Trypsin inhibition of Photosystem II

Abstract

1. Brief incubation of chloroplasts with trypsin accelerated (by uncoupling) while longer incubation inhibited the Hill reaction with 2,6-dichlorophenolindophenol (DCIP). When an uncoupler (NH 4Cl or methylamine · HCl) was present, or when the reaction was light-limited, even brief incubation with trypsin inhibited the reaction. 2. Electron transport from ascorbate and DCIP to methyl viologen, in the presence of 3,4-dichlorophenyl-1,1-dimethylurea (DCMU), was unaffected by trypsin treatment. 3. Addition of 0.35 M Tris-HCl to normal chloroplasts severely inhibited the reduction of DCIP: after a brief transient reduction of DCIP in high light, the inhibition was virtually complete. DCIP reduction was partially restored (approx. 50%) by the donor, 1,5-diphenylcarbohydrazide. Trypsin treatment abolished both the transient observed in the presence of 0.35 M Tris-HCl, and the partial restoration by 1,5-diphenylcarbohydrazide. 4. Inhibition of DCIP reduction by trypsin occurred two to three times faster with water as donor as with 1,5-diphenylcarbohydrazide. In both cases, the inhibition developed with first order kinetics. 5. Both treatment with 0.35 M Tris-HCl and with trypsin lowered the relative fluorescence yield of otherwise untreated chloroplasts to the same level. After the Tris treatment alone, but not after both trypsin and Tris treatments, 1,5-diphenylcarbohydrazide addition raised the fluorescence yield. Nor could the yield of trypsin-treated chloroplasts be raised by DCMU. 6. It is concluded that trypsin(i) uncouples, (ii) does not inhibit Photosystem I between plastocyanin and ferredoxin, and (iii) inhibits at least two sites of electron transport on the oxygen producing side of Photosystem II. A more rapid inhibition occurs between the sites of water oxidation and 1,5-diphenylcarbohydrazide oxidation, and a slower inhibition occurs between the sites of 1,5-diphenylcarbohydrazide oxidation and Photosystem II.