PARENT SESSION
Posters P7A Mechanisms of water oxidation. Abstracts (347-381)

Fast repetition rate laser fluorometer and role for bicarbonate in oxygen evolution by Photosystem II at site arginine 357 of CP43. Gennady Ananyev^*,1, Tuan Nguen¹, Rogier Willigen¹, Cindy Putnam-Evans², G. Dismukes¹, ¹ Princeton University, Princeton, NJ, USA² East Carolina University, Greenville, NC, USA

ABSTRACT- A time-resolved fast repetition rate laser fluorometer (FRRLF) ideally suited to detect changes in pigment light emission in photosynthetic samples over 10 orders in time from either environmental or laboratory induced perturbations will be described. It is configured for simultaneous oxygen measurements. The FRRLF features: 400 KHz modulated laser diode with 0.5 W optical power at wavelength 660 nm, avalanche photodiode detector (filtered at 685 nm for Chl), light saturation of electron transport in 20-40 us using a train of pulses, real-time sampling from individual pulses 50 ns, flash train repetition rates 130 Hz. Real-time presentation of PSII parameters: quantum efficiency, PSII functional cross section, primary quinone reoxidation rate, electron transfer rate via PSII and oxygen yield - Kok parameters. Application to the serine mutant R357S-CP43 from Synechocystis PCC 6803 is described. CP43 is an inner Chl-binding subunit of PSII. The recent crystal structure of a cyanobacterial PSII reveals that arginine 357 may be involved in binding a small anion. Oxygen evolving activity of R357S mutant cells are 10 % of the wild-type activity under continuous saturating illumination, but increased to 28 % using low light intensity, indicating a kinetic limitation during turnover. Bicarbonate depletion in cells and thylakoids decreases the flash oxygen yield by 4 fold in WT vs. 1.5 fold in mutant cells and is fully reversible. The mutant lacks period-four oscillations in oxygen yield and has low quantum efficiency in Chl fluorescence (Fv/Fm), reflecting specific dysfunction in water oxidation. Fitting to a Kok model reveals high double hits. PSII centers appear to no longer work independently in the mutant nor in bicarbonate-depleted WT cells, suggesting the sharing of charge accumulation between non-functional PSII reaction centers and functional RCs in the dimeric PSII complexes. In conclusion, arginine R357-CP43 serves as a likely binding site for bicarbonate/carbonate involved in catalysis of oxygen production. Supported by NIH and HFSP.

KEY WORDS: laser fluorometer, Synechocystis mutant, oxygen evolution, bicarbonate