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This long dark interval allows conversion of IM1 to form IM1?. The 1-s duration preflash does not allow a significant proportion of the sample to advance past IM1?, because the required dark process is much slower (k2 ? 70 s; Fig.?5). We estimate that no more than 1% of the total number of centers can progress past IM1? during the 1-s-long preflash. This experimental approach is designed so that the system must end up in either of the following two measurable states: 1), centers progress to IM1?; or 2), centers progress past IM1? to form completely assembled cluster (in S0 or S1) due to the higher quantum efficiency of the later photoassembly steps (see next paragraph). Centers that do not progress beyond IM0 or decay back to IM0 from IM1 or IM1? are not measurable until about the 40th flash ( Fig.?6B) because only inefficient [http://en.wikipedia.org/wiki/Arginase Arginase] photoassembly takes place during the single 60-��s flashes due to the much slower dark process (k2 ? 70 s). Data from Tamura et?al. (20?and?54) and others (30?and?35) established that subsequent photoassembly intermediates (IM2, and potentially IM3 and IM4) form with much higher quantum efficiencies than IM1?. As a consequence, [http://www.selleckchem.com/products/ly2157299.html LY2157299 purchase] are expected to be fully assembled by flash 2. Because there is a long dark time between preflash 1 and flash 2 (240?s), the few centers that assembled during the preflash [http://www.selleckchem.com/products/BAY-73-4506.html BAY 73-4506 manufacturer] are expected to decay back to the dark stable S?states, S1 or S0. This population is accounted for in the simulations. The integrated O2 signal during photoassembly is shown in Fig.?6B, using the optimal flash sequence and 1-s-long preflash to populate IM1 ( Fig.?6A). The data in Fig.?6B show that the population of fast photoassembling centers (those that are in IM1? or later) is ?24% of all PSII centers (compared with the steady-state O2 yield of intact PS II). After 20 flashes, a sharp decrease in the slope of O2 recovery occurs, which is an order of magnitude slower than the initial slope (for flashes 20�C100). The O2 recovery of apo-WOC-PSII without the long preflash, but otherwise using the same flash sequence (60-��s standard), is also given in Fig.?6B. It shows only the slow recovery phase due to centers starting from IM0. Thus the rapid phase (?24% of centers) corresponds to centers that efficiently assembled IM1 (during the preflash) and advanced to IM1? (in the dark following the 240-s adaption period) and beyond to fully assemble. The first 20 flashes (i.e., the first phase) of the photoassembly curve further resolve into two temporally distinct phases when expanded (Fig.?7A).