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The Thermodynamics and Kinetics of Electron Transfer between Cytochrome b6f and Photosystem I in the Chlorophyll d-dominated Cyanobacterium, Acaryochloris marina

2008; Elsevier BV; Volume: 283; Issue: 37 Linguagem: Inglês

10.1074/jbc.m803047200

1083-351X

Benjamin Bailleul, Xenie Johnson, Giovanni Finazzi, James Barber, Fabrice Rappaport, Alison Telfer,

Light effects on plants

We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P740) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319–13323), the midpoint potential of the electron donor P740 has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P740, via the cytochrome b6f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b6f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems. We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P740) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319–13323), the midpoint potential of the electron donor P740 has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P740, via the cytochrome b6f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b6f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems. Acaryochloris marina is an unusual cyanobacterium, because it contains mainly chlorophyll (Chl) 3The abbreviations used are: ChlchlorophyllcytcytochromeDCMU3-(3,4-dichlorophenyl)-1,1-dimethylureaDBMIB2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneP700 and P740chlorophyll dimer bearing the long-lived cation in photosystem I in Chl a and Chl d organisms, respectivelyPCplastocyaninPSI and PSIIphotosystem I and II, respectivelyRPradical pairMKmenaquinonePQplastoquinoneMES4-morpholineethanesulfonic acid. d. It was first isolated from a suspension of algae squeezed out of Lissoclinum patella, a colonial ascidian (1Miyashita H. Ikemoto H. Kurano N. Adachi K. Chihara M. Miyachi S. Nature. 1996; 383: 402Crossref Scopus (375) Google Scholar). It mainly grows as a biofilm on the undersurface of the ascidian beneath a symbiotic layer of a Prochloron, which is in the upper tunic of the ascidian (2Kuhl M. Chen M. Ralph P.J. Schreiber U. Larkum A.W. Nature. 2005; 433: 820Crossref PubMed Scopus (177) Google Scholar). Other varieties of Acaryochloris have been found on the underside of the thallus of red algae (3Murakami A. Miyashita H. Iseki M. Adachi K. Mimuro M. Science. 2004; 303: 1633Crossref PubMed Scopus (143) Google Scholar) and free living in a salt lake (4Miller S.R. Augustine S. Olson T.L. Blankenship R.E. Selker J. Wood A.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 850-855Crossref PubMed Scopus (130) Google Scholar). The general feature of its habitat is that it lives at relatively low light intensities, which are enriched in the far red region of the spectrum. Therefore, its oxygenic photosynthesis provides a typical example of adaptation to specific light conditions (2Kuhl M. Chen M. Ralph P.J. Schreiber U. Larkum A.W. Nature. 2005; 433: 820Crossref PubMed Scopus (177) Google Scholar), as evidenced by its pigment composition, where Chl d is predominant and the Chl a level is very low (5Marquardt J. Senger H. Miyashita H. Miyachi S. Morschel E. FEBS Lett. 1997; 410: 428-432Crossref PubMed Scopus (94) Google Scholar, 6Hu Q. Marquardt J. Iwasaki I. Miyashita H. Kurano N. Morschel E. Miyachi S. Biochim. Biophys. Acta. 1999; 1412: 250-261Crossref PubMed Scopus (75) Google Scholar). This pigment composition contrasts with the usual high level of Chl a found in other oxygenic phototrophes, as illustrated by the absorption spectrum of a suspension of A. marina cells, which is markedly red-shifted when compared with other Chl a-containing photosynthetic organisms. The extent of the red shift of the absorption spectrum of A. marina is of similar magnitude to that observed between Chl d and a in organic solvent (e.g. methanol) (7Manning W.M. Strain H.H. J. Biol. Chem. 1943; 151: 1-19Abstract Full Text PDF Google Scholar). chlorophyll cytochrome 3-(3,4-dichlorophenyl)-1,1-dimethylurea 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone chlorophyll dimer bearing the long-lived cation in photosystem I in Chl a and Chl d organisms, respectively plastocyanin photosystem I and II, respectively radical pair menaquinone plastoquinone 4-morpholineethanesulfonic acid. Not surprisingly, the unusual absorption spectrum of A. marina, due to the high level of Chl d, influences the spectroscopic signatures of the various cofactors involved in the primary photochemistry. For example, light-induced charge separation in photosystem I (PSI) is associated with a main absorption band (in the Qy region) bleaching maximally at 740 nm, indicating that the primary donor is composed of Chl d rather than Chl a. Hence, this pigment was named P740 (8Hu Q. Miyashita H. Iwasaki I.I. Kurano N. Miyachi S. Iwaki M. Itoh S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13319-13323Crossref PubMed Scopus (167) Google Scholar). Clearly, the energy of the quantum inducing charge separation by P740 is lower than that of P700 present in Chl a containing PSI (1.68 eV instead of 1.77 eV). This decrease by about 100 meV is significant when compared with the ∼800 meV required to drive electron transfer between the soluble electron donor to P740+ (plastocyanin) and the first soluble electron acceptor (ferredoxin). However, some specific strategies may have been developed by this organism to cope with this peculiar situation and maintain a photosynthetic activity compatible with its growth requirements. A possible rationale came from the study by Hu et al. (8Hu Q. Miyashita H. Iwasaki I.I. Kurano N. Miyachi S. Iwaki M. Itoh S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13319-13323Crossref PubMed Scopus (167) Google Scholar), who measured the midpoint potential of P740 and found that it was down-shifted by about 100 mV when compared with that of P700, the primary electron donor in PSI, where only Chl a is present. On this basis, they suggested that the lower midpoint potential of P740 would compensate for the lower energy of the absorbed quantum, thereby maintaining the reducing power of the excited state. Although such a tuning of the midpoint potential of P740 would allow a similar energetic picture for the electron acceptor side of PSI in A. marina and in Chl a-containing species, it would result in a significant thermodynamic pitfall on the donor side of PSI by decreasing the free energy gap between P740+ and its electron donors. For example, the midpoint potential of cytochrome (cyt) f, in Chl a-containing organisms is in the 320–370 mV range (9Hurt E.C. Hauska G. Eur. J. Biochem. 1981; 117: 591-599Crossref PubMed Scopus (305) Google Scholar, 10Kramer D.M. Crofts A.R. Biochim. Biophys. Acta. 1994; 1184: 193-201Crossref Scopus (50) Google Scholar, 11Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 49: 29342-29349Abstract Full Text Full Text PDF Scopus (149) Google Scholar). If cyt f in A. marina has the same midpoint potential, the driving force for electron transfer between cyt f and P740+ would be slightly uphill. This would lead to the accumulation of a significant fraction of P740+ under steady state illumination, which would obviously reduce the overall photochemical efficiency by closing most of the PSI photochemical traps. Alternatively, the midpoint potential of the redox-active cofactors involved in electron donation supply to P740+ (the Rieske FeS center, cytochrome f, and plastocyanin or cyt c6) would have to be tuned to compensate for the loss in oxidizing power of P740 so as to provide a driving force for maintaining efficient electron flux through PSI. These issues are addressed in the present work. We first determined the thermodynamic equilibrium constant of the electron transfer reaction between cyt f and P740 in actively growing cells. The equilibrium constant for this reaction was found to be ∼15, showing that the midpoint potential of cyt f is about 70 mV lower than that of the P740+/P740 couple. Consistent with this, the directly measured midpoint potentials of cyt f and of the P740+/P740 couples were 345 and 425 mV, respectively, in contradiction of the previous report by Hu et al. (8Hu Q. Miyashita H. Iwasaki I.I. Kurano N. Miyachi S. Iwaki M. Itoh S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13319-13323Crossref PubMed Scopus (167) Google Scholar) of a midpoint potential of 335 mV for the latter. The values that we have determined are similar to those found for the analogous redox cofactors in Chl a organisms for cytochrome b6f (9Hurt E.C. Hauska G. Eur. J. Biochem. 1981; 117: 591-599Crossref PubMed Scopus (305) Google Scholar, 10Kramer D.M. Crofts A.R. Biochim. Biophys. Acta. 1994; 1184: 193-201Crossref Scopus (50) Google Scholar, 11Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 49: 29342-29349Abstract Full Text Full Text PDF Scopus (149) Google Scholar) and P700 (12Nakamura A. Suzawa T. Kato Y. Watanabe T. FEBS Lett. 2005; 579: 2273-2276Crossref PubMed Scopus (29) Google Scholar, 13Witt H. Bordignon E. Carbonera D. Dekker J.P. Karapetyan N. Teutloff C. Webber A. Lubitz W. Schlodder E. J. Biol. Chem. 2003; 278: 46760-46771Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), suggesting that the midpoint potential of the primary electron donor in PSI is not necessarily affected by the chemical nature of the Chls that comprise it. This finding raises interesting questions concerning the overall energetics of PSI-driven charge separation in A. marina, which are discussed in this paper. Growth Conditions—A. marina cells were grown under low white light illumination (5–10 microeinsteins m–2 s–1) in a modified K+ESM medium (14Keller M.D. Selvin R. Claus W. Guillard R.R.L. J. Phycol. 1987; 23: 633-638Crossref Scopus (895) Google Scholar, 15Miyashita H. Adachi K. Kurano N. Ikemoto H. Chihara M. Miyachi S. Plant Cell Physiol. 1997; 38: 274-281Crossref Scopus (148) Google Scholar) supplemented with extra iron to a final concentration of 14 μm. The cyanobacterium was harvested in its midexponential phase, and the cells were resuspended in fresh medium at the desired density for experiments. Membrane Isolation—Thylakoid membranes were prepared from freshly harvested cells, which were resuspended in a medium containing 50 mm MES, pH 6.5, 20–25% glycerol (W/v), 10 mm CaCl2, 5 mm MgCl2, and 1 mm benzamidine. Cells were broken by several cycles of centrifugation in the presence of 0.1-mm glass beads (10 s), followed by a short (10 s) incubation in an ice bath. Unbroken cells were removed by a short centrifugation at 2000 × g. Thylakoid membranes were recovered from the supernatant, after an additional centrifugation at 80,000 × g for 30 min. The final pellet was resuspended in a 50 mm MES, pH 6.5, buffer, in the presence of 20 mm CaCl2 and 2.5 mm MgCl2. RNA Isolation and Reverse Transcription-PCR—RNA was isolated using a hot phenol acid method for Synechocystis sp. PCC 6803 4G. Ajlani, personal communication. adapted from Ref. 16Aiba H. Adhya S. de Crombrugghe B. J. Biol. Chem. 1981; 256: 11905-11910Abstract Full Text PDF PubMed Google Scholar. Total RNA was treated with 1 unit/μg RNA for 1 h with DNase (Promega). 350 ng of total RNA was used in a reverse transcription reaction according to the manufacturer's instructions (BcaBEST RNA PCR kit version 1.1; TaKaRa), using a specific reverse primer (5′-TTAGCCCTGAACCGTAATGG-3′) to the petE genomic DNA sequence (accession number: YP_001517679), and the entire volume was added to the subsequent PCR (94 °C 30 s, 57 °C 30 s, 72 °C 1 min for 35 cycles) using a specific forward primer (5′-TCAAATGGGGTCTTCTACGG-3′). Spectroscopy—Absorption and fluorescence spectra were measured with a laboratory-built spectrophotometer based on a diode array (AVS-USB 200; Ocean Optics). Excitation of fluorescence was at 470 nm unless otherwise indicated. Spectroscopic analysis was performed at room temperature, using home-made pump and probe spectrophotometers. Three different set-ups were employed. The kinetics of light-induced redox changes were measured with a LED-based spectrophotometer (JTS10, Biologic, France), having a time resolution of 10 μs (Figs. 2 and 5). Actinic flashes were provided by a dye laser at 690 nm, whereas measuring flashes were provided by a white LED (Luxeon; Lumileds) fitted with appropriate interference filters (10 nm full width at half-maximum). Alternatively, a second set-up having a time resolution of 10 ns (17Béal D. Rappaport F. Joliot P. Rev. Sci. Instr. 1999; 70: 202-207Crossref Scopus (84) Google Scholar) was employed, in which the actinic flashes were provided by a dye laser at 690 nm, whereas probe flashes were generated by an optical parametric oscillator pumped by an Nd:Yag laser (Figs. 3 and 4). The third set-up was for electrochemical redox titrations, which were measured as described previously (18Li Y. Lucas M.G. Konovalova T. Abbott B. MacMillan F. Petrenko A. Sivakumar V. Wang R. Hastings G. Gu F. van Tol J. Brunel L.C. Timkovich R. Rappaport F. Redding K. Biochemistry. 2004; 43: 12634-12647Crossref PubMed Scopus (35) Google Scholar, 19Alric J. Pierre Y. Picot D. Lavergne J. Rappaport F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15860-15865Crossref PubMed Scopus (78) Google Scholar) (Fig. 6). In this case, the actinic flashes were provided by a xenon flash lamp (giving saturating light pulses, 5-μs duration at half-height), passed through a Schott RG695 interference filter. Probe flashes were provided by a second xenon flash lamp (3-μs duration at half-height), filtered through a monochromator (Jobin Yvon). Titration of cyt f was performed measuring the absorption changes at 554 nm, with a base line drawn between 546 and 562 nm. Mediators were para-benzoquinone, diaminodurene, 2,5 dimethyl-benzoquinone, phenazine methosulfate, and menadione, at a concentration of 1 μm for each. Similarly, the redox potential of P740 was measured using the amplitude of the flash-induced absorption changes (at 740 nm) at various redox potentials, poised either electrochemically or chemically.FIGURE 5RT-PCR on total RNA isolated from A. marina to amplify the petE gene. Lane 1, 100-bp ladder. Lane 2, RT-PCR using petE-specific primers. Lane 3, no reverse transcriptase added to reaction as negative control. Samples were migrated for 1 h on a 1.4% agarose gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Light-induced absorption changes between 500 and 570 nm. DBMIB (5 μm) was added to slow down signal recovery between each flash and allow integration of the absorption signal. Absorption was measured 100 μs and 10 ms after the last flash. The rather featureless difference between those two spectra (triangles) is attributed to electrochromic bandshift.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Relative amplitude of the signal at 554 nm, measured 10 ms after a saturating flash as a function of the number of actinic flashes during a series of 10 closely spaced (150 ms) saturating pulses. Conditions were the same as in Fig. 3. DBMIB was present at a concentration of 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6A, in vivo measurements of P740 and cyt f oxidation under continuous illumination and consecutive rereduction. DBMIB (5 μm) was added to slow down reduction of both cytochrome f+ and P740+, in order to allow full equilibration between the redox partners. See "Results" for further explanation. Open circles, cyt f+; open squares, P740+. B, equilibrium plots for the components of the high potential chain in A marina. Relaxation signals after the continuous light (see A) of cytochrome f and P740 were normalized and plotted against each other (squares). Maximum amplitudes of redox changes, at t = 0, were set to 1. The two curves were fitted using Equations 2, 3, 4 (solid line) and the simulation (dashed line) corresponding to a redox potential of 335 mV for P740 (8Hu Q. Miyashita H. Iwasaki I.I. Kurano N. Miyachi S. Iwaki M. Itoh S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13319-13323Crossref PubMed Scopus (167) Google Scholar) and 345 mV for cyt f (our measurement).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Absorption and Fluorescence Emission Spectra—Cells of A. marina, in midexponential phase, were used to characterize the main properties of the intersystem electron transport chain in this cyanobacterium. Fig. 1, A and B, shows their absorption and fluorescence spectra, respectively. The room temperature absorption spectrum is characterized by a major peak at ∼712 nm, consistent with the pigment content of this organism, where Chl d is predominant (1Miyashita H. Ikemoto H. Kurano N. Adachi K. Chihara M. Miyachi S. Nature. 1996; 383: 402Crossref Scopus (375) Google Scholar). A second broad peak is seen in the 600–650 nm range, consistent with the presence of significant amounts of phycobiliproteins, which together with Chl d-binding Pcb proteins, provide a light harvesting system to Acaryochloris (5Marquardt J. Senger H. Miyashita H. Miyachi S. Morschel E. FEBS Lett. 1997; 410: 428-432Crossref PubMed Scopus (94) Google Scholar, 20Schiller H. Senger H. Miyashita H. Miyachi S. Dau H. FEBS Lett. 1997; 410: 433-436Crossref PubMed Scopus (53) Google Scholar, 21Chen M. Quinnell R.G. Larkum A.W. FEBS Lett. 2002; 514: 149-152Crossref PubMed Scopus (71) Google Scholar). The fluorescence emission spectra show a main peak at 724 nm at room temperature, which is shifted to 732 nm at 77 K. No significant emission from the phycobiliproteins or from detached Pcb proteins is seen, either under the conditions shown (chlorophyll-selected excitation at 470 nm; Fig. 1B) or after specific excitation of the phycobilins at 590 nm (data not shown). Thus, all of the light harvesting complexes are tightly coupled to the photochemical traps, in agreement with previous reports (22Boichenko V.A. Klimov V.V. Miyashita H. Miyachi S. Photosynth. Res. 2000; 65: 269-277Crossref PubMed Scopus (37) Google Scholar). Overall Photosynthetic Activity—The overall photosynthetic activity of A. marina was evaluated from the fluorescence induction curve shown in Fig. 1, C and D. The quantum yield of PSII, Fv/Fm = ((Fm – Fo)/Fm) (23Delosme R. Joliot P. Lavorel J. C. R. Acad. Sci. Paris. 1959; 249: 1409-1411Google Scholar, 24Butler W.L. Annu. Rev. Plant Physiol. 1978; 29: 345-378Crossref Google Scholar, 25Genty B. Briantais J.-M. Baker N.R. Biochim. Biophys. Acta. 1989; 990: 87-92Crossref Scopus (7229) Google Scholar) gave a typical value of ∼0.7 for Fv/Fm, which is in agreement with previous reports (20Schiller H. Senger H. Miyashita H. Miyachi S. Dau H. FEBS Lett. 1997; 410: 433-436Crossref PubMed Scopus (53) Google Scholar). This is higher than values for other phycobilisome-containing cyanobacteria (see Ref. 26Campbell D. Hurry V. Clarke A.K. Gustafsson P. Oquist G. Microbiol. Mol. Biol. Rev. 1998; 62: 667-683Crossref PubMed Google Scholar) for a review). In these organisms, the low Fv/Fm ratio is interpreted as reflecting either the presence of sustained energy spillover from PSII to PSI (27Biggins J. Bruce D. Photosynth. Res. 1989; 20: 1-34Crossref PubMed Scopus (127) Google Scholar), the loose excitonic connectivity between the phycobilisomes, or the photochemical traps (which is not the case here; see above), or the low PSII/PSI ratio (reviewed in Ref. 26Campbell D. Hurry V. Clarke A.K. Gustafsson P. Oquist G. Microbiol. Mol. Biol. Rev. 1998; 62: 667-683Crossref PubMed Google Scholar)). Neither of these phenomena significantly occur in A. marina, since the large Fv/Fm is incompatible with a low amount of PSII, and the shape of the fluorescence rise, measured in the presence of the PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), is clearly sigmoidal (Fig. 1C) (also see Ref. 20Schiller H. Senger H. Miyashita H. Miyachi S. Dau H. FEBS Lett. 1997; 410: 433-436Crossref PubMed Scopus (53) Google Scholar). This feature, which is commonly observed in photosynthetic eukaryotes but not in cyanobacteria, is interpreted as reflecting the progressive increase in the light harvesting capacity of a PSII photochemical unit as its PSII neighbors become photochemically inactive due to the reduction of the quinone acceptor QA (28Joliot P. Joliot A. C. R. Acad. Sci. Paris. 1964; 258: 4622-4625Google Scholar, 29Lavergne J. Trissl H.-W. Biophys. J. 1995; 65: 2474-2492Abstract Full Text PDF Scopus (226) Google Scholar). Such a feature is indicative of efficient energy transfer between closed and open PSII traps, without any significant competition by energy quenching through PSI photochemistry. Another important feature of the fluorescence data shown in Fig. 1 is the large difference between the level measured under steady state (Fs), noninhibited conditions (control in Fig. 1D) and the maximum level (Fm)(+DCMU in Fig. 1D). Since Fs is expected to reflect the steady state amount of reduced QA, this finding suggests that the limiting step in the reoxidation of the plastoquinone pool (i.e. in the electron flow downstream of PSII) is in the same range as the rate of its reduction (i.e. the number of electrons transferred by PSII per unit of time). As discussed by Joliot (28Joliot P. Joliot A. C. R. Acad. Sci. Paris. 1964; 258: 4622-4625Google Scholar, 30Rappaport F. Beal D. Joliot A. Joliot P. Biochim. Biophys. Acta. 2007; 1767: 56-65Crossref PubMed Scopus (53) Google Scholar), this can be empirically estimated from the time required to reach ∼66% of the Fm value, in the presence of DCMU. Since this value is ∼10 ms in the present experiment (Fig. 1D), it can be deduced that the number of electrons transferred per photosynthetic chain is in the 100 s–1 range (i.e. close to the maximum rate of PQH2 oxidation in vivo) (31Finazzi G. Furia A. Barbagallo R.P. Forti G. Biochim. Biophys. Acta. 1999; 1413: 117-129Crossref PubMed Scopus (141) Google Scholar). In other words, there seems to be no severe bottleneck in electron transfer downstream of PSII in A. marina. Kinetic Features of the Electron Flow Chain—Following on from above, we investigated further the properties of the electron transfer chain by directly measuring the photochemically induced turnover of the cytochrome b6f complex. Fig. 2 shows the time course of the changes occurring at 554 nm upon excitation with a single turnover saturating flash. At this wavelength, the actinic flash induces a rapid bleaching (see Fig. 2). The recovery kinetics, to the initial (dark-adapted) level, was inhibited by addition of the plastoquinone analogue, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) (32Trebst A. Methods Enzymol. 1980; 69: 675-715Crossref Scopus (276) Google Scholar), as expected if it reflects electron flow from PSII through the lumenal (Q0) site of the cytochrome b6f complex. Fig. 3 shows two spectra measured 100 μs and 10 ms after the last flash of a series of 10. Both spectra show pronounced bleach at 554 nm and a less pronounced one around 520 nm. Conversely, the absorption changes comprised between 100 μs and 100 ms (see difference spectrum in Fig. 3) were rather featureless, with a broad absorption increase peaking at 515 nm. The two bleaching bands, at 554 and 520 nm, are characteristic of the oxidation spectrum of c-type cytochrome (they correspond to the α and β bands of the reduced state, respectively; see Ref. 33Pettigrew G.W. Moore G.R. Rich A. Cytochromes c: Biological Aspects. Springer-Verlag, Berlin1987: 14-15Google Scholar). We tentatively ascribe the broad absorption change at 515 nm to an electrochromic band shift undergone by pigments embedded in the membrane upon generation of a transmembrane electric field due to PSI and PSII photochemical activity (34Witt H.T. Biochim. Biophys. Acta. 1979; 505: 355-427Crossref PubMed Scopus (435) Google Scholar). Interestingly, no absorption changes were observed at 554 nm between 100 μs and 10 ms, suggesting that there is no contribution of the electrochromic band shift at this wavelength, indicating that this wavelength may be used with confidence to follow the redox changes of c-type cytochromes. The observation of a light-induced oxidation of one or more c-type cytochrome(s) raises the question of the identity of these oxidized redox carriers. The absorption changes shown in Figs. 2 and 3 can be taken as the signature of the oxidation of either cyt f alone or with a soluble cyt c6. The latter is known to act as a soluble electron carrier between the cyt b6f complex and PSI in some cyanobacteria (35Baymann F. Rappaport F. Joliot P. Kallas T. Biochemistry. 2001; 40: 10570-10577Crossref PubMed Scopus (12) Google Scholar) (see Ref. 36Hope A.B. Biochim. Biophys. Acta. 2000; 1456: 5-26Crossref PubMed Scopus (196) Google Scholar for a review), and it cannot be ruled out as an electron carrier in A. marina, where two putative genes coding for cyt c6 are present in the genome sequence (37Swingley W.D. Chen M. Cheung P.C. Conrad A.L. Dejesa L.C. Hao J. Honchak B.M. Karbach L.E. Kurdoglu A. Lahiri S. Mastrian S.D. Miyashita H. Page L. Ramakrishna P. Satoh S. Sattley W.M. Shimada Y. Taylor H.L. Tomo T. Tsuchiya T. Wang Z.T. Raymond J. Mimuro M. Blankenship R.E. Touchman J.W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2005-2010Crossref PubMed Scopus (176) Google Scholar). On the other hand, a gene encoding for plastocyanin is also found in this cyanobacterium (37Swingley W.D. Chen M. Cheung P.C. Conrad A.L. Dejesa L.C. Hao J. Honchak B.M. Karbach L.E. Kurdoglu A. Lahiri S. Mastrian S.D. Miyashita H. Page L. Ramakrishna P. Satoh S. Sattley W.M. Shimada Y. Taylor H.L. Tomo T. Tsuchiya T. Wang Z.T. Raymond J. Mimuro M. Blankenship R.E. Touchman J.W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2005-2010Crossref PubMed Scopus (176) Google Scholar). To determine the nature of the soluble electron carrier between the cyt b6f complex and PSI, we measured the progressive increase in the absorption changes associated with the c-type cytochrome oxidation, at 554 nm, during a series of 10 flashes (Fig. 4). Again, DBMIB was added to prevent the rereduction of the cytochromes by plastoquinol (32Trebst A. Methods Enzymol. 1980; 69: 675-715Crossref Scopus (276) Google Scholar). As shown in Fig. 4, about 75% of the absorption changes were observed after the first flash in the series. This shows that a single charge separation at the level of PSI is sufficient to oxidize 75% of the c-type cytochrome. Thus, the pool of electron donor to P740+ which gives rise to an absorption change at 554 nm is not in excess with respect to the PSI content. Since the soluble electron carriers, such as cyt c6 or plastocyanin, are usually found in an ∼3:1 ratio with respect to PSI (see Ref. 36Hope A.B. Biochim. Biophys. Acta. 2000; 1456: 5-26Crossref PubMed Scopus (196) Google Scholar for a review)