Regular paper How fast can Photosystem II split water? Kinetic performance at high and low frequencies w Gennady Ananyev & G. Charles Dismukes* Department of Chemistry and Princeton Environmental Institute, Princeton University, Princeton, NJ 08544, USA; *Author for correspondence (e-mail: dismukes@princeton.edu; fax: +1-609-258-1980) Received 11 October 2004; accepted 2 December 2004 Key words: Arthrospira, Chlorella, Kok S-states; oxygen evolution, photosynthesis, Photosystem II, Spirulina, variable fluorescence, water oxidation Abstract Molecular oxygen evolution from water is a universal signature of oxygenic photosynthesis. Detection of the presence, speed and efficiency of the enzymatic machinery that catalyzes this process in vivo has been limited. We describe a laser-based fast repetition rate fluorometer (FRRF) that allows highly accurate and rapid measurements of these properties via the kinetics of Chl-a variable fluorescence yield (Fv) in living cells and leaves at repetition rates up to 10 kHz. Application to the detection of quenching of Fv is described and compared to flash-induced O 2 yield data. Period-four oscillations in both Fv and O 2 , caused by stimulation of primary charge recombination by the O 2 -evolving complex (WOC) within Photosystem II (PS II), are directly compared. The first quantitative calculations of the enzymatic parameters of the Kok model (a – miss; b – double hit; S-state populations) are reported from Fv data over a 5 kHz range of flash frequencies that is 100-fold wider than previously examined. Comparison of a few examples of cyano- bacteria, green algae and spinach reveals that Arthrospira m., a cyanobacterium that thrives in alkaline carbonate lakes, exhibits the fastest water-splitting rates ever observed thus far in vivo. In all oxygenic phototrophs examined thus far, an unprecedented large increase in the Kok a and b parameters occur at both high and low flash frequencies, which together with their strong correlation, indicates that PS II-WOC centers split water at remarkably lower efficiencies and possibly by different mechanisms at these extreme flash frequencies. Revisions to the classic Kok model are anticipated. Abbreviations: a, b – miss and double hit parameters in Kok model; Chl – chlorophyll; FRRF – fast repetition rate fluorometer; Fv – variable fluorescence; PS II – Photosystem II; T dark – dark interval between single turnover flashes; STF – single turnover flash with saturated light intensity; Y 1 …Y n – yield of quantum efficiency Fv/Fm on 1…nth single turnover flashes; WOC – water-oxidizing complex; Y Z – redox active tyrosine on the D1 protein Introduction It is our great pleasure to contribute to this special issue in honor of Professor Norio Murata. He has made historical contributions to the applications and understanding of fluores- cence phenomena in photosynthetic systems, including the analysis of fluorescence emission and excitation spectra of Chl-a in chloroplast organelles and the influence of environmental factors. These works form part of the foundation of this paper. w Dedicated to Professor Norio Murata on the occasion of his retirement. Photosynthesis Research (2005) 84: 355–365 Ó Springer 2005