application to mutagenesis at the donor side. Sixth Internatl. Conf. on Cell and Mol. Biol. of Chlamydomonas. Abstracts, p. 74.
  90. Minagawa, J. and Crofts, A.R. (1994) A robust protocol for site-directed mutagenesis of the D1 protein of Chlamydomonas reinhardtii: a PCR-spliced psbA gene in a plasmid conferring spectinomycin resistance was introduced into a psbA deletion strain. Photosynth. Research, 42, 121-132.
  91. Ermler, U., Fritzsch, G., Buchanan, S.K. and Michel, H. (1995) Structure of the photosynthetic reaction center from Rhodobacter sphaeroides at 2.65 Angstrom resolution - cofactors and protein-cofactor interactions. Structure 2, 925-936.
  92. Deisenhofer, J., Epp, O., Sinning, I. and Michel, H. (1995) Crystallographic refinement at 2.3-Angstrom resolution and refined model of the photosynthetic reaction center from Rhodopseudomonas viridis. J. Mol. Biol. 246, 429-457.