The Central Role of Protons in Establishing, Regulating, Controlling and Limiting the Energy Budget of Photosynthesis
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.
+ 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 = -(DH+)/F = DY - 59 mV(DpH) Dp is also called pmf or proton-motive force.
+ + 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)
+ + + + 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.
+ + 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 = -(DH+)/F = DY - 59 mV(DpH) = - 59 mV(DpH)
The proton gradient also plays a central role in the regulation of photosynthesis. + H - photochemistry ADP + Pi ATP heat Xanthophyll cycle qE quenching
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.
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
0.5 0.4 0.3 ddt (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
High-resolution structures provide an explanation for the proton pumping activity of the cytochrome bc1 (and related b6f complexes
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.
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
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