Reactivation of photosynthetic oxygen evolution in tris-inactivated inside-out photosystem II vesicles from spinach

Abstract

Purified inside-out photosystem II vesicles with retained capacity for oxygen evolution and a simple polypeptide composition were found to be more sensitive to washing with tris(hydroxymethyl)aminomethane (Tris) at alkaline pH than right-side out thylakoids. This suggests that the thylakoid membrane limits the penetration of the active unprotonated form of Tris.The inhibition of photosynthetic oxygen evolution by alkaline Tris-washing (pH 8.5) coincided with the release of a polypeptide with a molecular weight of 32,000 from the inside-out vesicles. Only this polypeptide was released with concentrations of Tris up to 200 mM, while higher concentrations removed additional polypeptides with a molecular weight of 34,000 and 56,000. About 70 96 of the membrane bound manganese is lost by washing the vesicles with 200 mM-Tris. Oxygen evolution was not inhibited nor were polypeptides released with Tris buffered at pH 6.5. From right-side out thylakoids the 32,000 molecular weight polypeptide could not be extracted into the ambient medium by Tris-washing.Restoration of oxygen evolution of Tris-inactivated inside-out and right-side out vesicles was possible with the aid of reduced dichlorophenol indophenol but without the addition of the 32,000 molecular weight polypeptide or manganese.Abbreviations: Chl = chlorophyll; DCMU = 3-(3,4-dichlorophenyl)-l,l-dimethyl urea; DCPIP = 2,6-dichlorophenol indophenol; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; TMBZ = tetramethylbenzidine; Tris = tris(hydroxymethyl)aminomethane.

A thylakoid polypeptide involved in the reconstitution of photosynthetic oxygen evolution

“…The amino acid composition of the isolated protein is very similar to that of a M r 32,000 polypeptide removed from inside-out vesicles of spinach by washing with "Iris (19,50,MOLLER and HENRY,unpublished) and to that of a M r 32,000 polypeptide obtained from spinach by acetone extraction (29) ( Table Ill). The pl of the isolated M r 23,000 polypeptide was 5.1 (section 3.6.).…”

“…A faint green band with an electrophoretic mobility slightly different from that of chlorophyll aprotein 1 was also observed in addition to four polypeptides at M r 23,000, 21,500, 19,000 and 13,500. Although some of the bands may contain more than one polypeptide species, the composition of these vesicles is remarkable simple and compares well with that of inside-out vesicles of spinach (18,19). The EPR characteristics of the preparation (6) thus agree with an essentially complete absence of photosystem I polypeptide components.…”

“…preparations used The polypeptide composition of purified photosystem I1 inside-out vesicles obtained from spinach was reported earlier (18,19). Inside-out photosystem II vesicles from barley ( Figure 3) had a similar composition although they were always contaminated to some extent with photosystem I components (20).…”

“…In thylakoids of normal side orientation, these polypeptides are electrostatically bound to the inner thylakoid surface and therefore not released into the medium upon washing with sodium chloride. The yield of inside-out vesicles is low, especially when vesicles of simple polypeptide composition are desired (18,19). The use of these vesicles as starting material for isolation of polypeptides located on the inner thylakoid surface will therefore only provide low amounts.…”

“…Tris, which is known to inhibit oxygen evolution at alkaline pH (13,19) was also excluded. Retention of any manganese originally bound to one of the isolated polypeptides would thus be favored.…”