The proton to electron stoichiometry of steady-state photosynthesis in living plants: A proton-pumping Q cycle is continuously engaged

Abstract

A noninvasive technique is introduced with which relative proton to electron stoichiometries (H ؉ ͞e ؊ ratios) for photosynthetic electron transfer can be obtained from leaves of living plants under steady-state illumination. Both electron and proton transfer fluxes were estimated by a modification of our previously reported dark-interval relaxation kinetics (DIRK) analysis, in which processes that occur upon rapid shuttering of the actinic light are analyzed. Rates of turnover of linear electron transfer through the cytochrome (cyt) b 6f complex were estimated by measuring the DIRK signals associated with reduction of cyt f and P700. The rates of proton pumping through the electron transfer chain and the CF O-CF1 ATP synthase (ATPase) were estimated by measuring the DIRK signals associated with the electrochromic shifting of pigments in the light-harvesting complexes. Electron transfer fluxes were also estimated by analysis of saturation pulse-induced changes in chlorophyll a fluorescence yield. It was shown that the H ؉ ͞e ؊ ratio, with respect to both cyt b6f complex and photosystem (PS) II turnover, was constant under low to saturating illumination in intact tobacco leaves. Because a H ؉ ͞e ؊ ratio of 3 at a low light is generally accepted, we infer that this ratio is maintained under conditions of normal (unstressed) photosynthesis, implying a continuously engaged, proton-pumping Q cycle at the cyt b 6f complex.steady-state electron and proton transfer ͉ chemiosmotic coupling ͉ cyclic electron transfer ͉ energy budget T he energy budget of a plant depends upon the ratio of protons pumped across the thylakoid membrane to electrons passed through photosynthetic electron transfer complexes (the H ϩ ͞e Ϫ ratio), which sets the stoichiometries of ATP and NADPH production for use in the Calvin-Benson cycle and other biochemical pathways (reviewed in refs. 1 and 2). There has been a long-standing debate about the magnitude of H ϩ ͞e Ϫ for green plant photosynthesis, particularly regarding the proton pumping reactions of the cytochrome (cyt) b 6 f complex. It is generally accepted (see review in ref.3) that for each electron transferred through the linear pathway, one proton is released into the lumen at the level of water splitting; another is transported across the thylakoid membrane by the reduction and reoxidation of plastoquinone (PQ) at the photosystem II (PSII) Q B site and the cyt b 6 f complex Q o site, respectively; and a third proton is pumped, at least under some conditions, by the turnover of a Q cycle associated with the oxidation of plastoquinol at the cyt b 6 f complex (see refs. 4-7 for reviews). However, in vitro measurements of H ϩ ͞e Ϫ ratios for linear electron transport in isolated thylakoids range from 2 (e.g., refs. 8-10) to 3 (e.g., refs. 11 and 12), and several groups have suggested that it changes from 3 to 2 with increasing light intensity (1,11,(13)(14)(15)(16)(17). Such indications of variable coupling ratios have led several groups to suggest that the Q cycle or its proton pumping rea...

Regulating the proton budget of higher plant photosynthesis

“…A similar (Ϸ6-fold) change in q E sensitivity was observed when both O 2 and CO 2 were lowered (to 1% and 50 ppm, respectively), but in this case, both changes in g H ϩ and increased partitioning of pmf into ⌬pH were invoked to explain the effect (18). In both cases, the ratio of v H ϩ ͞LEF remained essentially constant (within noise levels), indicating that contributions from CEF1 to proton flux were either small or remained a relatively constant fraction of those from LEF, as previously found for tobacco (25). On the whole, these results support a large role for Type II mechanisms in modulating q E sensitivity upon short term changes in CO 2 ͞O 2 levels, but they do not rule out smaller contributions from Type I mechanisms in balancing ATP͞NADPH output (12,16,28).…”

“…Recently, a series of in vivo probes of the pmf have been introduced (2,3,16,(25)(26)(27)(28), allowing contributions from Types I and II flexibility mechanisms to be directly assessed. These Abbreviations: CEF1, cyclic electron flow associated with PSI; ECS, electrochromic shift; LC, low CO2 (50 ppm CO2, 21% O2); LEF, linear electron flow; PS, photosystem; pmf, proton motive force; pmfLEF, pmf generated by LEF; qE, energy-dependent nonphotochemical quenching.…”

“…Recently, a series of in vivo probes of the pmf have been introduced (2,3,16,(25)(26)(27)(28), allowing contributions from Types I and II flexibility mechanisms to be directly assessed. These techniques are based on kinetic analyses of the ECS (26) of photosynthetic pigments, which yields absorbance changes proportional to changes in transthylakoid ⌬ (29).…”

In Vivo Changes of the Oxidation-Reduction State of NADP and of the ATP/ADP Cellular Ratio Linked to the Photosynthetic Activity in Chlamydomonas reinhardtii

“…The stoichiometry of one ATP per NADPH (or one ATP per two electrons; for review, see Witt, 1979) has been established in thylakoids isolated from higher plants, and more recent experiments have confirmed this stoichiometry (see, e.g. and Rumberg, 1999;Sacksteder et al, 2000). So, the measurements with isolated thylakoids consistently show lower ATP per two electrons ratios than that required for CO 2 assimilation.…”

“…Higher values have been calculated theoretically, combining reported values of H ϩ /electron and H ϩ /ATP ratios. However, such calculations are based on the still controversial values of H ϩ /electron ratios (see Berry and Rumberg, 1999;Sacksteder et al, 2000). So, the measurements with isolated thylakoids consistently show lower ATP per two electrons ratios than that required for CO 2 assimilation.…”