Gerald T. Babcock

A highly resolved, oxygen-evolving photosystem II preparation from spinach thylakoid membranes

Photosystem II of chloroplast thylakoid membranes provides light-generated oxidizing equivalents which ultimately oxidize water to oxygen. The system is known to contain a reaction center chlorophyll complex, P680, a quinone acceptor denoted as Q, a P680 donor molecule designated as Z and a highpotential cytochrome b-559 [l]. The relationship between these components and the presumed manganese protein thought to be the actual site of watersplitting is as yet poorly understood. Attempts to resolve this photoreaction from intact membranes of higher plant chloroplasts by detergent action have resulted in a variety of preparations ranging from reaction centers [2,3] to more complex assemblies with vesicular structure [3]. While these preparations are generally able to support electron transfer from an added donor to an exogenous acceptor, all higher plant preparations reported are unable to carry out oxygen evolution with high activity. Photosystem II may be isolated from membranes of the cyanobacterium Phormidium laminosum [4]. These preparations, in contrast to those from higher plants, retain high rates of oxygen evolution [4] and are enriched in EPR signal II with residual contamination from PS I, as shown by the presence of some signal I (P700’) [5]. membranes; here we report the properties of oxygenevolving PS II preparations obtained by detergent resolution of chloroplast thylakoid membranes.

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.

Proton and hydrogen currents in photosynthetic water oxidation

The photosynthetic processes that lead to water oxidation involve an evolution in time from photon dynamics to photochemically-driven electron transfer to coupled electron/proton chemistry. The redox-active tyrosine, Y(Z), is the component at which the proton currents necessary for water oxidation are switched on. The thermodynamic and kinetic implications of this function for Y(Z) are discussed. These considerations also provide insight into the related roles of Y(Z) in preserving the high photochemical quantum efficiency in Photosystem II (PSII) and of conserving the highly oxidizing conditions generated by the photochemistry in the PSII reaction center. The oxidation of Y(Z) by P(680)(+) can be described well by a treatment that invokes proton coupling within the context of non-adiabatic electron transfer. The reduction of Y(.)(Z), however, appears to proceed by an adiabatic process that may have hydrogen-atom transfer character.

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.

Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochrome aa3 and cytochrome bo

Cytochromeaa3 ofRhodobacter sphaeroides and cytochromebo ofE. coli are useful models of the more complex cytochromec oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the hemea3 (oro)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.

A hydrogen-atom abstraction model for the function of YZ in photosynthetic oxygen evolution

Recent magnetic-resonance work on YŻ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YŻ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.

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.

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.

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+.