Demetrios F. Ghanotakis

Calcium reconstitutes high rates of oxygen evolution in polypeptide depleted Photosystem II preparations

Exposure of highly resolved Photosystem II preparations to 2 M NaCl produces an 80% inhibition of oxygen‐evolution activity concomitant with extensive loss of two water‐soluble polypeptides (23 and 17 kDa). Addition of Ca2+ to salt‐washed PS II membranes causes an acceleration in the decay of Z⨥, the primary donor to P‐680+, and we show here that this acceleration is due to reconstitution of oxygen‐evolution activity by Ca2+. Other cations (Mg2+, Mn2+, Sr2+) are much less effective in restoring oxygen evolution. On the basis of these observations we propose that Ca2+, perhaps in concert with the 23 kDa polypeptide, is an essential cofactor for electron transfer from the ‘S’‐states to Z on the oxidizing side of PS II.

Water‐soluble 17 and 23 kDa polypeptides restore oxygen evolution activity by creating a high‐affinity binding site for Ca2+ on the oxidizing side of Photosystem II

Exposure of detergent‐isolated preparations of the Photosystem II complex to 2 M NaCl releases water‐soluble 17 and 23 kDa polypeptides; the inhibited rate of oxygen evolution activity is stimulated by addition of Ca2+ [(1984) FEBS Lett. 167, 127–130]. Reactivation of oxygen evolution activity by Ca2+ requires the presence of the ion in high (mM) non‐physiological concentrations. Using a new dialysis‐reconstitution procedure we have shown that rebinding of the 17 and 23 kDa polypeptides restores oxygen evolution activity only when the system has not been pretreated with EGTA. Removal of loosely‐bound Ca2+ from the salt‐extracted PS II complex and from the polypeptide solution, by dialysis against EGTA, blocks reconstitution of oxygen evolution activity even though the two polypeptides do rebind; restoration of Ca2+ to EGTA‐treated systems, after rebinding of the 17 and 23 kDa polypeptides, results in a strong reconstitution of oxygen evolution activity. The effect of rebound 17 and 23 kDa polypeptides is to promote high affinity binding of Ca2+ to the reconstituted membrane.

Isolation and characterization of an oxygen-evolving Photosystem II reaction center core preparation and a 28 kDa Chl-a-binding protein

An oxygen-evolving Photosystem II reaction center complex was characterized by using both biophysical and biochemical techniques. A low-temperature EPR study of this preparation has revealed that cytochrome b-559 has been converted to its low-potential form(s); although in the presence of Ca 2 + and CI- the PS II reaction center complex shows high rates of oxygen-evolution activity, cytochrome b-559 is not converted to its high-potential form. The same study also demonstrated that Ca 2+ and Cl-, not the 17 and 23 kDa proteins, are the cofactors required for the generation of the multiline signal which is associated with the S 2 state. Further solubilization of the PS II reaction center complex, followed by gel filtration chromotography, resulted in the isolation of a purified oxygen-evolving PS II reaction center core and a 28 kDa Chl-a-binding protein. The purified oxygen-evolving preparation contains polypeptides with molecular masses of 47, 43, 32, 30 and 9 kDa as well as the extrinsic 33 kDa polpeptide. These proteins, along with manganese, chloride and calcium, appear to form the simplest structure thus far reported to retain the enzymatic activity necessary for oxidation of water to molecular oxygen.

Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Abstract Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O 2 evolution. The preparation contains four managanese, one cytochrome b -559, one Signal II f and one Signal II s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z + is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.

STRUCTURAL ORGANIZATION OF THE OXIDIZING SIDE OF PHOTOSYSTEM II EXOGENOUS REDUCTANTS REDUCE AND DESTROY THE Mn-COMPLEX IN PHOTOSYSTEMS II MEMBRANES DEPLETED OF THE 17 AND 23 kDa POLYPEPTIDES

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca2+ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH2OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH2OH, a wide range of other small (H2O2, NH2NH2, Fe2+) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca2+.

Hydroxylamine as an inhibitor between Z and P680 in photosystem II

Upon addition of hydroxylamine to chloroplasts or photosystem II preparations, the EPR signal of Z⨥ disappears and a new signal is observed. From its shape and g‐value this signal is identified with the oxidized reaction center chlorophyll, P680+. The decay of P680+ occurs with a halftime of ⪅ 200 μs and apparently is the result of a back reaction with the reduced form of the primary acceptor, QA. This mode of hydroxylamine inhibition is reversible. These observations indicate that hydroxylamine, in addition to its well known inhibitory action on the oxygen evolving complex, is also able to disrupt physiological electron flow to P680 itself.

Isolation and characterization of the 47 kDa protein and the D1-D2-cytochrome b-559 complex

The 47 kDa polypeptide and a protein complex consisting of the D1 (32 kDa), D2 (34 kDa) and cytochrome b-559 (9 kDa) species were isolated from a Tris-washed Photosystem II core complex solubilized with dodecylmaltoside in the presence of LiClO4. Although the 43 kDa chlorophyll-binding protein is readily dissociated from the Photosystem II complex under our conditions, two cycles of exposure to high concentrations of detergent and LiClO4 were required for complete removal of the 47 kDa chlorophyll-binding protein from the D1-D2-cytochrome b-559 complex. Spectroscopic characterization of these two species revealed that the 47 kDa protein binds chlorophyll a, whereas the D1-D2-cytochrome b-559 complex shows an enrichment in Pheo a and heme on a chlorophyll basis. A spin-polarized EPR triplet can be observed at liquid helium temperatures in the D1-D2-cytochrome b-559 complex, but no such triplet is observed in the purified 47 kDa species. The zero-field splitting parameters of the P-680+ triplet indicate that the triplet spin is localized onto one chlorophyll molecule. Resonance Raman spectroscopy showed that: (i) beta-carotene is bound to the reaction center in its all-trans conformation; (ii) all chlorophyll a molecules are five-coordinate; and (iii) the C-9 keto group of one of the chlorine pigments is hydrogen-bonded. Our results support the proposal that the D1-D2 complex binds the P-680+ and Pheo a species that are involved in the primary charge separation.

Polyamines in the photosynthetic apparatus : Photosystem II highly resolved subcomplexes are enriched in spermine.

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.

Purification and properties of an oxygen-evolving reaction center complex from photosystem II membranes : A simple procedure utilizing a non-ionic detergent and elevated ionic strength

A method is reported for the isolation of a highly resolved oxygen‐evolving photosystem II reaction center preparation. This preparation can be separated from the more complex photosystem II membranes isolated by the procedure of Berthold et al. [(1981) FEBS Lett. 134, 231‐234] by use of octylglucopyranoside at elevated ionic strengths; the oxygen‐evolving material can be collected by centrifugation at relatively low g values (40000 × g) in yields estimated to be more than 80%. This new preparation lacks the 17 and 23 kDa extrinsic polypeptides; addition of calcium and chloride produces activities approaching 1000 μmol O2/h per mg chlorophyll. Although activity is maximal in the presence of 2,5‐dichloro‐β‐benzoquinone, the response of activity to ferricyanide and 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea indicates that the reducing side of photosystem II has been modified in this new oxygen‐evolving reaction center preparation.

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z+ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl2, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P+Q− recombination and a slow Z+ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl2. These results are interpreted as a light-induced release of strongly bound Ca2+ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca2+.