Alexander B. Hope

Electron transfers amongst cytochrome f, plastocyanin and photosystem I: kinetics and mechanisms

The review covers the theory and practice of the determination of kinetic constants for the electron transfer reactions in chloroplast thylakoid membranes between plastocyanin and cytochrome f in cytochrome bf complexes, and between plastocyanin and the reaction centre of photosystem I. Effects of ionic strength and pH are featured. The contribution of mutant studies is included. It is concluded that nearly all data from in vitro experiments can be interpreted with a reaction scheme in which an encounter complex between donor and acceptor is formed by long-range electrostatic attraction, followed by rearrangement during which metal centres become close enough for rapid intra-complex electron transfer. In vivo experiments so far cast doubt on this particular sequence, but their interpretation is not straightforward. Means of modelling the bimolecular complex between cytochrome f and plastocyanin are outlined, and two likely structures are illustrated. The complex formed by plastocyanin and photosystem I in higher plants involves the PsaF subunit, but its structure has not been fully determined.

Oxygen per flash from leaf disks quantifies Photosystem II

The quantity of functional Photosystem II (PS II) centres in vivo has been determined by the oxygen yield from leaf disks, exposed to 1% CO 2 and repetitive flashes at 4 Hz (xenon flash lamp, 3 μs), and compared with the diuron-binding capacity of thylakoids isolated from the corresponding tissue of several herbaceous species. With continuous background far-red light present during the repetitive flashing of leaves, a good correlation was obtained between the two assays of PS II, the concentration of diuron-binding sites being only slightly higher. Omitting the background far-red light reduced the estimate of functional PS II centres by only about 10%, even when the PS II/PS I ratio was nearly 2. It is suggested that PS I is able to turn over more than once per flash, thus preventing a backlog of electrons in the plastoquinone pool. The oxygen yield per flash provides a convenient, direct assay of PS II in vivo when conditions are selected so as to avoid limitation by PS I.

Electron Fluxes through Photosystem I in Cucumber Leaf Discs Probed by far-red Light.

Cucumber leaf discs were illuminated at room-temperature with far-red light to photo-oxidise P700, the chlorophyll dimer in Photosystem (PS) I. The post-illumination kinetics of P700+ re-reduction were studied in the presence of inhibitors or cofactors of photosynthetic electron transport. The re-reduction kinetics of P700+ were well fitted as the sum of three exponentials, each with its amplitude and rate coefficient, and an initial flux (at the instant of turning off far-red light) given as the product of the two. Each initial flux is assumed equal to a steady state flux during far-red illumination. The fast phase of re-reduction, with rate coefficient k1∼ 10 s−1, was completely abolished by a saturating concentration of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); it is attributed to electron flow to P700+ from PS II, which was stimulated to some extent by far-red light. The intermediate phase, with rate coefficient k1∼ 1 s−1, was only partly diminished by methyl viologen (MV) which diverts electron flow to oxygen. The intermediate phase is attributed to electron donation from reduced ferredoxin to the intersystem pool; reduced ferredoxin could be formed: (1) directly by electron donation on the acceptor of PS I; and/or (2) indirectly by stromal reductants, in line with only a partial inhibition of the intermediate phase by MV. Duroquinol enhanced the intermediate phase in the presence of DCMU, presumably through its interaction with thylakoid membrane components leading to the partial reduction of plastoquinone. The slow phase of P700+ re-reduction, with rate coefficient k1∼ 0.1 s−1, was unaffected by DCMU and only slightly affected by MV; it could be associated with electron donation to either: (1) the intersystem chain by stromal reductants catalysed by NAD(P)H dehydrogenase slowly; or (2) plastocyanin/P700+ by ascorbate diffusing across the thylakoid membrane to the lumen. It is concluded that a post-illumination analysis of the fluxes to P700+ can be used to probe the pathways of electron flow to PS I in steady state illumination.

The flash-induced turnover of cytochrome b-563, cytochrome f and plastocyanin in chloroplasts. Models and estimation of kinetic parameters

Flash-induced redox changes of cytochrome b-563, cytochrome f and plastocyanin (PC), and the electrochromic response from chloroplast suspensions in reducing conditions (added dichlorophenyldimethylurea, exogenous quinol, anaerobic) were measured in the time range 0–20 ms by deconvoluting absorbance changes at appropriate wavelenghts. No response attributable to cytochrome b-559 was observed. Various dimethylbenzoquinols with different substituents on the 6-position of the benzene ring gave similar kinetics for these processes. There was no significant effect on cytochrome b-563, f or PC kinetics of adding nonactin to decay the transthylakoid electric potential difference. These data, together with comparable data for proton deposition, were used in a parameter optimisation procedure, the Inverse Method, to produce rate coefficients for some of the partial reactions occurring when cytochrome f is oxidised by plastocyanin and quinol is subsequently oxidised by cytochrome b/f complexes. Models such as the Q-cycle or semiquinone (SQ) cycle were used to formulate differential equations describing the time-dependencies of various forms of the cytochrome b/f complex containing reduced or oxidised cytochrome f. Rieske centre, cytochrome b-563, and so forth. The Inverse Method minimised the error between data and corresponding model predictions by adjusting parameter values. A model with the two b-563 cytochromes not directly connected electronically was unsatisfactory; Q-cycle and SQ-cycle models could not be differentiated by the available data. A Q-cycle model gave a close match in all respects between data and model predictions using 8 rate coefficients. The following average, reduced rate-coefficients (s−1) were estimated for the chloroplast cytochrome b/f complex under the conditionsused: kpp (between cytochrome f and P 2000; kpp (reverse) 220; kOR (between quinol and Rieske centre) 200; kHQ (between cytochrome b-563 (high potential) and quinone at n-sites) 380; kQH (reverse) 150. The Rieske centre and cytochrome f appeared to be in rapid equilibrium, with an equilibrium constant of about 3, Rieske positive to f.

Effects of pH on the kinetics of redox reactions in and around the cytochromebf complex in an isolated system

Rate-coefficients describing the electron transfer reactions between P700 and plastocyanin, between cytochromef in cytochromebf complexes and plastocyanin, and between decyl plastoquinol and cytochromebf complexes were determined as a function of pH in the range 4–10 from flash-induced absorbancy changes at four wavelengths. The reactions between P700 and plastocyanin, and between cytochromef and plastocyanin were optimised when there was electrostatic interaction between ionised acidic groups in plastocyanin with a pKa of 4.3–4.7 and ionised basic constituents in P700 (assumed to be in the PSI-F subunit) and in cytochromef, with a pKb of 8.9–9.4. The basic groups are thought to be lysine rather than arginine. This mechanism agrees with that inferred from effects of ionic strength changes on rate-coefficients. The relation between the second-order rate-coefficient for decyl plastoquinol oxidation by thebf complex and pH was characterised by a pKa of 6.1. This is interpreted as showing that the anion radical form of that quinol, which has a pKa of 6, and which becomes progressively protonated when pH is changed from 7 to 5, is essential to reduce cytochromeb-563 (low potential) during quinol oxidation. Above pH 9, permanent effects were observed on this rate-coefficient, which were absent in the reactions between P700, plastocyanin and cytochromef.

Variable stoichiometries of photosystem II to photosystem I reaction centres

Prior to 1980 it was assumed that in the grana of plant chloroplasts the two photosystems (PS) were organized as a supercomplex. Then Andersson and Anderson (1980) proposed that PSI was totally excluded from the appressed membranes of the grana stacks, where most PS II complexes and their associated chlorophyll (Chl) a/b-proteins (LHC II) were located. Final proof for an extreme lateral heterogeneity in the distribution of P S I now comes from immunocytochemical studies which directly demonstrate that P S I is present only in non-appressed membranes (Vallon et al. 1986, Anderson and Goodchild 1987). Given that P S I and PS II complexes are structurally and functionally autonomous and that PS I is involved in both cyclic and non-cyclic electron transport, there is no need for equal or fixed numbers of P S I and PS II complexes (Anderson 1981, Anderson 1982), which were first assumed in the Z scheme and subsequently advocated by Whitmarsh and Ort (1984). Indeed, Melis and Brown (1980) first reported variable PS II/PS I reaction centre ratios in plant thylakoids. As summarized below, our research consistently shows variable stoichiometries of PS II/PS I reaction centres for plants grown either under controlled light conditions (1, 2 below) or in natural habitats (3, 4 below). (1) Growth irradiance: With increasing irradiance for growth, there is a marked increase in the amount of P680 per total chlorophyll; in contrast, the PT00/Chl ratios are unaltered. Hence there is an increase in the PS II/PS I reaction centre ratio with acclimation to increasing growth irradiance as shown in Table 1 (Leong and Anderson 1984, Chow and Hope 1987, Evans 1987, Chow et al. 1988, see also Wild et al. 1986). (2) Adaptation to altered irradiance: Following the transfer to high irradiance of pea plants fully adapted to low irradiance, marked modulations occur in the composition and function of their thylakoid membranes (Chow and Anderson 1987a, 1987b). The ratio of the PS II to PSI reaction centres increased from 1.25 to 1.7 within 6 days after transfer from low to high irradiance. (3) Shade-tolerant species: Shade-tolerant plants have lower PS II/PS I reaction centre ratios closer to unity when grown under shade or low light,

Ionic fluxes in cells of Chara corallina

Abstract The influx and efflux of Cl−, K+ and Na+ have been measured in a series of experiments with comparable cells of Chara corallina, under different experimental conditions. Several “state” of Chara cells have been found. In the state which is most often found, Cl− influx is light-stimulated and requires K+ or Na+, and K+ influx is light-stimulated and dependent on Cl− in the medium. These fractions of the Cl− and K+ influx are about 1 pmole · cm−2 · sec−1 on the average. Other states have been found in which neither Cl− nor K+ influx is light-stimulated, or in which Cl− influx is light; and K+-dependent while K+ influx is light- but not Cl−-sensitive. A residual Cl− influx in the dark is postulated to be part of an exchange system with an equal efflux of Cl−, and not active transport. Neither K+ influx nor Na+ efflux were sensitive to up to 1 mM ouabain in the medium, in contrast to reports that cells of Nitella translucens and Hydrodictyon africanum are ouabain-sensitive. A proposed passive component of the K+ influx was sometimes, but not always, consistent with observed changes in the potential difference and conductance of the plasmalemma.

A rapid, whole-tissue determination of the functional fraction of PSII after photoinhibition of leaves based on flash-induced P700 redox kinetics.

Assaying the number of functional PSII complexes by the oxygen yield from leaf tissue per saturating, single-turnover flash, assuming that each functional PSII evolves one oxygen molecule after four flashes, is one of the most direct methods but time-consuming. The ratio of variable to maximum Chl fluorescence yield (F(v)/F(m)) in leaves can be correlated with the oxygen yield per flash during a progressive loss of PSII activity associated with high-light stress and is rapid and non-intrusive, but suffers from being representative of chloroplasts near the measured leaf surface; consequently, the exact correlation depends on the internal leaf structure and on which leaf surface is being measured. Our results show that the average F(v)/F(m) of the adaxial and abaxial surfaces has a reasonable linear correlation with the oxygen yield per flash after varied extents of photoinactivation of PSII. However, we obtained an even better linear correlation between (1) the integrated, transient electron flow (Sigma) to P700+, the dimeric Chl cation in PSI, after superimposing a single-turnover flash on steady background far-red light and (2) the relative oxygen yield per flash. Leaves of C3 and C4 plants, woody and herbaceous species, wild-type and a Chl-b-less mutant, and monocot and dicot plants gave a single straight line, which seems to be a universal relation for predicting the relative oxygen yield per flash from Sigma. Measurement of Sigma is non-intrusive, representative of the whole leaf tissue, rapid and applicable to attached leaves; it may even be applicable in the field.

A reassessment of the use of herbicide binding to measure photosystem II reaction centres in plant thylakoids.

The binding of the herbicide atrazine to thylakoid membranes is often used to quantify Photosystem II reaction centres. Two atrazine binding sites, with high and low affinities, have been observed on the D1 and D2 polypeptides of Photosystem II, respectively (McCarthy S., Jursinic P. and Stemler A. (1988) Plant Physiol. 86S:46). We have observed that the accessibility of the low-affinity binding sites is variable, being limited in freshly isolated thylakoids or in fresh frozen-thawed thylakoids, but increasing during storage of the membranes on ice. In contrast, the accessibility of the high-affinity binding sites, which are titratable at low concentrations (< 500 nM) of herbicide, is much less variable, although the dissociation constant is greatly influenced by ethanol. We conclude that to quantify Photosystem II reaction centres by atrazine binding, it is sufficient and more reliable to assay only the high-affinity binding sites.

Proton uptake by the chloroplast cytochrome bf complex

The proton uptake, ΔHn+, associated with turnover of the quinone reduction site (here termed the Qn site — see abbreviations) of the chloroplast cytochrome bf complex has been kinetically resolved. It was approximately correlated with cytochrome f rereduction in a range of conditions. ΔHn+ was inhibited by the quinol oxidation site (here termed the Qp site — see abbreviations) inhibitors stigmateilin and DBMIB, but was unaffected by NQNO, an effector of the quinone reduction site (the Qn site), or by antimycin A. At external pH of 6 and 8, ΔHn+ was equivalent to about one proton per Photosystem I per flash, i.e., each turnover of the complex caused a proton uptake. At intermediate pH values on the first flashthis value was lower, but it rose to around 1 on subsequent flashes, provided that they were given sufficiently rapidly. Comparable measurements were made in the absence of nonactin so that a transmembrane potential difference was present during proton uptake. This potential, of the order of 30–50mV, had little effect on the extent of ΔHn+.