IMMOBILIZATION OF THE 3 EXTRINSIC PROTEINS IN SPINACH OXYGEN-EVOLVING PHOTOSYSTEM-II MEMBRANES - ROLES OF THE PROTEINS IN STABILIZATION OF BINDING OF MN AND CA2+

The three extrinsic proteins of 33, 23 and 17 kDa were immobilized by treating oxygen-evolving Photosystem II (PS II) membranes with a zero-length crosslinker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The 33 kDa and 17 kDa proteins were totally immobilized at 1% and 2% EDC, respectively. The 23 kDa protein was also immobilized in parallel to the 17 kDa protein at low concentrations of EDC, but a small fraction of the 23 kDa protein remained uncrosslinked at 3% EDC. Immobilization of the three proteins had no or only a minor inhibitory effect on oxygen evolution. The amount of Mn that was gradually released from urea/NaCl-washed PS II membranes was reduced from two to one per PS Il by immobilization of the 33 kDa protein and further to a lower level by crosslinking of the 23 and 17 kDa proteins. Stabilization of Mn by the latter two proteins was also suggested by release of a small amount of Mn from NaCl-washed membranes during prolonged incubation at 0-degrees-C. Reductant-induced inactivation of oxygen evolution in urea/NaCl-washed membranes, which is ascribed to reduction and subsequent release of Mn, was suppressed by more than 50% by immobilization of the 33 kDa protein and further to a small extent by that of the 23 and 17 kDa proteins. Immobilization of the 23 kDa (and 17 kDa) protein(s) rendered oxygen evolution significantly resistant to treatment with 1.5 M NaCl and at low pH levels. Thus, binding of a functional Ca2+ is stabilized by covalent binding of the protein against not only salt- but also acid-extraction.