Purification of a manganese-containing protein involved in photosynthetic oxygen evolution and its use in reconstituting an active membrane

Abstract

Extraction of thylakoid membranes with cholate in the presence of ammonium sulfate inactivated oxygen evolution and liberated a manganese-containing protein. This protein could be combined with preformed liposomes containing the depleted thylakoid membranes to restore 85% of the original oxygen-evolution activity. The protein did not affect the primary photochemical events of photosystem I or photosystem II, and it was required only for electron transport in which water was the electron donor. The protein has been purified to homogeneity and has an apparent molecular weight of 65,000 (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate). Atomic absorption revealed two atoms of manganese bound to each 65,000-dalton protein molecule. Treatment with alkaline Tris removed the bound manganese and rendered the protein incapable of restoring oxygen evolution; however, Tris treatment of the depleted membranes before reconstitution had no effect. Thus, this manganese protein is probably the site of Tris action in isolated chloroplasts and is at least part of the water-oxidation enzyme system. The chemical events leading to the photosynthetic evolution of oxygen are still largely a matter of speculation. Many aspects of the kinetics of oxygen evolution have been elucidated. A model involving the storage of oxidizing equivalents on an unknown enzyme (the "S-states") has been proposed and seems to have gained general acceptance (1). The photoactivation of an uncharacterized manganese-containing protein may be essential for oxygen evolution (2, 3). In addition, the specific requirement for manganese in the oxygen-evolving reaction has been demonstrated by inducing deficiency during growth or by depleting manganese by extraction, in both algae and higher plants (4, 5).A subchloroplast system in which depletion and restoration of oxygen evolution can be accomplished by the extraction and replacement of individual thylakoid components would be helpful in furthering our understanding of photosynthetic oxygen evolution. We present here a method for extracting a manganese-containing protein and using it to reconstitute an active system; our results provide evidence that this protein is required for photosynthetic oxygen evolution. METHODS AND MATERIALSChloroplasts were isolated from fresh market spinach and extracted with cholate (6). Briefly, specific chloroplast lipids and proteins were solubilized by using 0.05 M sodium cholate and 0.4 M ammonium sulfate at pH 8.0 with stirring for 15 min on ice. The suspension was then centrifuged at 144,000 X g for 90 min, and the supernatant was collected; the pellet was set aside. Saturated ammonium sulfate (pH 8.0) was then added to the supernatant to increase the concentration to 1.2 M. After incubation for 20 min on ice the precipitate was collected by centrifugation (10,000 X g, 10 min). This pellet was dissolved in about 3 ml of 0.2 M sucrose/0.02 M N-[tris(hydroxymethyl)methyllglycine (Tricine)-NaOH (pH 8.0)/S mM MgCl2, then adjusted to 0.84 M with sat...

A thylakoid polypeptide involved in the reconstitution of photosynthetic oxygen evolution

“…SPECTOR and WINGET reported on the isolation of a manganese-containing protein ofM r 65,000 involved in the reconstitution of oxygen evolution of depleted thylakoids (45). Although the results of this study could not be repeated (42,43), it is interesting to note that an M r 23,000 polypeptide constitutes a major component during the initial purification steps of the M r 65,000 polypeptide.…”

“…Much evidence suggest that manganese is essential in the oxygen evolving process (2,12,41) but its mechanism of participation and site of action remains unknown. SPECTOR and WINGET (45) reported the isolation of a cholate-extractable Tris-sensitive manganese-containing polypeptide of M r 65,000 which reconstituted oxygen evolution when added to the cholate-extracted thylakoid material after its incorporation into artificial liposomes. SAYRE and CHENIAE (42,43) were unable to isolate such a manganese-containing polypeptide.…”

“…NAKATANI and BARBER (38) using essentially the procedure of SPECTOR and WINGET (45) isolated a protein of molecular weight 230,000 composed of four identical subunits. This protein was also reported to restore oxygen evolution but otherwise resembled catalase.…”

Effect of competitive interactions between manganese and copper on cellular manganese and growth in estuarine and oceanic species of the diatom Thalassiosira1,2

“…Recent electron paramagnetic resonance measurements are consistent with there being two manganese ions present in the oxygen-evolving centers within the chloroplast, perhaps one for each of the two II20 molecules oxidized to produce one 0, molecule (Wydrzynski and Sauer 1980). In addition, a 65,000-dalton protein containing two bound manganese ions recently has been isolated from chloroplasts of spinach (Spector and Winget 1980). Extraction of this mangancse-protein complex from thylakoid membranes inactivated oxygen evolution.…”

“…Competitive binding models-Although manganese serves as a cofactor in a number of enzymes, its primary role in plants is in photosynthesis where it is an essential component of the oxygen-evolving centers of photosystem II (Eyster et al 1958;Cheniae and Martin 1969,197O;Diner and Joliot 1976;Spector and Winget 1980). Its exact function in oxygen evolution has not been fully elucidated, but it is thought to act as a redox catalyst in the oxidation of water by utilizing the redox couple between Mn(I1) and higher oxidation states (Wydrzynski and Sauer 1980).…”