Mitsue Miyao

Role of the 33-kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by chloride ions

Treatment of Photosystem II particles from spinach chloroplasts with Triton X‐100 with 2.6 M urea in the presence of 200 mM NaCl removed 3 polypeptides of 33 kDa, 24 kDa and 18 kDa, but left Mn bound to the particles. The (urea + NaCl)‐treated particles could evolve oxygen in 200 mM, but not in 10 mM NaCl. Mn was gradually released with concomitant loss of oxygen‐evolution activity in 10 mM NaCl but not in 200 mM Cl−. The NaCl‐treated particles, which contained Mn and the 33‐kDa polypeptide but not the 24‐kDa and 18‐kDa polypeptides, did not lose Mn or oxygen‐evolution activity in 10 mM NaCl. These observations suggest that the 33‐kDa polypeptide maintains the binding of Mn to the oxygen‐evolution system and can be functionally replaced by 200 mM Cl−.

Calcium ions can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution

Photosystem II particles were prepared from spinach chloroplasts with Triton X‐100, and treated with 1.0 M NaCl to remove polypeptides of 24 kDa and 18 kDa and to reduce the photosynthetic oxygen‐evolution activity by about half. Oxygen‐evolution activity was restored almost to the original level with 10 mM Ca2+, in a similar manner to the rebinding of 24‐kDa polypeptide. Other cations such as magnesium, sodium and manganese ions could not restore any oxygen‐evolution activity. These observations, together with a kinetic analysis, suggest that Ca2+ can be substituted for the 24‐kDa polypeptide in photosynthetic oxygen evolution in Photosystem II particles.

Heat inactivation of oxygen evolution in Photosystem II particles and its acceleration by chloride depletion and exogenous manganese

Abstract Heat inactivation of oxygen evolution by isolated Photosystem II particles was accelerated by Cl − depletion and exogenous Mn 2+ . Weak red light also accelerated heat inactivation. Heat treatment released the 33, 24 and 18 kDa proteins and Mn from the Photosystem II particles. The protein release was stimulated by Cl − depletion and exogenous Mn 2+ , and the Mn release was also stimulated by Cl − depletion. A 50% loss of Mn corresponded to full inactivation of oxygen evolution, whereas no direct correlation seemed to exist between the loss of any one protein and inactivation of oxygen evolution. Removal of the 24 and 18 kDa proteins from photosystem II particles only slightly decreased the heat stability of oxygen evolution.

Stoichiometry of components in the photosynthetic oxygen evolution system of Photosystem II particles prepared with Triton X-100 from spinach chloroplasts

Abstract The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b -559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b -563, cytochrome f , phylloquinone, α-tocopherol or α-tocopherylquinone.

Extrinsic membrane proteins in the photosynthetic oxygen-evolving complex

Abstract Preparations of photosystem II membranes, in which the active site of oxygen evolution is exposed to the bulk aqueous phase, have facilitated biochemical studies of the photosynthetic oxygen-evolving complex (the photosynthetic water oxidase complex). Described here are photosystem II membrane preparations highly active in oxygen evolution and three extrinsic membrane proteins for which the molecular properties and functions in oxygen evolution have been characterized.

Partial disintegration and reconstitution of the photosynthetic oxygen evolution system. Binding of 24 kilodalton and 18 kilodalton polypeptides

Abstract Treatment with 1 M NaCl almost totally removed two polypeptides of 24 and 18 kDa from the Photosystem II particles of spinach chloroplasts and reduced the oxygen-evolution activity by about half. Both polypeptides were able to rebind to the NaCl-treated particles in a low-salt medium. The rebinding of the 24 kDa polypeptide showed a saturation curve whose maximum level was close to that naturally occurring in the untreated particles. In parallel with the amount of rebound 24 kDa polypeptide, the oxygen-evolution activity was recovered. The 18 kDa polypeptide bound to the NaCl-treated particles without saturation. When the 18 kDa polypeptide was added to the particles previously treated with NaCl and then supplemented with a saturating amount of 24 kDa polypeptide, there appeared, in addition to the binding without saturation, another binding of the 18 kDa polypeptide with saturation to a maximum level close to that naturally occurring in the untreated particles. The 18 kDa polypeptide did not restore the oxygen-evolution activity. These findings suggest that there are specific binding sites; one for the 24 kDa polypeptide located on the Photosystem II particles, and the other for the 18 kDa polypeptide on the 24 kDa polypeptide.

The function of 33-kDa protein in the photosynthetic oxygen-evolution system studied by reconstitution experiments

Abstract Treatment of Photosystem II particles with 1.2 M CaCl2 released three proteins of 33, 24 and 18 kDa of the photosynthetic oxygen evolution system, but left Mn bound to the particles as demonstrated by Ono and Inoue (Ono, T. and Inoue, Y. (1983) FEBS Lett. 164, 252–260). Oxygen-evolution activity of the CaCl2-treated particles was very low in a medium containing 10 mM NaCl as a salt, but could be restored by the 33-kDa protein. When the particles were incubated in 10 mM NaCl at 0°C, two of the four Mn atoms per oxygen-evolution system were released with concomitant loss of oxygen-evolution activity. The 33-kDa protein suppressed the release of Mn and the inactivation during the incubation. These findings from reconstitution experiments suggest that the 33-kDa protein acts to preserve Mn atoms in the oxygen-evolution system. The 33-kDa protein could be partially substituted by 100 or 150 mM Cl− for the preservation of the Mn and oxygen-evolution activity. The Mn in Photosystem II particles enhanced rebinding of the 33-kDa protein to the particles.

The Cl− effect on photosynthetic oxygen evolution: interaction of Cl− with 18-kDa, 24-kDa and 33-kDa proteins

The interaction of Cl− with the extrinsic proteins of 18 kDa, 24 kDa and 33 kDa in the photosynthetic oxygen‐evolution complex was studied by comparing spinach photosystem II particles of different protein compositions. The 33‐kDa protein decreased the Cl− concentration optimum for oxygen evolution from 150 to 30 mM, and the 24‐kDa protein decreased it from 30 to 10 mM. The 18‐kDa protein did not change the optimum Cl− concentration, but sustained oxygen evolution at Cl− concentrations lower than 3 mM. The presence of the 24‐kDa and 18‐kDa proteins, but not each protein alone, markedly suppressed inactivation of oxygen evolution at a very low Cl− concentration and its restoration by readdition of Cl−.

Effect of urea on Photosystem II particles. Evidence for an essential role of the 33 kilodalton polypeptide in photosynthetic oxygen evolution

Abstract Treatment with concentrated urea of Photosystem II particles, which were depleted of two polypeptides of 24 and 18 kDa by a prior washing with 1.0 M NaCl, released a polypeptide of 33 kDa and reduced photosynthetic oxygen-evolution activity. Light stimulated the polypeptide release and inactivation. The degree of inactivation was proportional to the release of the 33 kDa polypeptide from the particles, both in the light and in the dark. These observations suggest that the 33 kDa polypeptide plays an essential role in the oxygen-evolution system. The urea treatment also released Mn from the particles. A quantitative analysis suggests that one molecule of 33 kDa polypeptide interacts with two Mn atoms in the oxygen-evolution system.

Partial reconstitution of the photosynthetic oxygen evolution system by rebinding of the 33-kDa polypeptide

Treatment with 2.6 M urea of the Photosystem II particles depleted of two polypeptides of 24 kDa and 18 kDa completely released a polypeptide of 33 kDa and eliminated the oxygen‐evolution activity. The 33‐kDa polypeptide rebound to the urea‐treated particles and partially reactivated the oxygen evolution. A quantitative analysis of the rebinding suggests tha there is a specific binding site for the 33‐kDa polypeptide on the membrane surface.