Photosynthesis Research 23: 1-27, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Minireview

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II* William J. Coleman

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 6 August 1987; accepted 9 December 1988

Key words." chloride, calcium, manganese, Photosystem II, oxygen-evolving complex, metalloenzyme Abstract

Chloride plays a key role in activating the photosynethetic oxygen-evolving complex (OEC) of Photosystem II, but the OEC is only one of many enzymes affected by this anion. Some of the mechanistic features of Cl involvement in water-splitting resemble those of other proteins whose structure and chemistry are known in detail. An overview of the similarities and differences between these Cl--binding systems is presented.

A. Introduction

A number of enzymes and proteins that catalyze acid-base reactions or that are involved in proton uptake and release (see Section H) require chloride as a cofactor. The oxygen-evolving complex (OEC) of Photosystem II (PS II) is such an enzyme (for reviews, see Critchley 1985, Renger and Govindjee 1985, Govindjee et al. 1985, Homann and Inoue 1986, Homann 1987, 1988a). The light-driven catalytic reaction has the following stoichiometry: 2H20 --* 02 + 4H + + 4e-,

(1)

with an average E °' (mid-point redox potential at pH 7.0) of +0.81 V per electron. Considerable effort has been directed toward understanding the electron transfer reactions on the donor side of PS II. Electrons removed from water by a transiently oxidized tetranuclear manganese-complex are transferred to an intermediate one-electron carrier (Z) which is the electron donor to P680, the reaction center chlorophyll (for a review, see Govindjee et al., 1985, Babcock, 1987, Renger, 1987). During the initial stages of the lightinduced charge separation, P680 is oxidized and * The literature survey for this Minireview was, for the most part, completed in 1987.

pheophytin (Pheo) is reduced. This event is followed by the reduction of the acceptor-side quinones (QA and QB; Vermaas and Govindjee 1981, Crofts and Wraight 1983). Coupling of the four-electron oxidation of water to the single-electron turnover of the central reaction center components (Z-P680-Pheo-QA) requires that the OEC be able to store four oxidizing equivalents at the Mn active site (see review by Wydrzynski 1982) during each catalytic cycle. The complete cycle for water oxidation has been represented as a series of several transient states ("S-states") of the OEC (Kok et al. 1970, Forbush et al. 1971, Fowler 1977, Saphon and Crofts 1977). Each transition is driven by the absorption of a photon by the reaction center chlorophylls; rapid charge separation generates a powerful oxidant (P680 +) that is ultimately reduced by electrons from water: e-

e-

e-

e-

02

T

T

T

T

T

hv

hv

hv

hv

So --* S~ ~ $2 --* $3 ~ H+

H+

($4)

~ So.

(2)

2H +

The higher S-states deactivate in the dark to create a stable distribution of centers having about 75% St and 25% So (Kok et al. 1970). Formation

o f the O - O b o n d most likely occurs during the S 2 ~ S 3 or $3 ~ ( 8 4 ) transition (see discussion in Babcock 1987). It is clear from the reaction mechanism (see F6rster and Junge 1985) that removal o f one o f the products (H ÷) is p r o b a b l y essential to the advancement o f certain S-states, if the rate o f the back-reaction is affected by H ÷ . Theoretical models for the overall mechanism o f 02-evolution and S-state turnover have recognized the importance o f having an efficient protonwithdrawing mechanism ( K u s o n o k i et al. 1980, Krishtalik 1986). Experimental evidence indicates that chloride (C1-) ions are required for both steady-state 02evolution (see, e.g., Kelley and Izawa 1978) and for the advancement o f particular S-states (Itoh et al. 1984, Theg et al. 1984). In addition, C1- appears to be intimately involved in the p H - d e p e n d e n t activation o f the enzyme ( G o r h a m and Clendenning 1952, H o m a n n et al. 1983, Critchley 1985). It is reasonable to expect that definable similarities might exist between the mechanism o f C1- action in the O E C and in other chloride-activated enzymes and proteins. A l t h o u g h the O E C is clearly a unique enzyme in terms o f its overall function, particular details o f its structure and mechanism that resemble other enzymes can be examined by comparative enzymology. ~ ~

Experimentally, the current challenge in this area o f photosynthesis research is to understand the mechanism o f C1- binding and to relate this binding to the subunit structure o f the enzyme and the kinetics o f O2-evolution. This review will focus on relevant similarities between the structures and catalytic mechanisms o f the O E C and other chloriderequiring enzymes, for the purpose o f exploring potential new areas o f investigation. It will emphasize fundamental enzymological aspects which directly relate to C l - action in proteins. Sections B - G describe what is k n o w n a b o u t h o w C1- activates oxygen evolution. Section H analyzes the properties o f a diverse set o f C1- binding proteins and compares them with those o f the OEC. Finally, Sections I and J present an evaluation o f the data and speculation on the nature o f Cl activation o f the OEC.

B. Structure of the OEC The polypeptide structure o f the O E C (Fig. 1) has been extensively reviewed (see, e.g., G h a n o t a k i s and Y o c u m 1985, H o m a n n 1987). F r o m studies o f inverted thylakoid vesicles, the complex is k n o w n to lie on the inner side o f the thylakoid m e m b r a n e s v~~

ii: Fig. 1. A model for the structure and organization of the central polypeptides of the oxygen-evolving PS I1 complex. P680 represents the reaction center primary donor Chls, Pheophytin (Pheo) is the primary electron acceptor, QAand QB are the bound quinone electron acceptors, and Z (a tyrosine residue) is the immediate electron donor to P680. Four manganese atoms (Mn), which accumulate the oxidizing equivalents needed to catalyze the water-splitting reactions, are located near the interface of the reaction center polypeptides and the 33 kD extrinsic polypeptide. Chloride and calcium binding sites are also situated on the lumenal (inner) side of the membrane. The structure surrounding these ions is known as the oxygen-evolving complex (OEC). The PS II core complex in spinach contains proteins with Mr43 and 47 kD (Chl binding proteins), 32 and 34 kD (D1 and D2 reaction center polypeptides, respectively), and 4 and 9 kD (cyt b559). The extrinsic polypeptides involved in O2-evolution have M r 33, 24, and 18 kD. Other proteins with M r 24, 22, 10 (2 proteins), and 5 kD have an unknown function. (After Murata and Miyao 1987).

(/kkerlund and Jansson 1981, Yamamoto et al. 1981,/kkerlund et al. 1982, Blankenship and Sauer 1974). Two intrinsic membrane polypeptides of Mr = 32 and 34 kD (known as the "D 1" and " D 2 " polypeptides, respectively; Chua and Gillham 1977, Rochaix et al. 1984) have been suggested to constitute the core of the PS II reaction center (Michel and Deisenhofer 1985, Trebst and Draber 1986, Nanba and Satoh 1987, Okamura et al. 1987). The D1 protein has been implicated in assembling the Mn cluster that catalyzes the water-splitting reactions (Metz and Bishop 1980, Metz et al. 1986, Diner et al. 1988). This catalytic Mn is responsible for generating a low-temperature flash-induced multline EPR signal at g = 2 that is characteristic of the $2 state (Dismukes and Siderer 1981, Brudvig et al. 1983, Miller et al. 1987, Styring et al. 1987). The OEC also contains several extrinsic polypeptides. Some of these, having Mr < 10kD (Ljungberg et al. 1986), have only recently been identified. The three largest (M,. = 18, 24, and 33 kD) have been actively studied in order to determine their precise roles in Oz-evolution. One possible role for these proteins involves the binding of Ca z+ , which is required for Oz-evolution (Ghanotakis and Yocum 1986). The presence of the 33 kD polypeptide is necessary for the other two polypeptides to bind to the membrane (Murata et al. 1983). The primary structure of the 33kD polypeptide has been determined by amino-acid sequencing (Ohoka et al. 1986). Recently, the putative amino-acid sequences of the 18 and 24kD polypeptides (as determined by sequencing their respective cDNAs) have also been reported (Jansen et al. 1987). Selective removal of these extrinsic polypeptides can be achieved by washing PSlI-enriched membrane fragments (known as PS II particles or membranes; Berthold et al. 1981, Kuwabara and Murata 1982) or inside-out thylakoid vesicles with high concentrations of various salts or denaturants (/~kerlund and Jansson 1981,/kkerlund et al. 1982). Washing with 1.0 M NaCI removes predominantly the 18 kD and 24 kD polypeptides, as well as some of the bound Ca 2+ , without affecting the functional Mn (Murata et al. 1983, Miyao and Murata 1986, Cammarata and Cheniae 1987). Washing with 1.0 M CaCI 2 removes all three polypeptides (Ono and lnoue 1983). Removal of the 33 kD polypeptide (along with the 18 kD and 24 kD polypeptides) accelerates the loss of Mn from the membrane

unless a high concentration of C1- (about 200 mM) is present in the medium (Miyao and Murata i 984a). Washing with 0.8 M Tris at pH 8.0 removes all of the extrinsic polypeptides (Yamamoto et al. 1981) as well as the functional Mn (Murata et al. 1983). Two other inhibitory treatments also disrupt the OEC. Incubation with millimolar concentrations of NHzOH, which removes a substantial amount of functional Mn, does not appear to affect the binding of the extrinsic polypeptides, although O2evolution is inactivated (Murata et al. 1983). Mild heating (3-5 min, 35-50°C) also removes Mn and inactivates O2-evolution, but its effect on polypeptide removal depends on the temperature (Nash et al. 1985). The 33 kD polypeptide appears to be the most essential extrinsic subunit (see Homann 1987). Although its amino acid sequence shows local sequence similarity with Mn-superoxide dismutase from E. coli (Oh-oka et al. 1986), it is not clear whether it binds functional Mn in situ (Hunziker et al. 1987, Miller et al. 1987). Two regions of the amino acid sequence also show local similarity with the Ca 2÷ binding sites in calmodulin and troponin C (Coleman et al. 1984, Coleman and Govindjee 1987), but beyond suggesting where Ca 2÷ could perhaps be bound, the significance of this resemblance is not known. The structural and functional linkage between Ca 2+ and C1- binding sites is currently an active area of research (Homann and Inoue 1986, Homann 1988b) and will be discussed in more detail in Sections C and E.

C. Number and location of C1- binding sites

Up to now, attempts to determine the number of C1- binding sites in spinach have relied on measurements of radioactive 36C1 binding (Theg and Homann 1982) and on Oz-evolution in a modulated electrode (Sinclair 1984). Both mesurements suggest that more than one C1- binding site is involved in Oz-evolution. Studies of the location of these binding sites have generally involved selective removal of the extrinsic polypeptides. Our knowledge of this aspect of the system is therefore limited to the polypeptide level. An estimate of the effect of polypeptide removal on the apparent affinity of the OEC for C1 can be

made by measuring the steady-state rate of 02evolution as a function of the concentration of C1added to CI depleted membranes. Miyao and Murata (1985) have shown that PSII membranes which lack all of the extrinsic polypeptides reach a low Vmax (about 30pmol 02 (mg Chl) -j h -~) at about 150 mM C1 . When the 33 kD polypeptide is present, Vmaxincreases to about 120#mol Oz (mg Chi)- ~h - J at 30 mM C1 . In untreated PS II membranes, Vm,xis greater than 300 #mol 02 (mg Chl) J h-~ at about 10 mM C1 . Similar results have been reported by Imaoka et al. (1984, 1986). The greatest contribution of the extrinsic polypeptides to the overall rate is in the range of low [CI ], particularly for the case of the 18 kD and 24 kD polypeptides (Andersson et al. 1984a). These two subunits exhibit the largest effect at low [C1-] when they are present together (Miyao and Murata 1985). Although part of this synergism reflects the fact that the 24 kD polypeptide (which binds to the 33 kD polypeptide) is needed to provide a binding site for the 18 kD polypeptide (Miyao and Murata 1983), it is nevertheless clear from these depletion/reconstitution studies that the 18 kD polypeptide contributes to C1- binding by increasing the apparent affinity of the'OEC for C1-. The stimulatory effect of the 18 kD polypeptide is greatest in the region of 0.1-1.0mM C1 ; that is, in the range of the apparent dissociation constant for activation by CI(K~,) in the thylakoid system. Its role in C1 stimulation of O2-evolution is therefore not trivial. Determining the true requirement for each of these polypeptides has been complicated, however, by the observation that various non-physiological concentrations of Ca 2÷ can apparently replace their function to a limited extent (Ghanotakis et al. 1984a, Miyao and Murata 1984b, Nakatani 1984). For example, in PS II membranes depleted of the extrinsic polypeptides, addition of 100mM NaC1 alone restores about 20% of the original activity. When this is supplemented with an additional 10 mM CaC12, nearly 40% of the original activity is restored (Kuwabara et al. 1985, see also Ono and Inoue 1984). In PS II membranes depleted of only the 18kD and 2 4 k D polypeptides, addition of 2 0 m M NaC1 alone restores only 26% of the original activity, whereas 10mM CaC12 restores 80% of the original activity (Ghanotakis et al. 1985). Even when the 18kD and 24kD polypeptides are added back after depletion, the presence of

is still a crucial factor, because if these two subunits are added back after having been dialyzed against E G T A to remove Ca 2+ , the two polypeptides bind to the membrane but do not stimulate O2-evolution (Ghanotakis et al. 1984b). Nevertheless these two polypeptides evidently enhance the binding of Ca 2+, reducing the apparent dissociation constant to the more physiologically relevant micromolar level (Ghanotakis et al. 1984b). Even in the absence of the 18 kD and 24 kD polypeptides, the OEC appears to have two binding sites for Ca 2+ (Cammarata and Cheniae 1987, H o m a n n 1988b, Boussac and Rutherford 1988). Recent experiments by H o m a n n (1988b) suggest that these two polypeptides exert their major direct effect by protecting the Ca 2+ binding sites, and have only an indirect effect on C1- binding. It is conceivable that no single polypeptide encompasses all of the C I - / C a 2÷ functions. Instead, it appears that there are multiple binding sites for C1and Ca 2÷ (Boussac et al. 1985, Cammarata and Cheniae 1987, Coleman et al. 1987b,c 1988, Katoh et al. 1987, H o m a n n 1988b, Boussac and Rutherford 1988) which may reside on different polypeptides. If the binding of these ions involves interactions between various subunits, then removal of any member of the complex could significantly decrease the apparent affinity of the remaining subunits for C1- or Ca 2÷. This property of the system would tend to create a "layering" effect; that is, removal of successive layers of extrinsic polypeptides (first the 18 and 24kD polypeptides and then the 33 kD polypeptide) would progressively reduce the apparent OEC affinity for these activators (and the maximum catalytic rate) without completely abolishing activity (see the discussion of the polypeptide depletion experiments above). Each layer would appear to contribute a percentage to the overall efficiency of 02-evolution. The innermost layer, if it directly involves the intrinsic polypeptides, cannot be physically removed without damaging the Mn active site and the reaction center structure. In this respect, both C1and Ca 2÷ appear to function over an extended region. Nevertheless, the extrinsic 33kD polypeptide seems to be the linchpin for the assembly of the C1 /Ca 2+ activatable complex. In addition to stabilizing at least half of the catalytic Mn associated with the intrinsic proteins (Kuwabara et al. 1985, C a 2+

Imaoka et al. 1986), this polypeptide appears to bind to both the 24 kD polypeptide (B. Andersson et al. 1984b, Ljungberg et al. 1984) and other PS II core polypeptides (Bowlby and Frasch 1986, Liveanu et al. 1986).

D. Relationship of CI- binding to Mn binding

In several published models for C1- activation of the OEC,C1- functions as a Mn ligand in the active site (Sandusky and Yocum 1983, 1984, Critchley and Sargeson 1984, Dismukes 1986). Experiments from various laboratories have indicated that C1depletion affects both the EPR properties of the Mn center (Damoder et al. 1986) and its accessibility to inhibitors, such as NH 3 (Sandusky and Yocum 1983, 1984, 1986). Damoder et al. (1986) found that the depletion of C1- from PS II membranes significantly reduces the amplitude of the low-temperature multi-line EPR signal attributed to the $2 state. Their analysis of the effect of Fcompetition on C1- enhancement of the $2 EPR signal yielded two dissociation constants for CI-, which they suggest reflects two C1- binding sites on the donor side of PS II. Although they raise the possibility that CI- binding to Mn could be involved in shifting the redox potential for Mn 4-- ~ Mn 3+ in aqueous medium (see Kambara and Govindjee 1985, Dismukes 1986), they concede that more evidence is needed to determine whether C1- binds directly to Mn. Using a different approach, Sandusky and Yocum (1983, 1984, 1986) studied the competition between NH3 and C1- by measuring steady-state O2-evolution in C1- depleted PS II membranes. Their results indicate that NH 3 inhibits CI- activation of O2-evolution by direct competition at the C1- binding site. Since amines have been shown to interfere with the reactions at the catalytic Mn (Velthuys 1975, Beck and Brudvig 1986, 1987, Andr6asson and Hansson 1987), it was proposed (Sandusky and Yocum 1983) that C1- might act as a bridging ligand between the Mn atoms, and that NH 3 inhibits the system by displacing CI . Critchley and Sargeson (1984) have also proposed a C1 bridging model, but it includes an S2-state that is not paramagnetic. Homann (1986) has reported that the protective effect of CI in the presence of amines and other Lewis bases is indirect, perhaps

involving changes in the tertiary and quaternary structure of the polypeptides. The most recent data of Sandusky and Yocum (1986) indicates that there are two binding sites for NH3, one at the catalytic Mn (i.e., the binding site for H20) and another which is competitive with C1-. The second site could allow NH3/CI- coordination to Mn without directly affecting H20 binding. Recent EXAFS (Extended X-ray Absorption Fine Structure) and EPR measurements indicate that chloride ions and other halides are not innersphere ligands to the Mn which is observed by these techniques. Using EXAFS, Yachandra et al. (1986a) have reported that PS II Mn in the S~-state does not show any involvement of C1- (or Br-) in the first coordination sphere. Likewise, by observing the EPR fine structure of Mn in the S2-state, Yachandra et al. (1986b) found no difference between C1 and Br-, which suggests that halides do not interact directly with Mn in this state. The EPR signal amplitude increased hyperbolically with the C1 concentration, however, indicating that a site to which C1- binds exerts an indirect effect on the EPR-active Mn. Surface-enhanced Raman scattering (SERS) spectroscopy also indicates that C1- does not bind to Mn in CaCI2washed PS II membranes, but instead produces an indirect alteration of the Mn environment (Seibert et al. 1988). A possible explanation for the discrepancies in all of these results might be that although CI- does not bind to Mn, ammonia binds both to Mn and to a C1 -binding amino acid side chain (e.g., a histidine imidazole). Another possibility is that C1- binds to Mn only in the higher S-states, as suggested by Sandusky and Yocum (1986).

E. The kinetics of CI- activation 1. Effect on the S-states

The effect of CI depletion on the advancement, stability, and deactivation of the S-states has been examined only during the last several years. Most reports indicate that the higher S-state transitions are primarily affected, although a few have suggested that So ~ Sj and St ~ $2 might also be involved (Govindjee et al. 1983, Damoder et al. 1986, Homann et al. 1986). The first studies of flash-O2

yields and delayed light emission by Muallem et al. (1980) and Muallem and Boszormenyi (1981) demonstrated that removal of CI stabilizes the $2 and $3 states by slowing down the rate of their deactivation. Itoh et al. (1984), monitoring Chl fluorescence yield changes, and Theg et al. (1984), monitoring fluorescence yield and delayed light emission, confirmed that C1- depleted thylakoids were capable of donating two electrons to the oxidized PS II reaction center (i.e., they were capable of generating the $2 state by taking one electron from Z and the second electron from the OEC). The latter group, however, questioned the earlier conclusion that the rate of deactivation is slowed by C1- depletion, since they observed a higher initial luminescence level and an accelerated luminescence decay in depleted samples in the 50 ms to 1 s interval after illumination. Other measurements of S-state turnover using thermoluminescence (Homann et al. 1986) and a modulated O2-electrode (Sinclair 1984) indicate that the $3 --. So reaction is effectively blocked by CI depletion. H o m a n n et al. (1986) suggest that earlier fluorescence measurements could not detect the very slow $2 ~ $3 transition in C1- depleted samples because of instrumental limitations. As Itoh et al. (1984) noted, the steady-state electron transfer from the OEC to Z ÷ changes from having a single half-time of 450 #s in C1 sufficient thylakoids to having biphasic kinetics with a fast component of 120ms and a slower component of several seconds in the CI- depleted case. Thus, H o m a n n et al. (1986) argue that Z ÷ was not capable of being re-reduced within the 60 ms interval between flashes, and that the $3 state was therefore never observed (see also Inoue 1987). Other thermoluminescence measurements, however, support the idea that the $2 --* $3 transition is blocked by CI depletion (Rozsa and Demeter 1987). This discrepancy has not yet been resolved. On the basis of EPR evidence, Ono et al. (1986, 1987) have argued that C1 depleted PSII membranes cannot advance beyond the $2 state, and that the $2 state produced after a single flash is abnormal (see also Yachandra et al. 1986b). This modified $2 state can be re-converted to a normal $2 state (i.e., one having a low-temperature multi-line signal at g = 2) simply by restoring C1 to the suspension medium. The light-induced g = 4.1 signal (also due to Mn) is not, however, affected by C1 depletion.

The low-temperature $2 EPR signal disappears when all of the extrinsic polypeptides have been removed, but is restored by the addition of 200 mM NaC1 (Styring et al. 1987) or 200mM NaC1 plus 15raM CaC12 (Miller et al. 1987). Vass et al. (1987) have shown that although depletion of C1 and extraction of the 33 kD and other extrinsic polypeptides reversibly block the advancement of the S-states, the two treatments have different effects on the stability of the S2QA and S2QB states, as determined by thermoluminescence. Only one study of C1- binding to the S-states has been done by 35C1- NMR: Preston and Pace (1985) observed C1- binding to C1- depleted PS II membranes from a halophyte (mangrove), and determined that high-affinity C1- binding involves primarily the $2 and $3 states. It is clear from all these studies that even if the deprotonation of water does indeed involve C1 directly, the question of which S-state transitions require anion binding still has not been answered. 2. Effect on steady-state electron transport Warburg and Liittgens (1944) were the first to demonstrate the "C1 effect" on 02-evolution in dialyzed chloroplasts. Many years later, a series of experiments by Boy6 et al. (1963), Hind et al. (1969), Izawa et al. (1969), and Heath and Hind (1969) eventually pinpointed the site of C1 action on the donor side of PS II in glycophytes. The same site for the CI effect was established in halophytes by Critchley et al. (1982). Hind et al. observed that C1- depletion significantly reduces the quantum efficiency of uncoupled electron transport. Heath and Hind reached a similar conclusion based on measurements of fluorescence yield. Izawa et al. showed, in addition, that CI- depletion reduces the maximum attainable rate of electron transport at a given light intensity. In C1 depleted PSII membranes lacking the 18kD and 2 4 k D polypeptides, addition of C1 improves the quantum efficiency, but does not enhance the maximum attainable rate of 02-evolution (Homann and Inoue, 1986). Addition of Ca 2÷ in the presence of sufficient C1 was found to raise the quantum efficiency much more dramatically than it raised the maximum rate of O2-evolution. Other studies, however, demonstrated a large effect of Ca 2+ on the maximum rate of O2-evolution in such preparations (see Sections B and C).

Kelly and Izawa (1978) used uncoupled, C1depleted thylakoids to obtain a kinetic estimate of the apparent activator dissociation constant (K~) for C1 of approximately 0.9 mM (see also Baianu et al. 1984). This value is probably somewhat higher than the actual value due to the presence of 2 mg ml-~ bovine serum albumin (a C1 binding protein; see Section H) in the suspension. Measurements by Homann (1988b), using PS II membranes, indicate that K~ may be as low as 20/~M at pH < 5. Estimates of K), by these methods are somewhat complicated by the fact that there is a measurable rate of O2-evolution in the absence of any added CI- (see Kelley and Izawa Fig. 5; Homann 1988b, Fig. 2). For the purposes of constructing double-reciprocal (Lineweaver-Burk) plots, this "endogenous" rate may introduce a significant error in estimating K~, and other parameters, and should not be overlooked (see, e.g., Reiner 1969, Allison and Purich 1983). In this respect, the key question of whether C1- is an essential or non-essential activator (see, e.g., Segel 1975) has only recently been addressed (Homann 1988b).

F. The CI- binding mechanism

A complete understanding of the mechanism of C1activation of the OEC cannot be achieved by studying the Oz-evolution kinetics alone. A thorough investigation of the binding properties of CI in this system will also be needed. In general, we would like to know the number and location of the C1 binding sites more precisely and the degree to which different sites might interact. More specifically, we would like to know: (a) the nature of the exchange between bound and free CI-; (b) the affinity/binding energy; (c) the relationship between anion size (or charge) and the ability of that anion to bind effectively and stimulate O2-evolution; and (d) the pK~'s of the groups involved in binding and the effect of C1- on these pK~'s. A useful way to approach these questions is by using 35C1-NMR. The initial 35CI-NMR studies of C1- binding to C1- depleted thylakoids were limited by low instrumental sensitivity, and thus thylakoids from various halophytes were used, since they have a very high C1- requirement for 02evolution (see Critchley et al. 1982, Baianu et al. 1984). These thylakoids apparently lacked the

18 kD and 24 kD polypeptides, however (B. Andersson et al. 1984b). Baianu et al. found that the exchange rate for CI between bound and free environments is rapid (> 10 4 S - l ) , and that the observed increases in 35C1-NMR linewidth upon CI- binding reflect contributions from quadrupolar relaxation. By measuring the 35C1-NMR linewidth as a function of the concentration of added CI-, it is possible to construct a C1- binding curve, which can be used to calculate the KA for C1- (see Baianu et al. 1984, reviewed in Coleman and Govindjee 1988). Values obtained for KA were 0.1 M and 0.14 M at 5°C and 25°C, respectively (after transformation from binding constants to dissociation constants). From these two constants it is also possible to calcuate a binding energy of 3 kcal mole-~ for this system, which indicates weak ionic binding. This is consistent with the known reversibility of C1- assocation with the membrane. Critchley et al. (1982) also analyzed the correlation between the relative effectiveness of various anions in stimulating Oz-evolution (which decreases in the order CI- > Br- ~ NO~ > I - ; Hind et al. 1969, Kelley and Izawa 1978) and their relative sizes and ionic field strengths. They concluded that ionic field effects and steric factors control the binding of anions, such that the anion must have a certain optimum charge and size in order to activate the enzyme. In PS II membranes, Homann (1985) and Homann and Inoue (1986) have reported a similar result. Homann found that anions such a s N O 3 have higher apparent dissociation constants than CI-, and are lost from the membrane at a faster rate. Homann and Inoue found, however, that removal of the 18kD and 24 kD polypeptides greatly diminishes the effectiveness of other anions relative to CI-, even in the presence of adequate Ca 2+ . An anion dependence of the low-temperature $2 EPR signal has also been reported (Damoder et al. 1986). The pH dependence of C1 activation of 02evolution in thylakoids and PS II membranes has been examined numerous times since the early 1950's. Most of these studies have been concerned with the effect of C1 depletion and re-addition on the activity vs. pH curves for Oz-evolution. Gorham and Clendenning (1952) made the crucial observation that the addition of C1- to C1- depleted thylakoids both raised the maximum rate of electron transport and shifted the pH optimum in their system from about 6.4 to about 6.8 at 20°C. The net

effect of this displacement is that the rate of 02evolution is greatly enhanced at alkaline pH. Since then, this effect has been confirmed in other systems (see, e.g., Critchley 1983, 1985). Massey (1953) has devised an explanation for how anion binding to a positive charge in the active site of an enzyme might be capable of inducing such a shift in the velocity-pH optimum. He has proposed that two catalytically active residues exist in such enzymes, and that at the pH optimum, one is protonated and the other is unprotonated. When the anion combines with a third positively-charged residue, the electrostatic effect of the (+)charge is screened, thereby raising the pK~'s of the two catalytic residues (Matthew and Richards 1982). The net effect is always an alkaline shift in the pH optimum for enzyme activity. Coleman and Govindjee (1985,1987) have incorporated Massey's idea into a hypothetical model for the catalytic action of Cl- in the OEC. Critchley et al. (1982) and Baianu et al. (1984) have used 35C1-NMR to measure directly the pH dependence of C1- binding in thylakoids from halophytes. Critchley et al. (1982) found that the excess 35C1-NMR linewidth, which reflects the fraction of C1- bound to the membrane, peaks at pH 7.2 in thylakoids from A vicennia germinans, and decreases on either side of this optimum. The pH dependence for O2-evolution shows a parallel trend. The activity vs. pH curve for these thylakoids also shows an alkaline shift as the [C1-] is increased (Critchley 1983). In the latter case, Critchley suggested that the steepness of the curve on the alkaline side indicates ionization of as many as five basic groups. However, since there is the possibility that treatment at alkaline pH causes irreversible damage to the C1- binding sites, additional tests of the pH stability of these preparations and spinach preparations are needed (see, e.g., Segel 1975, Cole et al. 1986). In glycophyte PSII membranes depleted of the 18 kD and 24 kD polypeptides, Homann (1988b) found that binding of less than 1 H + was sufficient to permit the binding of an activating Cl-ion. Other attempts have been made with preparations from spinach to characterize the pK~ of the group or groups involved in C1 binding, with the goal of determining the particular kind of amino acid involved (if the binding site is composed of amino acid residues). In spinach thylakoids,

however, complete CI- depletion is difficult to achieve. Since C1- deficiency can be induced by high pH treatment, the depletion process often involves incubation at high pH (Kelley and Izawa 1978, Theg and Homann 1982, Theg et al. 1982, Izawa et al. 1983). Chloride depletion is further accelerated in thylakoids by treatments with uncouplers such as EDTA (Izawa et al. 1969) or protonophores (Theg and Homann 1982, Theg et al. 1982), by competition with sulfate (Izawa et al. 1969), and by avoidance of illumination (Theg and Homann 1982, Theg et al. 1982). The likely explanation for these diverse phenomena is that the bound CI- ions are tightly associated with a sequestered pool of protons (Dilley et al. 1982, 1987, Theg and Homann 1982, Theg et al. 1982). Treatments which deplete this slowly-equilibrating pool therefore accelerate C1- release. The pKa for CIrelease in thylakoids is greater than 8.0, indicating that amine derivatives (Homann et al. 1983), such as lysine or the terminal alpha-amino groups of the polypeptides, might be involved. In the case of lysine, such a pK, would be anomalously low. This suggests that clusters of positively-charged residues or the proximity of bound cations (such as Ca 2+) might be involved in determining the observed pK, (see Section H). It is also interesting to note that addition of a small amount of Mg 2+ shifts the apparent pKa for CI release to lower pH (Homann et al. 1983, Homann 1985). Thorough C1 depletion of PSII membranes requries more delicate procedures than in thylakoids. Since the OEC polypeptides are not enclosed within the lumen, they are more susceptible to detachment at high pH (Chapman and Barber 1987). For this reason, pH- or anion-jump treatments are often employed (Homann 1985, Itoh and Iwaki 1986). Homann (1985) mesaured the apparent dissociation constant for C1- as a function o f p H in PS II membranes, and was able to show that C1- binding required protonation of a group having a pKa = 6.0. More recent measurements indicate that the pKa is actually 5.0 or lower (Homann 1988b). Such an acidic value for the pK~ seems to contradict the earlier observation in thylakoids that C1 release requires a pH > 8 (see discussion in Homann 1985). A discrepancy also appears when comparing the rapid rate of C1reactivation of the OEC with the relatively slow rate of C1 depletion (Homann 1985). This

9 apparent hysteresis may reflect slow conformational changes in the OEC (see Kurganov 1982). This effect may also be related to the slow changes that have been observed in the S2-state EPR signal in PS II membranes that have been dark-adapted for various periods of time before illumination (Beck et al. 1985). G. CI- effects on OEC conformation

It has long been known that mild heat treatment accelerates the dissociation of C1- from the OEC in C1- free medium (Hind et al. 1969). More recently, it was found that CI depletion accelerates the thermal inactivation of O2-evolution in thylakoids (Coleman et al. 1984, 1988) and PSII membranes (Nash et al. 1985, Coleman et al. 1988). Readdition of C1- to the thylakoids immediately before heating partially protects against the loss of activity, whereas other anions, such as Br-, N O 3 , and SO 2-, provide a lesser degree of protection (Coleman et al. 1984), in accordance with their ability to stimulate 02-evolution in unheated membranes. Krishnan and Mohanty (1984) have reported that the addition of C1- after heat treatment partially restores the Hill activity of thylakoid membranes. Chloride also appears to protect against inactivation by 0.8M Tris (Izawa et al. 1983). Protection by C1- against these two proteindenaturing treatments suggests that C1- interaction with a specific site or sites changes the energetics of unfolding or dissociation, and stabilizes the OEC against inactivation, by analogy with other ligandenzyme systems (see, e.g., Zyk et al. 1969, Gill et al. 1985). H. Comparisons with other Ci- binding enzymes and proteins 1. Introduction

As noted in Section A, C1 is involved in the mechanisms of numerous enzymes and proteins that catalyze hydrolysis, protonation/deprotonation, and proton transport. The objective of this section is to analyze several of these proteins and identify the features they may have in common with the OEC, in order to shed more light on the mechanism by which Cl- activates O2-evolution.

Two competing hypotheses which seek to explain how Cl- binds to the OEC are basically the same as those that have been proposed for other metalloprotein systems, namely, the "metal-site" hypothesis and the "protein-site" hypothesis. The controversy between the two will be emphasized in the subsequent discussion. As will be demonstrated by some of the examples, they are not always mutually exclusive for a given protein, and it is not always easy to conclusively prove one or the other. Potential binding sites for C1- in proteins include the amino acid side chains of histidine, lysine and arginine, the terminal alpha-amino groups, and a number of metal cofactors. Interactions between various charged side chains and between bound metal ions and these groups may also strongly influence the affinity of the binding site for C1-. Chloride ligation to a bound metal atom usually involves competition with the solvent water, and the observed affinity for C1- is relatively low. In cases where C1- binds directly to the protein, one must also consider the state of protonation of the positively-charged C1- binding groups, which depends on the charges present within the immediate environment of the binding site itself and on any linkage that may exist between the binding site and other regions of the protein. A complete description of CI- binding to any given site is therefore complicated by a number of other factors.

2. CI- binding to metal sites

There are a number of CI- requiring metalloenzymes that have been studied in detail (see Table 1). Myeloperoxidase (1), for example, is an iron chlorin-containing enzyme that has two C1binding sites. One of these sites, which is unaffected by pH, is believed to be the C1- substrate binding site (Andrews and Krinsky 1982). Chloride binding to the second site is pH-dependent, such that the site must be protonated (pKa ~ 4.5) before C1will bind. Chloride binding at this second site is competitive with H202 binding (Andrews and Krinsky 1982). Ikeda-Saito and Prince (1985) found that 0.1 M C1- affects the metal center by lowering the Em for the Fe(II)/Fe(III) redox transition from 143mV to 21mV at pH4.3, but has very little effect at pH 7.9. Chloride binding also appears to reduce the rhombicity of the high-spin

10 Table 1. A comparison between the C1- binding properties of various enzymes and proteins and those of the OEC. See Sections H and I for a detailed discussion.

1)

Enzyme/protein

Role of CI-

Nature of binding group(s)

Myeloperoxidase

Substrate

Unknown. Possibly chlorin iron Protonated amino acid residue or chlorin iron

Inhibitor

2)

Chloroperoxidase

Substrate Allosteric (?) inhibitor

Heme-bound intermediate Prontonated amino acid residue or heme iron

Equivalent features of the OEC

CI- effect on EPR spectrum of the metal center (restoration of g = 2 multi-line signal). Group with acidic pKa controls C1 binding near active site Group with acidic pKa controls C1- binding near active site

3)

Ferricytochrome c peroxidase

Weak inhibitor

Heme iron

Possible local change in structure around metal site due to C1- binding. Group with acidic pK, controls C1 binding near active site

4)

Horseradish peroxidase

Weak inhibitor

Heme iron

Group with acidic pKa controls anion binding near active site

5)

Cytochrome oxidase

Unknown. Affects redox interaction between metal centers

Cytochrome a3Cu B site

Possible C1- effect on redox properties of the PS II complex (as indicated by thermoluminescence)

6)

Alkaline phosphatase (Cd/Mn-substituted)

Possible activator, bridging ligand between metal centers

Metal center

Possible involvement of C1- as a bridging ligand between Mn. Low-field Mn EPR signal at g = 4.1

7)

Carboxypeptidase A

Inhibitor

Probaby Arg

C1- binding to groups with high pKa

8)

Alcohol dehydrogenase

Inhibitor/activator (depending on the nature/concentration of coenzyme and substrate

Zn or Arg

C1- binding to more than one site

9)

Ribonuclease A

Activator at low [C1 ]

Probably His (2) at active site

C1- induced shift in velocity-pH optimum. Catalytic effect of C1involving group(s) with acidic pKa's

Angiotensin converting enzyme

Allosteric activator or inhibitor (depending on the substrate)

Possibly Lys

C1 induced alkaline shift in velocity-pH optimum. C1- binding to groups with high pKa

(10)

11 Table 1. A comparison between the CI binding properties of various enzymes and proteins and those of the OEC. See Sections H and I for a detailed discussion.

Enzyme/protein

Role of C1-

Nature of binding group(s)

Equivalent features of the OEC

(I 1)

Pancreatic alphaamylase

Allosteric activator

Probably Lys

(12)

Serum Albumin

Unknown. Affects conformation, H ÷ uptake

His, alpha-amino, Lys

CI- induced alkaline shift in velocity-pH optimum. CI- binding to groups with high pK a. C1- induced suppression of H ÷ exchange. CI- induced conformational changes. C1- replacement by other monoanions. Linkage between CI-/Ca 2÷ binding. Protection by C1 against heat inactivation. Very low K~ for C1- (10 -4 to 10 -5 M) C1- binding to basic groups. C1- induced suppression of H ÷ exchange. CI- induced conformational changes. Linkage between C1-/Ca 2+ binding. Multiple C1binding sites

(13)

Hemoglobin

Allosteric effector. Stabilizes deoxy conformation

Alpha-amino, Lys, Arg (O2-1inked alkaline Bohr effect); His (alkaline Bohr effect)

CI- induced alkaline shift in reactivity at active site. C1 binding to basic groups. C1- induced suppression of H ÷ exchange. C1- induced conformational changes. Stabilization of conformation by C1-

(14)

Halorhodopsin

Anion transport

Probably Arg

CI- induced alkaline shift in pK~ at active site. C1binding to groups with high pKa. Anion effectiveness depends on size/charge

(15)

Erythrocyte Band 3 anion tranport protein

Anion transport

Possibly Arg

CI- binding to groups with high pKg. C1- induced conformational changes

EPR signal. These authors interpret the interaction between U 2 0 2 and CI- as a competition between the two ligands for direct binding to the Fe at the sixth coordination position. They also suggest that although C! displacement of the Em is inhibited by high pH, CI- and O H do not compete directly (or at least not to the same enzyme conformation), since the enzyme appears to have a substantially higher affinity for O H - . A similar conclusion was drawn earlier by Critchley et al. (1982) regarding

apparent competition between binding of C1- and O H - to the OEC in thylakoids from halophytes. Two C1 binding sites have also been proposed for a somewhat similar enzyme, chloroperoxidase (2; Table 1) (Lambeir and Dunford 1983). One of these sites, which binds C1- only at low pH, inhibits the formation of compound I (a transient oxoenzyme complex). Lambier and Dunford attribute this inhibition to direct binding of C1- to the sixth coordination position of the heine iron because of

12 the observed competition between C1- and C N - , an anion which is known to occupy that site. Chloride is also a substrate, however, since it reacts with compound I to form a second complex known as the halogenating intermediate (Libby et al. 1982, Sono et al. 1986, Dawson 1988). Sono et al. argue that this second CI- binds covatently to compound I to form the halogenating intermediate, and does not ligate directly to the iron. They also suggest that the inhibitory CI- ion interacts primarily with a protonated amino acid residue in the vicinity of the active site. The catalytic cycle for this enzyme has recently been reviewed (Dawson and Sono 1987). Inferricytochrome c peroxidase (3; Table 1), C1can only bind to the sixth position of the heme iron at pH < 5.5, when a nearby residue is protonated (Hashimoto et al. 1986). Protonation of this residue, which is believed to lie on the distal side of the heme, is proposed to cause a structural change in the heme environment (Anni and Yonetani 1988, Mauro et al. 1988, Smulevich et al. 1988a, b). This change involves movement of a key H 2 0 molecule in the sixth coordination position, such that C1access to the Fe is no longer blocked (Yonetani and Anni 1987). The nature of the interaction between the distal groups and the heme is currently being investigated by a number of methods (Ortiz de Montellano 1987, Yamazaki and Nakajima 1988, Smulevich et al. 1988a, b). In horseradish peroxidase (4; Table 1), C1- (but not N O 3 ) is suggested to bind directly to the sixth coordination position of the heme iron (Araiso and Dunford 1981). Nitrate is believed to bind instead to another nearby site on an amino acid residue whose protonation at low pH is required for binding. Binding of F - to the iron, however, depends on protonation o f a herne-linked acid group (pKa < 6; Dunford and Alberty 1967), and so this requirement may hold for C1 as well. At high pH, resonance Raman measurements indicate that OH may be coordinated to the iron (Sitter et al. 1988). The transition between the acid and alkaline forms of isozyme A (with carbon monoxide as a ligand) appears to involve deprotonation of a single distal group, probably a histidine imidazolium side chain (Lee et al. 1988). It is interesting to compare the CI binding properties of these peroxidases with those of the OEC. Although CI- is not involved as a substrate

in water oxidation, "hydrogen peroxide" has been proposed as a Mn-bound intermediate (see, e.g., Kamabara and Govindjee 1985, Renger 1987); it also appears to interact with the catalytic Mn in PS II membranes when the extrinsic polypeptides have been removed (Berg and Seibert 1987). The secondary C1- binding site on some of the peroxidases, which may involve the iron, may have relevance to C1- activation of the OEC, since pHdependent C1 binding to this site affects the redox and EPR properties of the metal center, at least in the case of myeloperoxidase. Chloride binding to the OEC likewise affects the EPR properties of the $2 state (see Section El), and may also influence the redox state of the complex (Vass et al. 1987). The pKa'S of groups which control C1- binding in the peroxidases are somewhat acidic, not unlike what has been observed for C1- activation of the OEC (Homann 1988b). Activation of C1- binding may perhaps occur directly (possibly by protonation of a distal imidazole), or indirectly (by protonation of a proximal carboxylate that interacts with the proximal histidine). It is important to remember, however, that the peroxidases are generally inhibited by C1- (Saunders et al. 1964). Another heme-containing enzyme, cytochrome c oxidase (5; Table 1), binds C1 specifically at the cyt a3/CuB site, but only when both metal centers are oxidized (Blair et al. 1986). This binding alters the redox interaction between the metal centers. Chloride activates the enzyme at low concentrations (50mM or less), and inhibits it at higher concentrations (Kadenbach et al. 1988). Cytochrome c oxidase also interacts with Ca 2+ at the cyt a site (Saari et al. 1980). Chloride binding to zinc-, cadmium-, and manganese-containing active sites has also been examined, although the metal coordination chemistry is not as well characterized as in the heme complexes. In alkaline phosphatase (6; Table 1), which in its native form is a Zn-enzyme, 35C1-NMR and ~3Cd-NMR were used by Gettins and J.E. Coleman (1984) to study CI binding to the ll3Cdsubstituted enzyme. For the phosphorylated, fully Cd-substituted enzyme, they concluded that C1 binds directly to one of the Cd ions by displacing H20. They also proposed that this displacement activates the enzyme by accelerating the dissociation of the product (phosphate). (This is in contrast to the inhibition observed in carbonic anhydrase,

13 where CI- binding at or near the Zn atom displaces the substrate O H - (or H20) that is essential to the reaction (Ward and Cull 1972).) An arginine at position 166, which lies near the two Zn atoms in the E. coli enzyme, is likely to assist in phosphate binding (Chaidaroglou et al. 1988). In a phosphatefree, partially Cd-substituted form of the enzyme, Gettins and Coleman suggest that CI- might be involved as a ligand between the two metal centers, which are 3.9 A apart. A similar bridging role for CI- in the OEC has been proposed (see Section D), but there is no extensive evidence for either model. 35CI-NMR studies of another Zn-enzyme, carboxypeptidase A (7; Table 1), led to the conclusion that the enzyme has two CI- binding sites with fairly low affinities, and that the active site Zn atom is the primary C1- binding site (Stephens et al. 1974, Stephens and Bryant 1976). Since C1- is actually an inhibitor of the enzyme, Williams and Auld (1986) examined the effect of CI- on the enzyme kinetics, and found that CI- is a partial competitive inhibitor of the substrate. Because the observed inhibitor dissociation constant ( ~ 50 mM) was much less than the lowest dissociation constant obtained by 35C1-NMR, they concluded that CI- binds primarily to a subtrate-binding amino acid residue in the active site (Arg 145) and not directly to the Zn. Chloride binding to a third Zn enzyme, liver alcohol dehydrogenase (8, Table 1), has also been suggested to occur at the catalytic metal ion in the horse enzyme in the presence o f N A D ÷ at low pH (Maret and Zeppezauer 1986). In the absence of the coenzyme N A D ÷ , C1- most likely binds to Arg 47 in the native horse enzyme, since 35C1-NMR measurements do not indicate direct ligation to Zn (I. Andersson et al. 1979). Chloride activation of the human enzyme is greatly reduced by an Arg ~ His mutation at position 47 in the flz subunit (Buhler and Von Wartburg 1984, Erhig et al. 1988). The influence of anions on the N A D H - L A D H association reaction also may involve global as well as local electrostatic shielding effects (Lively et al. 1987). These examples underscore the difficulty in unequivocally demonstrating C1- binding to protein-linked metal ions (to the exclusion of other potential sites), even in isolated, purified proteins. Well-characterized examples of C1- binding to Mn proteins are relatively scarce. One unusual case involves the Mn(II)-substituted form of alkaline

phosphatase (Haffner et al. 1974). The lowtemperature X-band EPR spectrum of this protein displays both a g = 2 signal and a prominent signal at g = 4.3, which is characteristic of a d 5 transition metal in a ligand environment with a large rhombic (tetrahedral) distortion (Reed and Markham 1984). Similar low-field EPR signals have been observed in organo-metallic Mn complexes with C1- as a contributing ligand (Dowsing et al. 1969). A low-field signal has been observed in the OEC S2-state at g = 4.1 (Casey and Sauer 1984), but it is thought to involve only the higher valence states of Mn (dePaula et al. 1986, Aasa et al. 1987). EPR data also indicate that C1- is unlikely to bind to the EPR-observable Mn in this state (Yachandra et al.1986b), and that C1- depletion does not eliminate the g = 4.1 signal (Ono et al. 1986). The conclusion that can be drawn from these comparisons with respect to the OEC is that reversible CI- binding to the catalytic Mn center would not be expected to stimulate water oxidation in those S-states where bound H 2 0 (and possibly H202 and O H - ) is involved, since CI- is capable of displacing H 2 0 from the metal coordination sphere (although mechanisms have been discussed in which ligand exchange could be advantageous for controlling the oxidation potential of Mn (Dismukes 1986)). Oxidation of C1- might also be a problem. Stable coordination of C1 as a bridging ligand between Mn atoms is a theoretical possibility, but it is not supported by observations of the Mn ligand environment in the S~ and $2 states (see Section D).

3. CI- binding to amino acid residues In addition to the enzymes described in the previous section, there are a number of transport proteins and enzymes that are activated by C1binding to particular amino acid residues. This section will focus on seven well-known C1 binding proteins and compare them to the OEC with respect to the composition of their Cl- binding sites, the C1- dependence of H + binding, and any C1mediated conformational changes. The interaction of pancreatic ribonuclease A (RNase A; 9, Table 1) with various anions has been extensively studied. The enzymatic activity has

14 been shown to be sensitive to the ionic strength of the suspending medium. Kalnitsky et al. (1959) found that increasing the ionic strength (primarily by increasing the C1- concentration) at pH 5.3 activates the enzyme when the ionic strength is less than 0.2. At higher ionic strength, the enzyme is inhibited. (The OEC has a similarly high salt requirement when the extrinsic polypeptides are removed (Miyao and Murata 1985).) Increasing the ionic strength above 0.1 also results in a downward shift in the pH optimum of RNase A, as well as a decrease in activity (Kalnitsky et al. 1959, Irie 1965). These studies suggest that CI binding to the protein alters the ionization state of critical residues in the active site. On the basis of calculations from electrostatic models, the active site of RNase S (a catalytically active subfragment of RNase A) is predicted to have two partially occupied anion binding sites at pH6.0 (Matthew and Richards 1982). Earlier measurements of H ÷ and C1- binding had indicated that at pH 6.6, 0.5 moles of CI- ions are bound per mole of RNase A. When the basic residues are protonated at pH 4.5, 2.0 moles of Clare bound (Saroff and Carroll 1962). The positive electrostatic potential giving rise to the two anion binding sites is suggested to result from contributions by several basic amino acid residues in the vicinity, notably Hisl2, Hisll9, and Lys41 (Matthew and Richards 1982). The clustering of charges within the active site of RNase A has a significant effect on the protonation state of these basic residues. The anomalously low pKa of Lys41, for example, has been attributed to the presence of nearby Arg39 (Carty and Hirs 1968). Jentoft et al. (1981) modified Lys41 by ~3Cmethylation and observed its ~3C-NMR chemical shift as a function of pH. They concluded that a conformational change involving the movement of Lys 41 may be coupled to ionization of a particular histidyl residue, probably His l2. The apparent pKa's of His 12 and His 119 are also predicted to be pH dependent, as a consequence of the alteration in their electrostatic environment (Matthew and Richards 1982). Chloride binding alters their protonation states still further. As the [C1-] is increased, the affinity of these two residues for H ÷ increases, consistent with their involvement in CI- binding (reviewed in Matthew and Richards 1982). The degree to which CI- binding raises the pK, of either

His 12 or His 119 is predicted to depend on the distance from each His residue to each of the C1- binding sites. The protonation state of these two histidyl residues is extremely important to the observed enzyme activity, since His 12 is proposed to act as a proton acceptor and His 119 as a proton donor in the catalytic mechanism (Roberts et al. 1969). Other effects on C1- binding at low pH have also been observed. Protonation of carboxylate groups by lowering the pH below 4.5 leads to the binding of additional C1- ions (Loeb and Saroff 1964). Loeb and Saroff attributed this linkage between H ÷ and C1- at low pH to the existence of charge pairs within discrete clusters of acidic and basic residues. They proposed that neutralization of the acidic residues by H ÷ allows CI to bind to the positivelycharged residues. The complex pH-dependent CI- binding behavior in RNase A demonstrates the important contribution of electrostatic interactions between H + , C1 , and protonatable amino acid residues to the activity of the enzyme. Thus, depending on the ionization state required for catalytic activity, C1may either activate or inhibit catalysis at an enzyme active site. Another enzyme whose activity can be enhanced by the presence of C1- is the angiotensin converting enzyme (ACE; 10, Table 1), a Zn-containing hydrolase (exopeptidase) involved in blood-pressure regulation (for a review, see Cushman and Ondetti 1980). In one study, C1- was found to raise Vmaxby over four-fold and lower the substrate Km by sixfold (Rohrbach et al. 1981). Overall, the enzyme requires about 100mM C1 for maximal activity (Tsai and Peach 1977), although the C1- requirement depends on the nature of the substrate. The role of C1- as either an essential or non-essential activator also depends on the particular substrate (Bunning and Riordan 1983). As with RNase A, C1- binding affects the height and position of the activity-pH optimum, shifting the curve to the alkaline side. Unlike RNase, however, ACE is still fully active at [C1-] above 0.1 M. The activity-pH curve for ACE is also considerably broadened at high [C1 ]. The K~ for C1shows a pH-dependent increase between pH6.0 and 9.0 (changing from 3.3mM to 190mM), and a critical lysine residue has been implicated in the binding (Shapiro and Riordan 1983). The precise mechanism by which CI- activates the enzyme is

15 not known, but may involve stabilization of a particular conformation at an allosteric site (Tsai and Peach 1977, Shapiro and Riordan 1984). Pancreatic alpha-amylase (PAA; 11, Table 1) is a soluble enzyme that hydrolyzes oligosaccharides and requires both C1- and Ca 2÷ as cofactors (for a review, see Karn and Malacinski 1978). The enzyme has an extremely high affinity for both of these ions. Calcium, which appears to stabilize the protein structure (Caldwell and Kung 1953) binds at both a low-affinity and high-affinity site. The latter has a K D of 5 x 10 -12 M (Payan et al. 1980). Calcium alone does not activate the enzyme in the absence of C1- (Caldwell and Kung 1953). This has its parallel in the OEC. The high-affinity Ca 2+ binding site has been identified in the recent 2.9 ,~resolution X-ray crystal structure of PAA (Buisson et al. 1987). The site is unusual for C a 2+ -binding proteins in that it creates an ionic bridge between two different protein domains. The role of C a 2+ in this enzyme may be to maintain the proper orientation of the catalytic residues in the active site cleft. Chloride seems to function as an allosteric activator of PAA, with a KD = 0.3mM at 25°C (Levitzki and Steer 1974). It raises Vmax 30-fold, but has no effect on the substrate KM. Chloride binding appears to trigger a small, localized conformational change which suppresses the exchange of 26 protons and increases the apparent affinity of the enzyme for Ca 2÷ by 240-fold. In a later study, Lifshitz and Levitzki (1976) determined that there is one CI binding site per molecule, a site which probably consists of a lysine epsilon-amino group with a pK~ = 9.1. Calcium binding near this residue was suggested to explain the low pKa. When this lysine is selectively modified, C1 binding is abolished, but substrate binding is not affected. The persistence of a low basal activity following this modification suggests that C1- is a non-essential activator (Lifshitz and Levitzki 1976). The crystal structure of PAA indicates that the critical lysine may be Lys257, although Arg 195 and Arg 337 are also nearby (Buisson et al. 1987). The position of the C1 binding site on a local symmetry axis (near the active site) may explain the wideranging effects of this anion on PAA activity. Chloride binding protects the enzyme against inactivation by heating at 40°C (Caldwell and Kung 1953). This also has its parallel in the OEC (see Coleman et al. 1984, 1988, Nash et al. 1985). As in the OEC, the function of C1 can be replaced

by other monoanions, although they exhibit reduced effectiveness as activators (Levitzki and Steer 1974). The dependence of the activity on the anionic size is similar to what has been observed in spinach thylakoids (see Critchley et al. 1982), and may reflect the relative strength of the electrostatic interaction between the anions and charged groups within the protein (see the discussion for halorhodopsin, below). Unlike the OEC, the pH-activity curve for PAA is fairly broad (Wakim et al. 1969), indicating that the pKa's of the active site groups are well separated. Chloride binding does, however, shift the optimum to substantially higher pH, in addition to raising the relative velocity. The behavior of C1- in PAA bears some similarity to the C1-/OEC system, especially with respect to the interdependence of CI- and Ca 2+ (see, e.g., Cammarata and Cheniae 1987, Coleman et al. 1987c, Section J) and the suppression of H ÷ exchange by CI- binding. Other similarities include the low apparent dissociation constant for CI (0.3 mM), the basic pKa for the C1- binding site, and C1- induced resistance to heat inactivation. These features most closely resemble what is observed for binding of the "bulk" C1- to thylakoids (Homann 1983) when the extrinsic polypeptides are present (see Sections, B, C, E, F). Albumins from human and bovine serum (HSA and BSA, respectively; 12, Table 1) are examples of the most basic class of CI- binding proteins, and have been exhaustively studied as model systems for ion binding to proteins (see Peters 1985, Ocon and Reboiras 1987, Ocon et al. 1987). In 1949, Scatchard and Black reported that the binding of CI- by HSA shifts its isoionic point (see Vesterberg 1971) to higher pH, due to electrostatic interactions between the negatively charged ion and positivelycharged amino acid residues in the protein. Thus, the net effect of C1- binding is to protonate acidic groups on the protein and thereby raise the pH of the suspending medium (Louvrien and Sturtevant 1971), a process which tends to preserve the electroneutrality of the macromolecule (Ifft and Vinograd 1966). Chloride binding has been suggested to cause a conformational change in the protein that leads to a "tightening-up" of the structure (Louvrien and Sturtevant 1971). The magnitude of the observed shift in the isoionic point also depends on the nature of the monoanion employed (Scatchard and Black 1949). The protein contains such a large number of CI

16 binding sites that they can only be grouped into classes (Scatchard and Yap 1964). Class 1 has a KD = 1.4mM and consists of one group with a pK~ -- 8.0 (possibly an alpha-amino group). Class 2 has a KD = 16mM and consists of four groups with a pKa = 6.1 (probably imidazole). Class 3 has a KD = 100mM and consists of 22 groups with high pKa'S. Chloride binding to the lower affinity sites has also been measured by 35C1-NMR (Norne et al. 1975). BSA/HSA appears to undergo several changes in conformation as the pH is lowered (Peters 1985). When the pH is lowered below 5, additional C1binding sites appear, possibly as a result of exposure of sequestered basic groups whose carboxylate partners have been protonated (Saroff 1959). A conformational change in the neutral region appears to be affected by Ca 2+ (Harmsen et al. 1971), which is known to bind to the protein (Edsall et al. 1950, Saroff and Lewis 1963). This transistion may involve a shift in the pKa'S of imidazoles involved in internal salt bridges (Harmsen et al. 1971). The capacity of the OEC to bind C1at multiple sites in response to acidification of the thylakoid lumen appears to be fundamentally related to the binding phenomena observed in the albumins, although the overall architecture of the OEC is evidently much more complicated. A thorough summary of the extensive literature on Cl binding to hemoglobin (Hb; 13, Table l) is beyond the scope of this review, and therefore only the key elements will be discussed here. Chloride binding to this protein is intimately involved in a phenomenon known as the alkaline Bohr effect (for a review, see Perutz et al. 1980, 1987). The effect can be summarized as follows: As the pH falls from 9 to 6, the oxygen affinity of Hb drops, and the protein is converted from the oxy to the deoxy form. Simultaneously, however, the H + affinity rises, enabling the protein to absorb H ÷ as 02 is released. Chloride appears to stabilize to both H ÷ uptake and the oxy ~ deoxy conversion by binding preferentially to the deoxy form (Benesch et al. 1969). Chloride binds to at least three residues in deoxy Hb: (1) Val 1~, where it may be interlocked in a salt-bridge between the Val l~ or-amino group and the guanidinium group of Arg 14let on the other a-chain (Arnone et al. 1976, O'Donnell et al. 1979). This binding shifts the pKa of Val l~t upward in

deoxy Hb (O'Donnell et al. 1979). (2) Lys 82fl (Arnone 1972, Nigen and Manning 1975, Bonaventura et al. 1976, Adachi et al. 1983). (3) His 146fl (Rollema et al. 1975, Adachi et al. 1983, Matsukawa et al. 1984). Chloride binding to the highaffinity sites (Val 1ct/Arg 141 ~, His 146fl; KD = 0.01M) and to other, weaker sites (KD = 10M; possibly Lys82fl; see Perutz et al. 1980) has been measured by 35C1-NMR (Chiancone et al. 1972, 1975). The first two sites are believed to be the most important for the oxygen-linked binding of Cl (Amiconi et al. 1981, Currell et al. 1986, Di Cera et al. 1988). Chiancone et al. (1975) suggest that the involvement of C1- in the binding of H ÷ is fairly direct. For example, they propose that lowering the pH to 6.0 enhances C1- binding to deoxy Hb because the carboxyl group of Asp 94fl, which is normally salt-bridged to His 146fl, is partially neutralized, enabling C1 to bind to a site on His 146fl. Chloride binding to a basic group also would be expected to raise the pKa of that group, thereby stabilizing H ÷ binding (Rollema et al. 1975, O'Donnell et al. 1979). Studies of human hemoglobin have found that increasing the Cl concentration from 10-3M to 2 M shifts the maximum O2-pressure required for half-saturation to higher pH (Amiconi et al. 1981). In hemoglobins of certain mammals other than man, the effect of C1- on the pH-dependent 02 affinity can be very pronounced (Bonaventura et al. 1974, Fronticelli et al. 1984). In lemur Hb, for example, C1 reduces the 02 affinity at pH > 6 to nearly the same extent as the effector 2,3-diphosphoglycerate (Bonaventura et al. 1974). Chloride binding to an invertebrate (Arcid) terameric Hb has also been studied by 35C1-NMR (Chiancone et al. 1988). In this unusual protein, C1- binds to the tetrameric form with high affinity (possibly to Arg and Lys) and induces polymerization of the tetramers. This anion-linked association may explain why a maximum is observed in plots of the excess 35C1 linewidth as a function of [Cl ]. Linewidth maxima have also been observed in 35C1N M R measurements of thylakoids and PS II membranes from spinach (see Section J). The degree of similarity between the C1- binding properties of Hb and those of the OEC is not easily determined, since it is not yet known whether C1 might control the rate of O2-evolution by an allosteric mechanism, or whether amino acid salt-brid-

17 ges are involved at all in C1- binding (although recent 35CI-NMR results raise these possibilities; Coleman et al. 1987b, c, 1988). However, because of the well-characterized linkage between H ÷/C1binding and the affinity of the metal site for a substrate ligand, hemoglobin offers a potentially valuable system for future comparison. The pHdependent interaction between His146/3 and Asp 94fl is particularly interesting in light of recent evidence that a carboxylate residue could control access to the C1- binding site at the active center of the OEC (Homann 1988b, see Section I). Halorhodopsin (HR; 14, Table 1) and the erythrocyte Band 3 anion transport protein (Band 3 protein; 15, Table 1) are membrane-bound translocators of Cl- and other anions. HR is a light-driven pump which moves C1- across the bacterial membrane (Schobert and Lanyi 1982). 35C1-NMR binding studies revealed that the protein has two C1- binding sites: a high affinity site with KD = 135-150mM and a low-affinity site with Ko >> 1 M (Falke et al. 1984a). One of the sites (Site I) is relatively non-specific for C1- and will bind many other monovalent anions (Schobert and Lanyi 1986). This site is near, but not on, the retinal Schiff base chromophore (Steiner et al. 1984, Maeda et al. 1985, Schobert and Lanyi 1986). Chloride binding to this site raises the pKa of the Schiff base by about 2 units (Steiner et al. 1984, Schobert and Lanyi 1986, Schobert et al. 1986) and stabilizes the 580 nm intermediate in the photocycle (Steiner et al. 1984, Lanyi and Vodyanoy 1986). Both the binding constant of the anion and the observed ApKa of the Schiff base decrease sharply as the Stokes radius of the anion increases (Schobert and Lanyi 1986). These effects are best explained by an electrostatic interaction between the anion and the protein which depends on the size of the anion and which raises the pKa of the Schiff base (Schobert and Lanyi 1986). The other anion binding site (Site II) has a greater specificity for C1- (Steiner et al. 1984, Schobert and Lanyi 1986). Displacement of CIfrom Site I by other anions may block access to this second site (Steiner et al. 1984). Both sites may involve buried arginine residues on the extracellular side of the protein (Lanyi et al. 1988). Chloride binding to the Band 3 protein (for a review, see Passow et al. 1980, Jay and Cantley 1986) has been studied by a variety of methods,

including amino acid modification and 35C1-NMR. This protein translocates C1 across the red cell membrane by a sequence of conformational changes. An important group controlling the activity of the protein is a specific lysine residue known as Lys a, which is located near the outward facing (extraceilular) surface and which has an epsilon-amino group with a lower pKa (8.5) than those of most other Lys groups in the protein. This Lys and another nearby Lys (Lysb, pKa = 8.8) form part of a cluster of five basic amino acid residues. Due to their close proximity, their pKa's are shifted downward from the pKa of 10.6 that would be expected for typical epsilon-amino groups (Passow et al. 1980). Although this culster may be involved in the electrostatic accumulation of CI-, its direct involvement as part of the substrate binding/translocating site is not clear (Knauf and Grinstein 1982, Passow et al. 1982). The pH behavior of the substrate site is more consistent with the presence of a guanidinium group of arginine (for a discussion, see Wieth et al. 1980, Knauf and Grinstein 1982). Another positivelycharged region near the protein surface has been suggested to function as a modifier site which regulates the overall activity (Passow et al. 1982, Knauf and Grinstein 1982, see also Macara and Cantley 1981). The role, if any, of the positivelycharged residues on the cytosolic surface has not been determined (Jay and Cantley 1986). Falke and Chan (1985) examined C1- binding to the inward- and outward-facing transport sites by 35C1-NMR and determined a KD for C1 of about 90mM. Other monovalent anions, such as I - , HCO3, and F - , compete with C1- at these sites (with relative affinities in the order I > HCO3 > C1- > F- (Falke et al. 1984b)). These and other 35C1-NMR studies failed to identify a modifier site, and therefore a ping-pong mechanism with a single C1- binding site has recently been proposed (Falke et al. 1984b, c, 1985, Falke and Chan 1985). Although the OEC is not involved in transmembrane C1- transport, clustering of basic residues has been invoked as a possible explanation for C1sequestering within the thylakoid lumen (Homann 1983, Coleman and Govindjee 1987). The accessibility of these OEC sites to exchange with the bulk aqueous phase has also been examined by 35C1NMR in spinach, and was found to have a depen-

18 dence on the C1- concentration (see Section J) that is more complex than in the Band 3 protein or halorhodopsin. The greater complexity of the spinach PS II system is consistent with the possibility of a greater number of sites and a more intricate C1- binding mechansim. N M R measurements of mangrove thylakoids, however, show a simple dependence of excess 35C1 linewidth on [CI-] at high concentrations (Section F), which may reflect the absence of some of the extrinsic polypeptides in the halophyte preparations. Linewidth measurements at lower [C1-] in halophytes would provide an interesting comparison.

I. Conclusions

Comparison of what is currently known about CIbinding to various proteins with what is known about C1- behavior in the OEC provides several insights into the possible structure and operation of the photosynthetic system. Table 1 summarizes the C1- binding properties of the proteins described in Section H and compares them with the known properties of the OEC. It seems clear from this analysis that the most likely location for reversible, pH-dependent, high-affinity C1- binding (that activates, rather than inhibits, the enzyme) is a basic amino acid residue or residues, and not a metal center (see discussion in Sections D and H2). Only the direct association between C1 ions and protein side chains has been found to generate an alkaline shift of the activity-pH optimum, a phenomenon which has been described in general theoretical terms by Massey (see Section F), and which has been observed in thylakoids by G o r h a m and Clendenning. This effect is seen in nearly all of the proteins reviewed in Section H2. Every one of these proteins is believed to bind C1- at a basic amino acid residue. Upon more detailed examination, however, it is also clear that the pH-dependent behavior of C1 in spinach thylakoids and PS II membranes does not seem to resemble any one of the binding sites from these other proteins. In this respect, Homann's paradoxical finding that C1 activation has a pKa < 5, but that C1 release has a pK~ > 8 (Section F) may be an important clue. Resolution of this problem may be possible if one speculates that the OEC C1 binding/activation mechanism actu-

ally involves a hybrid structure. Thus, the activesite effect of C1- in the OEC may involve binding at or near groups with slightly acidic pKa's. These groups could be, for example, histidyl residues which, as in RNase A, are involved in H + abstraction (Coleman and Govindjee 1985, 1987, Homann 1987) and H + transfer (giving rise to a C1- sensitive, bell-shaped pH optimum). In the case of the OEC, however, the H + would be abstracted from water and then transferred out of the active site, possibly to another protein. Since C1- binding is capable of stabilizing a positive charge (H+), it could assist in stabilizing transiently-protonated bases, thereby allowing the S-states to advance as H + is released. (For earlier discussions, see Govindjee et al. 1983, Crofts and Wraight 1983). Another alternative, which Homann (1988b) has suggested, is that the acidic pKa observed for C1 binding reflects protonation of a carboxylate side chain which controls access to the positively-charged CI- binding site. The OEC might also possess a second set of sites, with greater resemblance to those in angiotensinconverting enzyme, alpha-amylase, serum albumin, and hemoglobin, containing C1- binding residues with high pK,'s (lysyl and arginyl). As in these proteins, C1 binding to such sites in the OEC might involve the sequestering of H +, indicating that inter- and/or intra-subunit salt-bridges are involved. The presence of a number of these secondary sites would explain why actual removal of most of the C1 from the OEC requires high pH treatment. A pK~ for these groups of ~ 8 would be consistent with organization into positively-charged clusters (as in Band 3) and/or with the proximity of bound Ca 2+ (as in alpha-amylase). A role for anions such as C1- in raising the pK~'s of protonatable groups at the active site may explain the observation that activity depends on the size of the anion, since, as was proposed for halorhodopsin, the magnitude of the electrostatic interaction between the anion and the bound positive charge may determine the degree to which the pK~'s in the vicinity are raised (see Matthew and Richards 1982, for RNase A). Finally, it should be noted that the stabilization against denaturation which is contributed to the OEC by C1 binding is typical of the effects of C1binding to alpha-amylase and some other CI- activated enzymes (see also Suzuki and Suzuki 1974).

19

J. Recent developments and future prospects

Acknowledgements

35Cl-NMR spectroscopy has recently been used to study C1- binding directly in C1- depleted spinach thylakoids and P S I I membranes. Measurements indicate that C1 binds to multiple sites within the O E C , since plots o f the excess 35C1- linewidth versus [C1-] contain several maxima. F r o m these d a t a it was concluded that a d d i n g C I - to C I depleted samples exposes binding sites that were otherwise inaccessible to exchange with the bulk C1- in solution (Coleman et al. 1987b, c). These linewidth maxima, which m a y reflect C I - induced c o n f o r m a t i o n a l changes, disappear after mild heating (Coleman et al. 1988) or Tris treatment ( C o l e m a n et al. 1987c). H y d r o x y l a m i n e treatment, however, does not p r o d u c e m u c h o f an effect (Coleman et al. 1987c). Measurements using polypeptide-depleted PS II m e m b r a n e s indicate that C I - binding takes place at both extrinsic and intrinsic sites (Coleman et al. 1987a, c, see also C o l e m a n and Govindjee 1985, 1987). The extrinsic site m a y consist o f a set o f sequestered d o m a i n s on the 33 k D extrinsic polypeptide. Calcium is required for high-affinity CI binding to the extrinsic d o m a i n s when the 18 k D and 24 k D polypeptides have been removed. The intrinsic site, which was examined by removing the extrinsic polypeptides, m a y involve C1- binding to the D 1 / D 2 proteins (Coleman and Govindjee 1987, C o l e m a n et al. 1987c). The experiments discussed in Sections A - G o f this review d e m o n s t r a t e that m u c h is k n o w n a b o u t the kinetics o f C l - activation o f the O E C and a b o u t the polypeptides involved in C1- binding. However, the c o m p a r i s o n with other C1- binding proteins in Section H illustrates h o w difficult it is to specify the location o f the binding sites and to elucidate the biochemical role o f CI without direct measurements o f C1- binding. A better understanding o f the function o f C1- in oxygen evolution will require m o r e detailed binding studies, in conjunction with modification o f the relevant polypeptides by proteolysis, a m i n o acid modification, and site-directed mutagenesis. F u r t h e r characterization of the relationship betwen C I - , Ca 2+, and M n binding sites will also be needed.

I thank Professor H. S. G u t o w s k y for encouragement and support during the preparation o f this review. I a m grateful to Professor Govindjee for helpful discussion, and for inviting me to submit this review to Photosynthesis Research.

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