Charles F. Yocum

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.

Oxygenic photosynthesis: the light reactions.

I: Introduction C. Yocum, D. Ort. II: Thylakoid Membranes. A: Structure. 1. Introduction, composition, heterogeneity in structure and composition A. Staehelin. 2. Evolution of thylakoid structure J.K. Hoober. 3. Evolution of thylakoid structure G.R. Wolfe, J.K. Hoober. B: Synthesis and Assembly of Thylakoid Membranes. 1. Thylakoid membrane development and assembly A. Webber, N. Baker. 2. Development of thylakoid membrane stacking L. Mustardy. 3. Biosynthesis of thylakoid membrane lipids R. Douce, J. Joyard. 4. Thylakoid membrane proteins: synthesis, import, processing, insertion C. Robinson. III: The Photosynthetic Apparatus. A: Oxygen Evolution. 1. Introduction to O2 evolution and the O2-evolving complex and role of extrinsic polypeptides T. Bricker, D. Ghanotakis. 2. Mechanism of O2 evolution: charge accumulation, Mn oxidation, S-state cycle, roles of Ca2+ and C1- D. Britt. 3. Proton release during water oxidation W. Junge. B: Photosystem II. 1. Introduction to PSII reaction center function, composition and structure K. Satoh. 2. Primary electron transfer: Z-->QA B. Diner, G. Babcock. 3. Plastoquinone reduction: 2e- gate, proton uptake, role of Fe, herbicide binding C. Wraight. 4. Role of cytochrome B559 J. Whitmarsh, H. Pakrasi. 5. PSII heterogeneity J.-M. Briantais, J. Lavergne. C: Photosystem I. 1. Introduction to PSI reaction center function, composition and structure R. Nechustai. 2. Primary electron transfer: P700 Fx R. Malkin. 3. Ferredoxin reduction and reactions of reduced ferredoxin: NAPD, thioredoxins, nitrite reductase, etc. D. Knaff. 4. Status report on crystal structure of PSI reaction center H. Witt. D: Components of Intersystem Electron Transfer. 1. Introduction to cytochrome b6f complex function and composition and structure G. Hauska. 2. Mechanism of H+ and e- transfer W. Cramer. 3. Plastocyanin: structure, location, diffusion, electron transfer mechanism E. Gross. 4. Status report on crystal structure of cytochrome f W. Cramer. E: Coupling Factor. 1. Introduction to coupling factor function and composition and structure R. McCarty. 2. Catalytic mechanism: role of subunits, nucleotide binding, interaction of CF1 with CF0 M. Richter. 3. Regulation of coupling factor activity J. Mills. 4. Status report of structure of CF1 E. Boekema. F: Light Harvesting Complexes. 1. Introduction, survey and nomenclature D. Simpson, J. Knotzel. 2. Structure, protein and pigment composition of LHC II, LHC I and other CAB species E. Pichersky, S. Jansson. 3. Excitation energy transfer: functional aspects of CAB proteins, spillover, etc. A. Melis. 4. Carotenoids: location and function H. Yamamoto, R. Bassi. IV: Molecular Biology/Genetics of the Photosynthetic App

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.

Vibronic coherence in oxygenic photosynthesis

Photosynthesis powers life on our planet. The basic photosynthetic architecture consists of antenna complexes that harvest solar energy and reaction centres that convert the energy into stable separated charge. In oxygenic photosynthesis, the initial charge separation occurs in the photosystem II reaction centre, the only known natural enzyme that uses solar energy to split water. Both energy transfer and charge separation in photosynthesis are rapid events with high quantum efficiencies. In recent nonlinear spectroscopic experiments, long-lived coherences have been observed in photosynthetic antenna complexes, and theoretical work suggests that they reflect underlying electronic-vibrational resonances, which may play a functional role in enhancing energy transfer. Here, we report the observation of coherent dynamics persisting on a picosecond timescale at 77 K in the photosystem II reaction centre using two-dimensional electronic spectroscopy. Supporting simulations suggest that the coherences are of a mixed electronic-vibrational (vibronic) nature and may enhance the rate of charge separation in oxygenic photosynthesis.

Calcium activation of photosynthetic water oxidation

As a consequence of investigations on the resolution and reconstitution of oxygen-evolution activity. Ca 2+ has also been identified as a cofactor for the reaction. The following sections assess the data currently available on Ca 2+ function in PS II, identify the areas where divergent results have been obtained, and compare properties of Ca 2+ binding to well-characterized proteins with that is known about the properties of the binding and action of the metal in PS II

THE CHLORIDE REQUIREMENT FOR PHOTOSYNTHETIC OXYGEN EVOLUTION ANALYSIS OF THE EFFECTS OF CHLORIDE AND OTHER ANIONS ON AMINE INHIBITION OF THE OXYGEN-EVOLVING COMPLEX

Abstract In the presence of Cl−, the severity of ammonia-induced inhibition of photosynthetic oxygen evolution is attenuated in spinach thylakoid membranes (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343). A further examination of this phenomenon using steady-state kinetic analysis suggests that there are two sites of ammonia attack, only one of which is protected by the presence of Cl−. In the case of Tris-induced inhibition of oxygen evolution only the Cl− protected site is evident. In both cases the mechanism of Cl− protection involves the binding of Cl− in competition with the inhibitory amine. Anions (Br− and NO−3) known to reactive oxygen evolution in Cl−-depleted membranes also protect against Tris-induced inhibition, and reactivation of Cl−-depleted membranes by Cl− is competitively inhibited by ammonia. Inactivation of the oxygen-evolving complex by NH2OH is impeded by Cl−, whereas Cl− does not affect the inhibition induced by so-called ADRY reagents. We propose that Cl− functions in the oxygen-evolving complex as a ligand bridging manganese atoms to mediate electron transfer. This model accounts both for the well known Cl− requirement of oxygen evolution, and for the inhibitory effects of amines on this reaction.

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.

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.