Although oxygen-evolving PS I1 preparations have been available for some time [20,21], these early preparations were not widely used. The isolation by Stewart and Bendall [22] in 1979 of a purified oxygen-evolving complex from the cyano- bacterium (blue-green alga) Phormidium laminosum marked the beginning of re- newed interest in separating the PS II/OEC from the other multi-subunit protein complexes in the membrane.
The effort has proceeded in two stages. In the first, thylakoids were stripped of the PS I, Cyt bd and CFdCF, complexes, and membrane fragments containing only PS IIiOEC and the Chl aib light-harvesting complex (LHC) were isolated [23-25].
The essence of these preparation methods, which are generally quick (3-4 h from spinach leaves to the isolated fragments) and require only inexpensive reagents, involves stacking the thylakoids with divalent cations before and during detergent extraction. The stacking apparently leads to lateral concentration of PS II/OEC and LH C in the grana; selective solubilization of the stroma leads to an easily pel- leted membrane fraction enriched to greater than 95% in PS I1 components.
The polypeptide and electron transfer cofactor compositions of typical prepa- rations of these membrane fractions have been summarized (e.g. Refs. 26, 27) and Seibert and co-workers have carried out a comparative biochemical analysis of several different PS I1 preparations [28]. The Chl content is typically 225-250 per PS 11, compared to 400 ChliPS I1 in thylakoids [26,27]. Accompanying the reduc- tion in the ChliPS I1 ratio is a concomitant increase, in good preparations by a fac- tor of two [29,30], in the oxygen rate on a per Chl basis. The pH optimum for oxygen evolution in the isolated fragments is shifted to acidic values. = p H 6 [24].
This reflects the fact that the membrane no longer provides a permeability barrier and that the water-splitting machinery, which is associated with the inner surface, equilibrates with the prevailing solution pH. A similar pH optimum is observed for oxygen evolution in sealed thylakoid membranes which have been everted dur- ing their isolation [31]. These observations have been incorporated into recent re- finements of the procedures for preparing PS IUOEC fragments as the detergent solubilization produces more active particles when carried out at acidic pH [29,30].
Although the high rates of 0, evolution in the membrane fragments suggest that the PS IIiOEC units survive the detergent extraction, modification to the acceptor side of PS I1 has occurred. The exogenous acceptor requirements for maximal 0, activity are more stringent, as a lipophilic quinone with moderately high redox po- tential (e.g., dichlorobenzoquinone [24]) is necessary at fairly high concentrations (-300 p M ) and van Gorkom and co-workers have provided evidence which sug- gests altered electron transfer in the QAQB region [32]. These observations suggest that the QB binding niche is accessible to detergent which may compete with en- dogenous PQ and exogenous acceptor for the site [33]. An additional complication is apparent in work by Petrouleas and Diner [34] and Zimmermann and Ruther- ford [3S], which shows that certain exogenous acceptors are able to oxidize the Fe2+
associated with Q A and QB.
The second stage in the resolution of the minimal PS II/OEC unit is in progress.
Non-oxygen-evolving PS I1 cores, stripped of the LHC, had been isolated and characterized in a number of laboratories (e.g. Refs. 3 6 3 8 ) . Recently several groups have described procedures by which a core preparation which retains high rates of 0, evolution may be isolated either from higher plants [39-421 or from thermophilic cyanobacteria [43]. These procedures involve solubilizing PS IIiOEC membrane fragments with non-ionic detergents and separating the core complex from L H C polypeptides by column chromatography [39,40], density gradient cen- trifugation [4O,43], or salt fractionation and conventional centrifugation [41,42].
The core preparations have not yet been characterized in as great detail as the 0,-evolving membrane fragments. Nonetheless, several of their key properties are apparent (Table 1). The chlorophyll content is approximately the same as in non- 0,-evolving PS I1 cores. Reported oxygen rates (=lo00 pmoles 0,img Chl per h), however, are not as high as one might expect from the enhancement in their P- 680 content. A likely cause for this behavior is modification to the PS I1 reducing side reactions as for the cruder PS I1 preparations. Inoue and co-workers [40] note that ferricyanide as an acceptor, in the presence of digitonin, provides maximal 0, rates and that these rates are insensitive to DCMU. Ghanotakis and Yocum find
Properties of PS IIiOEC core complexes
~ ~~
Procedure (Ref.) Organism Polypeptidesa Maximal 0, rates ChliPS I1 MniPS I1
(pmoles 0,img Chl Per h)
Tang, Satoh [39] Spinach 47, 43. 33h, 30'. (23.17)", 22, 10' 150 - -
Satoh et al. [43] Synechococcus 47. 40, 35h. 30', 18. lo' 300-400 50 3.2
Ikeuchi et al. [40] Spinach 47, 43'. 33h. 34, 32, 30. (23.17)", 10' 55CL850 60 4.0 Ghanotakis and Yocum [41] Spinach 47, 43', 33h, 34, 32, (23,17)''. 20, 10' 900-950 60 4.0 Ghanotakis et al. [42] ( A ) Spinach 47, 43. 33h, 34, 32, (23.17)". 20, 10, 9' 100CL1100 M) 4.0
( B ) Spinache 47. 43. 33h, 34, 32, (23,17)", 9' 1100 60 4.0
MW from gel electrophoresis (kDa).
Extrinsic polypeptide.
Diffuse, may contain more than one polypeptide.
Not isolated in these procedures but thought to be associated with PS IIiOEC in higher plants (see text).
May be lower molecular weight polypeptide(s) present [67].
Runs as two bands owing to occurrence of proteolytic fragment.
Obtained by subjecting preparation A to gel filtration chromatography.
reducing side properties more similar to those of the starting material [41]. A dif- ference between these two preparations is that the former lacks a polypeptide in the 20 kDa range (Table l), which suggests a role for this subunit in acceptor side reactions (Section 2.1.1).
2.1, Polypeptide composition and function in the P S IIIOEC
Given the diversity of laboratories involved in their isolation, the PS IIiOEC core preparations summarized in Table 1 show good consistency in terms of polypep- tide content. Moreover, their composition is similar to that found in non-oxygen- evolving PS I1 cores [36-381 with the exception that they contain the =20 kDa polypeptide mentioned above and the water-soluble 33 kDa polypeptide impli- cated in maintaining the Mn content of the preparations.
2.1. I . Intrinsic polypeptides.
Although there is good agreement as to composition, the function of the various intrinsic polypeptides, particularly of the 47, 34 and 32 kDa subunits, is currently under hot debate. Both the 47 and 43 kDa peptides bind Chl. The 43 kDa may be removed without loss of photochemistry, however, and several groups have pro- vided data which suggest that the 47 kDa contains the binding site for the reaction center Chl [44-501. In this model of the PS I1 core, the intrinsic 32 kDa polypep- tide, which is usually referred to as D-1 and which has been established as a locus of herbicide action in PS I1 [Sl], is postulated as providing the binding site for the secondary quinone acceptor QB. The intrinsic 34 kDa polypeptide, D-2, had been implicated by Metz, Bishop and co-workers in manganese binding [52,53], as they had observed decreased Mn levels in Sceiiedesmus mutants which appeared to contain an unprocessed form of the polypeptide (recent developments in this as- signment are discussed below). Thus one view of the PS IIIOEC core associates P-680 with the 47 kDa polypeptide. QB and herbicide binding with D-1, and man- ganese binding and the water-splitting site with D-2.
The principal alternative model for the roles of the intrinsic PS I1 polypeptides assigns t o the 47 kDa protein only a role in light-harvesting and places the reaction center components in D-1 and D-2. Three lines of argument have been used to support this hypothesis. First, there is sequence homology between D-1 and D-2 [54] and between D-1 and D-2 and the L and M subunits of the bacterial reaction center [5S]. Secondly, the crystal structure of the bacterial reaction center in Rho- dopseudomonas viridis (Chapter 3) shows clearly that L and M form the core of the structure and are involved in binding the photochemically relevant BChls, the quinones and the bridging, acceptor side iron [%I. This led Michel, Deisenhofer and co-workers to suggest an analogous role for D-1 and D-2 in PS 11. Trebst has considered this possibility in detail [57,58] and notes that if D-1 folds so that it crosses the membrane only five times (not seven as proposed [59]) in analogy with the known folding of L and M, then the location of amino acid replacements which confer herbicide resistance may be rationalized. He also points out that function- ally important residues (e.g. the histidines involved in Fe binding) in L and M oc-
cur in similar positions in membrane-spanning helices in D-1 and D-2 in his folding scheme. Thirdly, there is remarkable similarity in the chromophore organization and photochemical routes in the bacterial reaction center compared to PS I1 [17,18,60]. Although it breaks down in certain aspects of the properties of the pri- mary donors in the two systems, most strikingly in the apparent 90" rotation of P- 680 in PS I1 relative to P-870 in bacteria (Ref. 61; see also 62), the analogy re- mains strong enough to suggest that the polypeptide organization in the two sys- tems is also similar. Combining this idea with the Metz/Bishop data implicating D- 2 (or D-1, see below) in manganese binding, one arrives at a model in which these two polypeptides, most likely in concert with the water-soluble 33 kDa subunit, not only bind the photochemical core of PS I1 but are also the locus of the water- splitting process. An interesting addition to this hypothesis is to identify the 20 kDa polypeptide mentioned above with the bacterial reaction center H subunit which also promotes electron transfer in the QAQB region [63].
The logic of the D-1ID-2 model is satisfying. Unfortunately, there are few data yet available to support it. Trebst has carried out trypsin digestion experiments and notes rapid disappearance of the 47 kDa polypeptide on SDS gels even though O2 evolution remains active. This is not strong evidence for the D-1ID-2 model, how- ever, as it is possible that the cleaved but still membrane-bound fragments retain activity [S7]. In an interesting series of developments, it appears as if the 34 kDa polypeptide implicated in Mn binding and thought to be the D-2 polypeptide may actually be the D-1 subunit [64]. Affinity labeling work had shown that the MetziBishop peptide binds herbicide [65] and thus presumably QB. These data suggest, then, that D-1 is involved with cofactors which participate in electron transfer reactions on both the oxidizing and the reducing side of PS 11. The data are weak, however, in showing that D-1 actually binds Mn; its lack of processing in the mutant may simply prevent Mn binding at its normal, but distant, site. Ex- periments are proceeding in a number of laboratories to test the folding patterns for D-1 suggested by Rao et al. [S9] and by Trebst [58]. Data from Edelman's lab apparently conflict with Trebst's model [66], but more recent work provides sup- port for the five-helix model, albeit with somewhat different surface-exposed do- mains (R. Sayre, B.. Ande rson and L. Bogorad, Cell, in the press). While the sit- uation with respect to the functional roles of the intrinsic PS I1 polypeptides remains ambiguous, specific hypotheses are available and testable. (Recent data reported by Ki. Satoh at the 7th International Photosynthesis Congress (Providence, RI, August 1986) indicate that D-1 and D-2 constitute the reaction center polypeptide complex.)
In addition to the heavier intrinsic polypeptides of the PS II/OEC core, several lighter polypeptides are also isolated with the complex [67]. Two of these, with MW = 6 and 10 kDa, are associated with cytochrome b-559. This heme species, although redox active, has not been shown to undergo light-induced electron transfer reactions at rates relevant to PS IIiOEC function. Consequently its role in water splitting remains enigmatic. Herrmann, Cramer and co-workers have re- cently sequenced genes which code for both the 6 and the 10 kDa polypeptides [68,69]. Hydropathy plots show one membrane-spanning region for each and an
analysis of the EPR indicated that the axial ligation for the isolated, low-potential form of the protein, and most likely for the native, membrane-bound high-poten- tial form as well, involves two histidines. As both the 10 kDa and the 6 kDa poly- peptides contain only a single histidine, these data suggest that the h-559 heme crosslinks separate 10 and/or 6 kDa polypeptides to form the holoprotein 1701.
2 . 1 . 2 . Extrinsic polypeptides
One peripheral polypeptide with MW = 33 kDa is isolated with the PS IIiOEC core (Table 1). Two other extrinsic peptides with molecular masses of 17 and 23 kDa have been implicated in the 0,-evolving process, although they may be re- placed in the core preparations by high concentrations of Ca2+ and C1- salts with preservation of 0,-evolution activity. The involvement of these three polypeptides in 0, evolution was first suggested by the Tris extraction experiments on so-called inside-out chloroplast vesicles by Akerlund et al. [71] and by cholate extraction of chloroplasts by Sayre and Cheniae [72]. A flurry of activity ensued and the situ- ation with respect to these polypeptides is now reasonably clear. The biochemical properties of these polypeptides are discussed in detail in two recent reviews [7,8].
The 33 kDa polypeptide was originally purified and characterized by Murata and co-workers [8,73]. The N-terminal [74] and complete amino acid sequences [75]
have been determined. An interesting aspect of this work, given the evidence sup- porting a role for this peptide in promoting Mn binding in the PS IIiOEC core (81, is the similarity of part of its sequence to a region in Mn-superoxide dismutase that contains an aspartic acid ligand to the manganese [75]. In the 33 kDa polypeptide, however, this residue is replaced by cysteine, an unlikely candidate for an Mn li- gand in the light of X-ray absorption fine structure (XAFS) data (Section 2.2.2) and the highly oxidizing potential maintained in intermediate oxidation states of the OEC. Although the 17 and 23 kDa peripheral proteins have been isolated and characterized in some detail [7,8], only the N-terminal sequence for the 17 kDa has appeared 1761.
A major function of these extrinsic polypeptides is to promote interaction be- tween anion and cation cofactors and the PS IUOEC core. (A second function - retardation of stored charge dissipation in the OEC - is discussed in Section 3.1.) Selective depletion of the lighter two polypeptides may be achieved by washing inside-out thylakoid vesicles [71] or PS I1 particles 177-801 with NaCI; depletion of the 33 kDa polypeptide occurs upon washing with Tris, urea or divalent cations [7,8,81]. If the counterion in the latter treatment is chloride, Mn is retained in the PS IIiOEC core. On peptide depletion and removal of residual Ca2+ and C I ~ , ox- ygen evolution is inhibited 1821. Activity may be restored, provided Mn has not been perturbed, by readdition of CaZi and C1- [7,8,83]. The half-saturating con- centrations of the ionic cofactors are lowered dramatically if the 17 and 23 kDa peptides are also rebound. The polypeptide rebinding process is complex but may be summarized as follows. The 33 and 23 kDa peptides rebind directly to the hy- drophobic core [80]; Mn promotes 33 kDa rebinding [84] while the 33 kDa facil- itates 23 kDa binding 1851. Binding of the 17 kDa polypeptide apparently occurs only when the 23 kDa is in place [80]. At least two attempts have been made to
identify the intrinsic polypeptides to which the extrinsic 23 and 33 kDa peptides bind. Lundberg et al. used antibody techniques to implicate polypeptides of 10, 22 and 24 kDa [86], while Bowlby and Frasch used photoaffinity-labeled 33 kDa to identify 22, 24, 26, 28, 29 and 31 kDa polypeptides in the binding [87]. Some of the polypeptides in the latter study clearly arise from LHC components and their labeling may be fortuitous. Both sets of experiments were done on preparations more complex than those in Table 1, and it appears that repeating these experi- ments with the more resolved preparations will be useful. These have already proven useful in evaluating whether given polypeptides are fundamental to 0, ev- olution; for example, Table 1 indicates that a 10 kDa polypeptide isolated by Tris extraction [88] is probably not essential. Many authors have used the data sum- marized above to draw models for the polypeptide/cofactor organization of PS I1 (e.g. Refs. 7, 8, 16, 19 and 89).
While the identity of the polypeptides present in the PS IUOEC core complex is now reasonably well-established, the stoichiometries of these species remain un- certain. Little is known regarding the concentration ratios per PS WO E C unit of the intrinsic polypeptides and controversy exists regarding the stoichiometries of the extrinsic subunits (71. In the latter case, however, the work is reaching agree- ment on between 1 and 2 copies of each of the water-soluble polypeptides per P- 680. More quantitative work, most profitably with I4C labeling, will be necessary.
2.2. Electron transfer components
Photon absorption in the PS II/OEC leads to charge separation in the PS I1 re- action center to generate the oxidized reaction center, P-680' (Ref. 18; Chapter 4, this volume.) The simplest scheme for subsequent electron transfer steps in- volves only the intermediate carrier, Z, and the Mn ensemble at the water-split- ting site:
P-680+ - Z - (Mn),
The questions of branched pathways and of additional intermediates are often raised (Section 3.1), but only the components noted above have been detected directly as entities with distinct functional and spectroscopic properties (Table 2).
2.2.1. P-680 and Z
As opposed to P-700+ in PS I and to the cation radicals of the bacterial reaction centers, P-680' is difficult to trap in its oxidized state - even at low temperatures its lifetime following photogeneration is only 3-4 ms [90] - and chemical oxidation so far has not been possible owing to the high P-680' midpoint potential [l]. Con- sequently the battery of techniques, particularly magnetic resonance, which has proven fruitful in unraveling the structures of the other reaction center chloro- phylls has not been applied to P-680. Its spin-polarized triplet has been detected [61,91] and its unexpected parallel orientation with respect to the membrane plane postulated. The zero-field splitting parameters are almost identical to those of
Selected properties of PS IIIOEC components
Species Detection Identification Function Stoichiometry Binding site
P-680 OpticaVEPR Exciton-coupled Chl n pair Primary donor 1 47 kDa or D-I. D-2
(IPS 11)
D EPR Plastoquinone cation radical ? 1 47 kDa or D-1, D-2
Z OpticalIEPR Plastoquinone cation radical Intermediate electron 1
carrier
47 kDa or D-1, D-2
Cyt b-559 Optical/EPR Fe2+/3+ low-spin protoheme ? 2 6, 10 kDa
Mn OpticaliEPRIXAS Multinuclear cluster(s) Water oxidation 4 Interface 33 and D-1 or
D-2 A discussion of controversies and uncertainties, as well as references. for the information summarized is given in the text.
e w
'W
monomer Chl. Davis et al., arguing from redox-potential considerations and axial ligation effects on Chl EPR linewidths, proposed that the unpaired electron in P- 680' is localized on a single Chl macrocycle [92]. The 'hole-localized' model for the oxidized P-680' cation radical does not necessarily contradict the conclusion reached by den Blanken et al. that two interacting Chls contribute to the singlet and triplet properties of the reduced P680 species [93].
The oxidized form of the Z species was identified as an organic radical with an EPR lineshape identical to that of the well-known PS I1 radical which gives rise to the so-called Signal I1 EPR spectrum [94,95]. Its stoichiometry is one per PS I1 in both thylakoids and PS I1 particles [SS]. EPR [96,97] and optical [98,99] data are consistent in suggesting that Z is a hydroquinone species, most likely plastohydro- quinone, which is one-electron-oxidized to form the hydroquinone cation radical during its reaction with P-680'. Such a structure, i.e. PQH2+, is consistent with the high redox potential ( >+ 1.0 V) required for Z t in its reaction with the water-split- ting redox center [loo]. EPR on oriented membranes was used to assign the ma- jor, partially resolved hyperfine splittings to the 2-methyl group of the plastoqui- none moiety and to suggest an orientation for the radical such that its ring plane is perpendicular to the thylakoid membrane plane [ 101,1021.
Despite the congruence of the EPR and optical data, some results conflict with these interpretations. Neither Takahashi and Katoh [lo31 nor de Vitry et al. [lo41 have been able to find sufficient amounts of noncovalently bound plastoquinone- 9 in PS I1 preparations. In principle there should be three: one for the Q A accep- tor, one for Z and one for the stable Signal II species usually designated Dt. The former group finds 2 PQiPS II while the latter finds only 1.15. Several explana- tions are possible, including: (a) either Z or D is covalently bound PQ or noncov- alently bound but modified in the isoprenoid chain; (b) the Z and D concentra- tions have declined during purification (there is some indication that this occurs [89]); and (c) either Z or D or both are not quinones. The assigned 2-methyl origin of the partially resolved splittings in the Zt/Di E PR spectrum has been chal- lenged, as this causes difficulty in simulating the EPR spectrum and in understand- ing the rotational properties of the methyl group. Brok et al. have reinterpreted the spectral properties by retaining the PQH," identification but assigning major splittings to the 1,4-hydroxyl protons and to the isoprenoid methylene protons. In this interpretation the radical is tilted away from a perpendicular orientation with respect to the membrane plane [105,106]. The characters of the molecular orbitals implied by this model, however, are difficult to understand: it may be that E N - D O R spectroscopy simply overestimates the magnitude of the -CH, coupling [ 107,108]. Thus, although it has been widely accepted that plastoquinone cation radicals are involved in oxidizing-side PS I1 electron transfer, the situation is not as clear as one would like.
2.2.2. Manganese
A substantial body of work had associated manganese with water splitting [5,10,109]
and with the development of Kok's S-state model it was generally assumed that the S-state transitions correspond to valence changes in a functional manganese