Fig. 3 Theoretical Akt inhibitor and empirical fractions of closed RCs. PMS concentrations were 10, 60, and 150 μM, light intensities were 53, 166, 531, and 1028 μmol/m2/s. For 150 μM of PMS, the LOXO-101 nmr lowest light intensity gave a P700+ fraction which was too low to quantify, therefore this data point is not reported PMS is a fluorescence quencher To avoid the introduction of artifacts in the measurements the

reducing agent used to re-open the PSI RC should not by itself have an effect on the fluorescence. To investigate whether this is the case for PMS, we added it to a LHCII solution. Figure 4 shows the result. Addition of oxidized PMS did not affect the fluorescence intensity; however, as soon as it was reduced by NaAsc the intensity rapidly dropped. This effect was independent of the light intensity used. NaAsc itself did not reduce the fluorescence yield. Adding NaAsc first followed by PMS initially gave a similar result; however, for the higher PMS concentrations the solution rapidly became turbid. This turbidity was also observed in absence of Lhc complexes, and can possibly be explained by the aggregation of PMS. The addition of PMS followed by NaAsc reduced the fluorescence intensity by a factor of 2 for 10 μM, 18

for 60 μM, MLN2238 up to a factor of 64 for the highest concentration. The absorption of reduced PMS at these concentrations is below 0.05/cm for wavelengths longer than 500 nm, thus direct absorption of either excitation or emission light by PMS cannot explain the results. Therefore, it has to be concluded that PMS is quenching the chlorophyll emission. To investigate whether this is a general property, 60 μM of PMS and 40 mM of NaAsc were also added to PSII membranes (BBY’s, Berthold et al. 1981) and the PSI antenna complex Lhca1/4. In both

the cases, the fluorescence was strongly quenched, by 11 and 15 times, respectively. We also tested whether N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) is also quenching the LHCII emission. This is another reducing agent, which we found capable of reducing P700+ with a rate of 33/s at 2 mM concentration. Unfortunately, also TMPD was found to quench the LHCII emission. Fig. 4 Fluorescence emission of LHCII and PSI followed in time during others the addition of PMS and NaAsc. For LHCII, the excitation was at 630 nm and the emission was detected at 680 nm; for PSI, the excitation was at 500 nm and the emission was detected at 725 nm. Excitation of PSI at 630 nm gave similar results We proceeded to investigate the effect of PMS on the emission of PSI. Addition of 10 μM reduced PMS decreased the fluorescence intensity by 23%. Based on the excitation power of ~250 μmol/m2/s (at 500 nm), the 1.5 times larger PSI extinction coefficient at 500 nm compared with 635 nm, and the reduction rate of 36/s.