1. 1. In fresh chloroplasts ascorbate and other reducing agents, if present in sufficient concentration, fully replace water as the System II oxidant and can sustain maximum rates of 1000–1200 equiv/chlorophyll per h.
2. 2. None of the studied donors proved entirely specific for System II; to a variable extent all could react with the oxidant of System I. We therefore considered only the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(DCMU)-sensitive fraction of the observed rates as pertinent.
3. 3. Normal fresh chloroplasts contained 3 Mn/200 chlorophyllsII and showed flash yields of approx. 1 O2/1600 chlorophylls. This indicates that each System II trapping and O2-evolving center contains three Mn atoms.
4. 4. O2 evolution capacity is abolished when about 2/3 of the total Mn pool is removed by way of Tris or hydroxylamine extraction, i.e. upon removal of two of the three Mn atoms normally present per reaction center. Between the limits of 1 Mn per trap and 3 Mn per trap O2 evolution capacity is linear with Mn content.
5. 5. Mn removal affects the rates of O2 evolution in strong light and in weak light (quantum yield) in the same fashion. This indicates that complete O2 reaction centers are inactivated.
6. 6. With Mn removal the capacity for donor (ascorbate or p-phenylenediamine) photooxidation in strong light declines in a very similar fashion as the O2 evolving capacity. However, after removal of 2/3 of the Mn pool (by Tris or hydroxylamine extraction) 15–20% of the maximum rate remains (100–250 equiv/chlorophyll per h) as previously noticed by other workers. Secondly, the rate in weak light (quantum yield) of these photooxidations remains unaffected by Mn removal. This shows that for donor photooxidation the larger of the two Mn pools is not essential.
7. 7. Complete removal of Mn (< 1 Mn/4000 chlorophylls) led to 90–95% loss of donor photooxidation in strong light.
8. 8. Removal of 2/3 of the Mn left a low fluorescence yield (variable fraction = 0) which could be fully restored by adding DCMU. After complete removal of Mn (< 1 Mn/4000 chlorophylls) DCMU enhanced the yield of the variable fluorescence to only 1/2 the maximum level but the full maximum could be restored by chemical reduction. This indicates that fluorescence quencher of System II, Q, is not affected by Mn removal.
9. 9. Of the three Mn associated with each trapping center, one is linked more closely to the center than the other two. While all three are essential for O2 evolution, artificial donors can enter with various rate constants at several loci on the oxidant side of System II.
Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; Q, the quencher of System II fluorescence; F0, the invariant low level of fluorescence observed at onset of illumination; Fmax, maximum level of fluorescence; DCIPH2, 2,6-dichlorophenolindophenol, reduced form; DH2, a reductant capable of donating electrons to light-induced oxidants; A pool, the large electron acceptor pool in association with Q of System II; PMS, N-methylphenazonium ion (phenazine methosulfate)
