Derek S. Bendall

CYCLIC PHOTOPHOSPHORYLATION AND ELECTRON TRANSPORT

Abbreviations DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU, 3-(3,4dichlorophenyl)-1,1-dimethylurea; DNP-INT, 2-iodo-6-isopropyl-3-methyl-2â€2,4,4â€2trinitrodiphenyl ether; DSPD, disulfosalicylidenepropane-1,2-diamine; FQR, ferredoxinplastoquinone reductase; FNR, ferredoxin-NADP reductase; HQNO, 2-heptyl-4hydroxyquinoline N-oxide; HQNOBr, 2-heptyl-3-bromo-4-hydroxyquinoline N-oxide; 1

CYCLIC ELECTRON TRANSPORT IN CHLOROPLASTS THE Q-CYCLE AND THE SITE OF ACTION OF ANTIMYCIN

Cyclic electron transport systems have been set up in broken chloroplasts, with photochemically reduced ferredoxin or 9,10-anthraquinone-2-sulphonate as cofactor. In good agreement with the literature, only the ferredoxin-catalyzed pathway was found to be inhibited by antimycin; but both pathways were found to have a slow electrogenic reaction, both were inhibited by the cytochrome b-563 oxidation inhibitor 2-heptyl-4-hydroxyquinoline N-oxide (the inhibition being strongest at limiting light intensity), and the two pathways had the same proton/electron stoichiometry at limiting light intensity. It is concluded that a Q-cycle can occur in cyclic electron transport with either cofactor; and therefore that the site of action of antimycin in chloroplasts is not within the Q-cycle, as it is believed to be in mitochondria and bacteria. Instead, a ferredoxin-quinone reductase is proposed as the site of action of antimycin in the ferredoxin-catalyzed cyclic pathway. It is also concluded that the data presented here are consistent with the suggestion that the Q-cycle in photosynthetic electron transport is a facultative one, its degree of engagement depending on competition between the Rieske centre and cytochrome b-563 for reducing equivalents from plastosemiquinone.

The kinetics and thermodynamics of the reduction of cytochrome c by substituted p-benzoquinols in solution

1. The mechanisms by which p-benzoquinol and its derivatives reduce cytochrome c in solution have been investigated. 2. The two major reductants are the species QH- (anionic quinol) and Q.- (anionic semiquinone). A minor route of electron transfer from the fully protonated QH2 species can also occur. 3. The relative contributions of these routes to the overall reduction rate are governed by pH, ionic strength and relative reactant concentrations. 4. For a series of substituted p-benzoquinols, the forward rate constant, k1, of the anionic quinol-mediatd reaction is related to the midpoint potential of the QH-/QH. couple involved in the rate-limiting step, as predicted by the theory of Marcus for outer-sphere electron transfer reactions in a bimolecular collision process. 5. A mechanism for the biological quinol oxidation reactions in mitochondria and chloroplasts is proposed based upon the findings with these reactions in solution.

Emission of methane from plants

It has been proposed that plants are capable of producing methane by a novel and unidentified biochemical pathway. Emission of methane with an apparently biological origin was recorded from both whole plants and detached leaves. This was the first report of methanogenesis in an aerobic setting, and was estimated to account for 10–45 per cent of the global methane source. Here, we show that plants do not contain a known biochemical pathway to synthesize methane. However, under high UV stress conditions, there may be spontaneous breakdown of plant material, which releases methane. In addition, plants take up and transpire water containing dissolved methane, leading to the observation that methane is released. Together with a new analysis of global methane levels from satellite retrievals, we conclude that plants are not a major source of the global methane production.

The redox potentials of the b-type cytochromes of higher plant chloroplasts.

1. In fresh chloroplasts,three b-type cytochromes exist. These are b-559HP (lambda max, 559 nm; Em at pH 7, +370 mV; pH-independent Em), b-559LP (lambda max, 559 nm; Em at pH 7, +20 mV; pH-independent Em) and b-563 (lambda max, 563 nm; Em at pH 7, -110 mV; pH-independent Em), b-559HP may be converted to a lower potential form (lambda max, 559 nm; Em at pH 7, +110 mV; pH-independent Em). 2. In catalytically active b-f particle preparations, three cytochromes exist. These are cytochrome f (lambda max, 554 nm; Em at pH 7, +375 mV, pK on oxidised cytochrome at pH 9), b-563 (lambda max, 563 nm; Em at pH 7, -90 mV, small pH-dependence of Em) and a b-559 species (lambda max, 559 nm, Em at pH 7, +85 mV; pH-independent Em). 3. A positive method of demonstration and estimation of b-559LP in fresh chloroplasts is described which involves the use of menadiol as a selective reductant of b-559LP.

Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity

Absorbance changes of ferredoxin measured at 463 nm in isolated thylakoids were shown to arise from the activity of the enzyme ferredoxin-plastoquinone reductase (FQR) in cyclic electron transport. Under anaerobic conditions in the presence of DCMU and an appropriate concentration of reduced ferredoxin, a light-induced absorbance decrease due to further reduction of Fd was assigned to the oxidation of the other components in the cyclic pathway, primarily plastoquinone. When the light was turned off, Fd was reoxidised and this gave a direct quantitative measurement of the rate of cyclic electron transport due to the activity of FQR. This activity was sensitive to the classical inhibitor of cyclic electron transport, antimycin, and also to J820 and DBMIB. Antimycin had no effect on Fd reduction although this was inhibited by stigmatellin. This provides further evidence that there is a quinone reduction site outside the cytochrome bf complex. The effect of inhibitors of ferredoxin-NADP+ reductase and experiments involving the modification of ferredoxin suggest that there may be some role for the reductase as a component of FQR. Contrary to expectations, NADPH2 inhibited FQR activity; ATP and ADP had no effect.

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.

Production and HPLC analysis of black tea theaflavins and thearubigins during in vitro oxidation

Abstract A model fermentation system has been designed which utilizes pure catechins and partially purified polyphenol oxidase (EC 1.14.18.1 from green tea shoots. HPLC analysis of the products formed during in vitro oxidation has demonstrated a close similarity between this system and in vivo oxidation occurring during factory fermentation. Furthermore, changes in theaflavin and thearubigin levels, revealed by time courses of fermentation, show in vitro and in vivo systems to be qualitatively similar, although the former system produces considerably higher levels of both components. The model fermentation system, therefore, appears to be a suitable experimental system for studying the formation of theaflavin and thearubigin pigments under strictly controlled conditions. In preliminary experiments the theaflavins have been identified on HPLC profiles by enzymic oxidation of the relevant catechin pairs. Similarly, major coloured components other than theaflavins, which are considered to be thearubigins, have been shown to be formed by the oxidation and reaction of two gallocatechins (epigallocatechin and epigallocatechin gallate). The model fermentation system, in conjunction with HPLC as described in this paper, provides a means whereby precise data on theaflavin and thearubigin formation can be obtained and, in the case of the thearubigins, one which could yield additional structural information.

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.

Reactions at 77°K in Photosystem 2 of green plants

Abstract 1. 1. Light-induced changes of absorbance in the region 520–580 nm have been studied in chloroplast preparations and leaves from higher plants at the temperature of liquid nitrogen (77°K). Reactions occurring at this temperature are close to a primary photochemical process. 2. 2. Two absorbance changes were observed that represent respectively the photooxidation of cytochrome b-559 hp (high-potential component, E′0 = +0.37 V) and the photoreduction of a new pigment, P546. Both reactions have action spectra that show a “red drop” and are therefore associated with Photosystem 2. 3. 3. P546 is revealed by absorption of the oxidized form at 546 nm at 77°K in a spectrum of the difference: oxidized minus reduced. The pigment has no Soret band and is thus neither a cytochrome nor a chlorophyll. 4. 4. We suggest that cytochrome b-559 hp may be involved in the photochemical oxidation of water by Photosystem 2 and show how this might occur in terms of a linear four-step mechanism for oxygen evolution. 5. 5. Photooxidation of cytochrome f at 77°K could not be detected.