Photosynthesis Research 81: 153–163, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Regular paper
Kinetics of reactions around the cytochrome bf complex studied in intact leaf disks W.S. Chow1,∗ & A.B. Hope2 1 Photobioenergetics
Unit, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, ACT 2601, Australia; 2 School of Biological Sciences, Faculty of Science and Engineering, Flinders University, G.P.O. Box 2100, Adelaide, SA 5001, Australia; ∗ Author for correspondence (e-mail:
[email protected]; fax: +61-2-61258056)
Received 20 October 2003; accepted in revised form 6 January 2004
Key words: cytochrome bf complex, electrochromic signal, kinetics, leaf disks, Q-cycle
Abstract Electron transfers in the photosynthetic electron transport chain including the cytochrome (cyt) bf and Photosystem (PS) I complexes were studied in leaves of several plant species by measuring flash-induced absorbancy changes at specific wavelengths. The electrochromic signal (ECS), indicative of a trans-thylakoid membrane electric field, consisted of a fast phase arising from charge separation in both photosystems, and a slow rise usually interpreted as charge transfer in the cyt bf complex (part of the Q-cycle). The amplitude of the slow phase of the ECS was frequently greater than could be accounted for by the withdrawal of an electron from cyt bf via plastocyanin (PC) by oxidised P700 in PS I. The ‘extra’ slow ECS, variable depending on the number of turnovers and plant species, can be attributed to a variable operation of proton-pumping activity of the cyt bf complex. The redox kinetics of cyt f and b were obtained by deconvolution of the signals at three or four wavelengths. Rates of cyt b reduction were very high, and never the same as the onset kinetics of the slow ECS. The cyt f signal suggests that a fraction of the oxidised cyt f was re-reduced only slowly in the time of 5 s between consecutive flashes. Leaf discs in far-red light were given single-turnover flashes to measure the rates of P700ox reduction and reoxidation. To simulate the redox kinetics of the ECS, cyt f, cyt b and P700 it was assumed that a Q-cycle normally operated in bf complexes; reasonable values for the appropriate rate coefficients, and for the equilibrium constants for the cyt f/PC and P700/PC reactions were chosen. Close similarity of the observed data with those predicted from the simulation was obtained for cyt b, P700 (far-red light experiments) and the ECS, but not for cyt f. The results contribute to an understanding of photosynthetic electron transfers in vivo. Abbreviations: chl – chlorophyll; cyt – cytochrome; cyt b(H) and cyt b(L) – high- and low-potential cyt b, respectively; DCCD – dicyclohexylcarbodiimide; ECS – electrochromic signal; FNR, ferredoxin–NADP reductase; FR – far-red light; ISP – iron sulfur protein; MV – methyl viologen; P700 – the special chlorophyll pair in PS I; PC – plastocyanin; PQ – plastoquinone; PS – photosystem; STF – single-turnover flash Introduction Electron transfers between components of the electron transfer chain including the cytochrome bf complex of plant chloroplasts have been much studied using isolated chloroplasts, some intact algal cells and isolated multi-protein complexes (reviewed by
Hope 1993; Cramer et al. 1996; Hauska et al. 1996). Fewer data exist relating to the intact leaf (Kramer and Crofts 1990; Kramer and Sacksteder 1998; Joliot and Joliot 2002; the last two refer to kinetic changes during or following steady illumination). The Q-cycle (Figure 1) is a description of the oxidation/reduction of plastoquinol/plastoquinone at specific sites, Qo and
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Figure 1. A representation of the conventional Q-cycle in the thylakoids of higher plants and algae. In (1) and (2), PQH2 is oxidised at the Qo site near the lumenal side of the bf complex. As a result, two protons are deposited in the lumen, one electron reduces the oxidised ISP (FeS+ ) and the second very promptly reduces the high-potential cyt b to bH − via cyt bL . In (3) and (4), a second PQH2 is oxidised; in this case the second electron quickly reaches the Qr site, with the formation of PQH2 from proton uptake from the stroma. FeS+ is regenerated by PS I turnover, as pictured on the right and by the * sign in the left hand blocks. In the steady-state, each electron reducing P700+ via PC and cyt f is associated with two protons being deposited in the lumen.
Qr , on the cytochrome bf complex. While the concept of a mandatory Q-cycle has had a few challenges (Girvin and Cramer 1984; Cramer et al. 1996), it has become widely accepted (Sacksteder et al. 2000). However, niggling doubts remain, about an apparent discrepancy between the rates of cytochrome f re-reduction and cytochrome b reduction, the large difference being said to be incompatible with the notion of concerted reduction (Kramer and Crofts 1993) of the low- and high-potential paths in the bf complex by plastoquinol. A second ‘discrepancy’ is thought to be the observation, so far only in single-celled green algae (Joliot and Joliot 1998, in Chlorella lacking PS II; Zito et al. 1998; Deniau and Rappaport 2000; Joliot and Joliot 2001; Finazzi 2002; the last four studies used wild-type and mutant Chlamydomonas), of a slow electrochromic signal too large to be accounted for in
the Q-cycle model. The extra electrogenicity has been proposed to be due to a proton pump acting in parallel with the trans-membrane electron movement hitherto understood to lead to the slow electrochromic phase. The extra charge translocation was enhanced in the presence of DCCD (Joliot and Joliot 1998) which is assumed to block proton channels and reveal slow electrochromic processes otherwise made cryptic by fast decay of the total ECS. The putative proton pump is envisaged as operating via a gated proton channel in the bf complex (e.g., see Zito et al. 1998; Finazzi 2002). We have extended our study of leaf disks (Chow and Hope 1998) partly because of the possibilities of damage in isolated chloroplasts, and that in mutant cells the normal processes may have been modified in the bf complexes as well as in the target area of mutation (for example in deliberately PS II-modified
155 cells). With isolated complexes put together to form an electron transfer system driven by light (Hope 2000), the result is not a normal, complete electron transfer chain, and in addition, the effective retention of the dimeric conformation of the bf complexes is always in some doubt. These difficulties are absent when using leaf disks freshly cut from a healthy plant. Therefore, to inquire into the above ‘discrepancies’ in vivo, we have used leaf disks to investigate (1) electron transfer kinetics and (2) putative proton-pumping around the cyt bf complex.
Materials and methods Plants of Cucumis sativus (cucumber) were grown in a controlled growth chamber (Kim et al. 2001). Phaseolus vulgaris (dwarf bean), Vicia faba (broad bean) and Glycine max (soy bean) were grown in glasshouses with somewhat more variation in growth conditions. A few observations were made using leaves of a garden border plant, Centranthus ruber. Leaf sectors about 30 mm × 30 mm were placed in a cuvette such that the front surface was about 45◦ to the measuring beam from one of a series of interference filters of 520, 542, 554, 564, or 575 nm centre wavelength (1–2 nm bandwidth at half-maximum transmission). Dark adaptation for 30–40 min followed. A laser flash (Hope et al. 1992), or flash from an EG&G flashlamp filtered through an LP 630 red filter, struck the leaf disk at an angle 90◦ from the leaf surface. A photomultiplier tube protected by a BG-18 red-rejecting filter detected changes in absorbancy of the leaf, in the millisecond time range, due to the single-turnover flashes, given at 0.2 Hz. Usually 32 flashes were accumulated at each of four of the wavelengths, the data digitised and stored, and deconvoluted to yield component-related absorbancy changes all as previously described (Chow and Hope 1998). For the purposes of curve-fitting, all such data sets were ‘linearised’, by subtraction of the baseline before the flash, assumed to be linear, though slowly drifting upwards (increased A). In many experiments, no significant signal (
