Alison C. Stewart

Preparation of an active, oxygen-evolving photosystem 2 particle from a blue—green alga

The supply of atmospheric oxygen is largely dependent on a unique biological process, the photooxidation of water by photosystem 2 of plants and algae. Attempts to understand the mechanism of this reaction have been hampered by the lack of a pure, oxygen-evolving PS2 particle which could be subjected to detailed physical and chemical studies; most attempts to prepare such a particle have failed because of the extreme lability of the 02-evolving enzyme to many of the techniques, particularly detergent treatment, that are used for the extraction and purification of membrane-bound proteins. In recent years digitonin has been used in the preparation of increasingly pure PS2 particles from higher plant chloroplasts [l] and from the blue-green alga Synechococcus cedrorum [2]. Some of these preparations show high rates of electron transport from artificial electron donors, but all are completely inactive in 0, evolution. This report describes the purification of a highlyactive, O2 -evolving PS2 preparation from membranes of the thermophilic blue-green alga, Phormidium laminosum.

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Abstract Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum . Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria ( Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis ). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl 2 or MgCl 2 inhibited O 2 evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O 2 evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O 2 evolution in parallel.

Purification of Photosystem II particles from Phormidium laminosum using the detergent dodecyl-β-d-maltoside. Properties of the purified complex

The purification and properties of a new oxygen-evolving Photosystem (PS) II particle from the thermophilic blue-green alga Phormidium laminosum are described. The activity of the lauryldimethylamine N-oxide PS II-enriched supernatant described previously (Stewart, A.C. and Bendall, D.S. (1979) FEBS Lett. 107, 308–312) was found to be stabilized for several days at 4°;C by the addition of a second detergent, dodecyl-β-d-maltoside (lauryl maltoside). The lauryl maltoside/lauryldimethylamine N-oxide extract could be fractionated by sucrose density gradient centrifugation. Very high rates of oxygen evolution, typically 1900–2400 μmol O2/mg chlorophyll a per h at pH 7 with dimethylbenzoquinone and ferricyanide as acceptors, were observed for the lowest green band from the gradient. This fraction contained cytochromes b-559 (high-potential) and c-549, but was completely devoid of P-700 and cytochromes b-563 and f. The purified oxygen-evolving particles comprised seven major polypeptides (Mr 58 900, 52 400, 43 200, 33 900, 30 000, 16 000 and 15 000) and approximately five minor polypeptides. The particles contained 3–4 Mn atoms per reaction centre and had a chlorophyll antenna of approx. 50 chlorophyll a. The fast phase of fluorescence induction curves in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) could be described by an exponential, suggesting that no energy transfer was occurring between the PS II units responsible for this phase. Comparison of the area above the fluorescence induction curves in the absence and presence of DCMU suggested an acceptor pool size of 2–3 equivalents per centre.

Kinetics of reduction of the primary donor of Photosystem II. Influence of pH in various preparations

The kinetics of reduction of P-680, oxidized by a flash, have been measured with chloroplasts treated with Tris, hydroxylamine or cholate, with PS-II particles from Phormidium (Tris-treated) and with subchloroplast particles prepared with digitonin or Triton. In all cases the electron transfer from D1 to P-680+ has similar rates, influenced similarly by pH and D1 has a one-electron capacity.

The EPR spectrum of iron—sulphur centre B in photosystem 1 of Phormidium laminosum

The photosystem I complex of plant photosynthesis contains bound iron-sulphur centres which appear to act as terminal electron acceptors for electrons released from P700 in the primary photoreactions [ 1,2]. The centres can be observed in the reduced state by low temperature electron paramagnetic resonance (EPR) spectroscopy. In photosystem I preparations from chloroplasts, and green and blue-green algae, the centre which is first reduced, either photochemically by illumination at low temperature, or by chemical reduction, has a spectrum at g = 2.05, 1.94, 1.86. This was termed centre A [3]. On further chemical or photochemical reduction the spectrum changes to a more complex one with features at g = 2.05, 1.94, 1.92 and 1.89 [4]. This change was interpreted as due to reduction of a second centre, B [3]. The g = 1.86 feature was assumed to have shifted due to an interaction with centre A. Intensity measurements indicated that centres A and B and P700 were present in equivalent amounts [2]. However the spectrum of reduced centre B alone could not be extracted by subtraction of the spectrum of centre A from the fully reduced spectrum. During EPR spectroscopic measurements of membrane preparations and photosystem I particles from the thermophilic blue-green alga Phormidium laminosum, we noted a signal at g = 2.065,1.935 and 1.882 (kO.002) in samples reduced in the dark with dithionite. Chemical and photochemical reduction experiments indicated that it has the properties expected of centre B. The signal shows changes on reduction of centre A (including a shift of the g = 2.065 peak),

Glycerol stabilizes oxygen evolution and maintains binding of a 9 kDa polypeptide in photosystem II particles from the cyanobacterium, Phormidium laminosum

High concentrations of glycerol (⩾20% ) stabilize oxygen evolution in photosystem (PS) II particles from the thermophilic cyanobacterium, Phormidium laminosum. Treating PS II particles with lower glycerol concentrations inhibits activity and, in parallel, detaches a 9 kDa polypeptide from PS II. These results suggest that the 9 kDa polypeptide is essential for PS II activity, and that it is bound to PS II primarily by hydrophobic interactions that are strengthened in vitro by glycerol. The 9 kDa polypeptide is distinct from cytochrome b‐559 and does not appear to be associated with manganese or with the calcium or chloride requirements for oxygen evolution.

EPR characteristics of oxygen-evoloving Photosytem II particles from Phormidium laminosum

The EPR characteristics of oxygen evolving particles prepared from Phormidium laminosum are described. These particles are enriched in Photosystem II allowing EPR investigation of signals which were previously small or masked by those from Photosystem I in other preparations. EPR signals from a Signal II species and high potential cytochrome beta-559 appear as they are photooxidised at cryogenic temperatures by Photosystem II. The Signal II species is a donor close to the Photosystem II reaction centre and may represent part of the charge accumulation system of water oxidation. An EPR signal from an iron-sulphur centre which may represent an unidentified component of photosynthetic electron transport is also described. The properties of the oxygen evolving particles show that the preparation is superior to chloroplasts or unfractionated alga membranes for the study of Photosystem II with a functional water oxidation system.

The chlorophyll—protein complexes of a thermophilic blue-green alga

Valuable information about the structure and function of the photosynthetic apparatus in photosynthetic bacteria, blue-green algae and higher plants has been gained from fractionation of the photosynthetic membranes to separate the light-harvesting and reaction-centre pigment-protein complexes which comprise the photosystems. Many reports describe preparations of the reaction centre pi~ent-protein complex of PSI from both blue-green algae and higher plants, and also the higherplant light-harvesting cl-d a/b-protein complex [l-6]. These preparations have normally been obtained by detergent treatment of the photosynthetic membranes folfowed by techniques such as sucrose gradient centrifugation, ion-exchange chromatography or SDSPAGE to separate the pigment-protein complexes. However, only in recent years has comparable progress been made in the purification of the PS2 reaction centre [6-81. Satoh [8] has now obtained a highly purified preparation from spinach c~oroplasts by digitonin extraction, sucrose density gradient centrifugation, ion-exchange chromatography and isoelectric focussing. In addition, the development of much milder procedures for SDS-PAGE has enabled the amount of free c~orophyll generated during electrophoresis to be kept to a m~imum [9-l 2 3 and several laboratories have now reported finding a band on SDS-polyacrylamide gels which may correspond to

[28] Oxygen-evolving photosystem II particles from Phormidium laminosum

Publisher Summary This chapter focuses on the oxygen-evolving photosystem II particles from Phormidium laminosum. Photosystem II preparations that retain the capacity for photosynthetic oxygen evolution have been obtained from a number of thermophilic cyanobacteria. The first such preparation is obtained from the lysozyme-sensitive filamentous strain, Phormidium laminosum. Treatment of Phormidium thylakoids with the nonionic detergent lauryldimethylamine oxide (LDAO), which had been used to purify reaction centers from purple photosynthetic bacteria, preferentially solubilized photosystem II; centrifugation then yielded a supernatant that was depleted in photosystem I and enriched in photosystem II-catalyzed O2 evolution. The chapter describes the preparation of the crude LDAO particles and the purified lauryl-β-D-maltoside (LM) particles. The details given are those that one currently use and differ in certain respects from those given in the original publications. Polypeptides corresponding to the 16- and 23-kDa extrinsic proteins of chloroplast photosystem II seem to be absent, and neither the LM particles nor spheroplast extracts from Phormidium laminosum cross-react with antibodies to the higher plant proteins.

Generation and isolation of cyanobacterial inside-out thylakoid vesicles

A method has been designed to prepare inside-out thylakoid vesicles from a cyanobacterial species (Phormidium laminosum). Everted thylakoid vesicles could be generated by Yeda press treatment after induced membrane pairing. Membrane pairing was induced either by addition of high concentrations of Mg2+ ions or by lowering the pH of the fragmentation media. The inside-out vesicles were isolated by aqueous polymer two-phase partition. The membrane orientation was determined by proton translocation studies and freeze-fracture electron microscopy.