The Central Role of Protons in Establishing, Regulating, Controlling and Limiting the Energy Budget of Photosynthe

1. The Central Role of Protons in Establishing, Regulating, Controlling and Limiting the Energy Budget of Photosynthesis

2. The photosynthetic apparatus must: • Efficiently store solar energy in chemical bonds • Provide the correct ratio of products (NADPH, ATP) • Avoid over-excitation of the reaction centers which can lead to photodamage.

3. + H An electrochemical gradient of protons is the essential energetic intermediate in ATP synthesis ADP + Pi NADPH ATP The energetic intermediate is an electrochemical gradient of H+. In the formulation of Mitchell, Dp = -(DH+)/F = DY - 59 mV(DpH) Dp is also called pmf or proton-motive force.

4. + + H H NADPH n ADP + Pi ATP H+/e- The Ratio of NADPH:ATP produced by photosynthesis is determined by: 1) The ratio of H+ pumped per e- transferred (H+/e-) 2) The ratio of H+ transferred through the ATP synthase: ATP produced (n)

5. + + + + H H H H Chemiosmotic Coupling in Mitochondria ATP synthesis (endergonic) electron transfer (exergonic) e- O2 Cl- SH2 ATP ADP + Pi DY ATP Cl- In this case, the pH of the matrix and IMS are nearly the same, and essentially all pmf is stored as DY.

6. + + H H Cl- ADP + Pi DY ATP Cl- It is commonly believed that, in chloroplasts, the Dy component collapses as Cl- ions move in response to the electric field. Consequently, it is widely thought that pmf in thylakoids is stored solely as a DpH Dp = -(DH+)/F = DY - 59 mV(DpH) = - 59 mV(DpH)

7. The proton gradient also plays a central role in the regulation of photosynthesis. + H - photochemistry ADP + Pi ATP heat Xanthophyll cycle qE quenching

9. The relationships between the proton gradient and regulatory phenomena are fundamental to understanding how plants convert energy and respond to the environment. • Several new discoveries have altered our view of these relationships: • Structure/function of • cytochrome bc complexes • ATP synthase • The ability to probe key energy conserving reactions in vivo in the steady-state.

12. 6 1976: Mitchell introduces the Q cycle 5 3: Low light 2: High light 4 - 3 /e + H 2 1 0 1965 1970 1975 1980 1985 1990 1995 Historical Perspective of the H+/e- 1/3 reduction of ATP Year

13. 0.5 0.4 0.3 ddt (a.u.) 0.2 0.1 0.0 0.00 0.01 0.02 0.03 d (Electron Pool) / dt (a.u.) Linear H+/e-: Electron Pool

14. High-resolution structures provide an explanation for the proton pumping activity of the cytochrome bc1 (and related b6f complexes

15. How large of a DpH is required to sustain photosynthesis? 4 • Assuming that pmf is in the form of DpH and using a H+/ATP of n=4 1-5, the DpH required to sustain measured values of DGATP range between 1.7 and 2.2, suggesting the minimal lumen pH required ranges from 5.4 to 5.9. • If a steady state Dycomponent exists (DpH=0.2 to 0.7), see 6a&b, this would increase the minimal pH range to 5.6 and 6.6.

16. What is the value of n?

17. 80 f 80 Pea Tobacco Reduction half-time (ms) 60 Cytochrome Cucumber 60 40 Cytochrome Reduction half-time (ms) 20 40 5.5 6.0 6.5 7.0 7.5 8.0 pH 20 0 0 500 1000 1500 2000 2500 3000 -2 -1 m Light Intensity ( mole m sec ) Comparison of cytochrome f rereduction rates in intact leaves from pea (squares), tobacco (circles) and cucumber (triangles) with those from isolated thylakoids suspended at different pH values, adapted from12 (Inset). Observed half-times ranged from 20ms to 28ms for the entire range of light intensitities up to 2800 umoles m-2 s-1. Comparison to thylakoid data (dotted lines in inset12) suggests that the lumen pH is regulated so that it remains above ~ pH 6. 3b f

18. 4 6 8 5 7 Stromal pH 7.8 n=44 n=4 + Dy6 Upper and Lower Bounds of Lumen pH Estimated by in situ Probes PC2 Degrades PSII2 OEC loses Ca2+ VDE1 B6f3 ATP5 activation qE quenching7

20. Summary