Yasusi Yamamoto

Release of polypeptides from highly active O2-evolving photosystem-2 preparation by this treatment

Oxygen evolution in higher plants is thought to be mediated by an enzyme system containing membranebound manganese where positive charges might be accumulated by illuminating the chloroplasts and oxidation of water occurs subsequently [ 1±3]. The enzyme system is so labile that the isolation and characterization of the enzyme complex have not met much success. Several attempts were made to identify the polypeptide(s) involved in the O2-evolution enzyme complex by SDS-PAGE, using plant and algal mutants with deletions in PS-2 [4-6]. These results showed a total or partial loss of polypeptides or a change in the electrophoretic mobility of 32 -36 × 103 M r polypeptide bands, associated with the mutation. An Mn-protein of 65 X 103 M r isolated by cholate treatment of chloroplasts was also reported to be involved in the O2-evolution enzyme [7]. Here, we isolated a highly active Oz-evolving PS-2 preparation from spinach chloroplasts using a low concentration of digitonin and Triton X-100, and examined the effect of Tris treatment on the preparation. The subsequent release of polypeptides from the PS-2 preparation was analysed by SDS-PAGE and the result was compared with that obtained by Tris treatment of unfractionated broken cloroplasts.

Purification and molecular properties of 3 polypeptides released from a highly active O2-evolving photosystem-II preparation by Tris-treatment

Tris‐treatment of a highly active O2‐evolving photosystem‐II preparation induced release of 3 polypeptides (M r 33 000, 24 000 and 18 000), concomitant with inhibition of O2 evolution [FEBS Lett. (1981)_133. 265‐268]. The 3 polypeptides were purified with the use of electrofocusing. Isoelectric points of the proteins were 5.1, 6.5 and 9.2 in order of decreasing M r value. Only a trace amount of histidine, cystein and methionine were detected in these proteins. Based on the amino acid compositions, polarity indexes of the proteins were calculated to be 47–49%, suggesting the 3 proteins to be hydrophilic.

Association of the 33-kDa polypeptide with the 43-kDa component in photosystem II particles

The organization of polypeptides and Mn atoms in the oxygen‐evolution system was studied by analyzing the effects of protease treatment on photosystem (PS) II particles from spinach chloroplasts. Hydrolysis of the 47‐ and 43‐kDa polypeptides of the PS II core complex had similar profiles of dependence on trypsin concentration in the treatment of control PS II particles and NaCl‐washed particles, which were devoid of the peripheral 24‐ and 18‐kDa polypeptides. In CaCl2‐washed particles, which were depleted of the 33‐kDa polypeptide besides the 24‐ and 18‐kDa polypeptides, the 43‐kDa polypeptide was much more sensitive to trypsin than the 47‐kDa polypeptide. Chymotrypsin treatment gave similar results. These findings suggest that the 33‐kDa polypeptide is associated to the 43‐kDa polypeptide and shields it from tryptic attack. Changes of the amount of Mn in the PS II and salt‐washed particles on protease treatment indicated a heterogeneity of binding sites of Mn in the PS II particles

Isolation of an Mn-carrying 33-kDa protein from an oxygen-evolving photosystem-II preparation by phase partitioning with butanol

An Mn‐containing 33‐kDa protein was isolated by phase partitioning with 50% n‐butanol from an O2‐evolving photosystem‐II preparation. The Mn content in the 33‐kDa protein increased when 1 mM potassium ferricyanide and 0.4 mM diaminodurene were present as oxidants during the butanol treatment and the following dialysis. Under these conditions, 0.1–0.25 atom Mn was detected in one 33‐kDa protein molecule. EPR spectra of the Mn protein showed that the Mn atoms were bound to the protein.

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O2-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O2-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.

Association of two manganese atoms with the reaction center of Photosystem II in a highly active O2-evolving Photosystem II preparation

Abstract The stoichiometry of manganese atom and the Photosystem (PS) II reaction center was determined in a highly active O2-evolving PS II preparation from spinach chloroplasts. Two manganese atoms were shown to be associated with a reaction center of PS II. Judging from the sensitivity to various treatments which specifically inhibited O2 evolution, the two manganese atoms seem to be the minimum and essential amount of manganese required for the O2-evolution activity of the subchloroplast preparation.

Effect of surface potential on P-700 reduction in chloroplasts

Abstract The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP. At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations. In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl. The Debye-Huckel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.

Analysis of the relationship between the extrinsic 30-kDa protein, manganese and oxygen evolution in the thylakoid of Chlamydomonas reinhardtii grown under manganese-deficient conditions

Abstract The content and stability of an extrinsic protein in the thylakoid of a green alga Chlamydomonas reinhardtii which has a relative molecular weight of 30 000 and corresponds to the ‘33-kDa’ protein involved in oxygen evolution in spinach chloroplast were studied with the cells grown in a manganese-deficient medium. The amount of the 30-kDa protein in the thylakoid did not change even when the content of manganese in the membrane was decreased significantly and the activity of oxygen evolution was reduced concomitantly under manganese-depleted conditions. The stability of the 30-kDa protein in the membrane, which was determined by the susceptibility of the protein to the treatment with an alkaline buffer and sonication, also showed no significant change between the cells grown with and without manganese. The thylakoid membrane of the cells grown under manganese-deficient conditions was devoid of the loosely bound manganese atoms located at the hydrophilic environment of the membrane probably at the interface between the 30-kDa protein and the reaction center complex of Photosystem II, judging from the partitioning behavior of manganese atoms on butanol/water phase partitioning of the thylakoid. These results suggest that the manganese atoms involved in oxygen evolution are not required for the binding of the 30-kDa protein to the Photosystem II membrane.

Highly efficient purification of the 33-, 24-, and 18-kDa proteins in spinach photosystem II by butanol/water phase partitioning and high-performance liquid chromatography

The 33-, 24-, and 18-kDa proteins involved in photosynthetic oxygen evolution were purified from spinach photosystem II particles by butanol/water phase partitioning and high-performance liquid chromatography with a silica-based cation-exchange column. With this procedure a significant improvement was made in the time required for the purification and also in the amount and purity of the proteins. The N-terminal sequence of amino acid was determined for the purified proteins. Partial degradation of the proteins, which sometimes occurred in the purification, was not detected in the new procedure.

ORGANIZATION OF THE O2-EVOLUTION ENZYME COMPLEX IN A HIGHLY ACTIVE O2-EVOLVING PHOTOSYSTEM-II PREPARATION

Publisher Summary This chapter discusses the organization of the O2-evolution enzyme complex in a highly active O2-evolving photosystem-II (PS-II) preparation. O2 evolution in chloroplast is believed to be catalyzed by an enzyme complex containing manganese (Mn) at its catalytic site. In a study discussed in the chapter, a highly active O2-evolving PS-II preparation was first isolated from the membranes of a thermophilic blue–green alga Phormidium laminosum, by treatment with laurylamine oxide followed by a Sepharose 6B column chromatography. As the O2-evolution enzyme system of chloroplasts from higher plants seems to be not stable enough to withstand the relatively strong detergent treatment and long preparation procedure, low concentrations of digitonin and Triton X-100 was used for the isolation of a highly active O2-evolving PS-II preparation from spinach chloroplasts. The results suggest that the 33-, 24-, and 18-kDa polypeptides, and two Mn atoms are involved in the O2-evolution system. They are probably located on the outer side of the membrane vesicles on the PS-II preparation and released from the membranes by treatments that inhibit O2 evolution specifically. However, the extent of the release of the three polypeptides and Mn varied depending on the treatment employed.