Calcium Binding Studies of Photosystem II Using a Calcium-Selective Electrode

• Published in: Biochemistry (Easton) 1998-02, Vol.37 (6), p.1532-1539
• Main Authors: Grove, Geoffrey N, Brudvig, Gary W
• Format: Article
• Language: English
• Subjects: Binding Sites; CALCIUM; Calcium - metabolism; Edetic Acid - metabolism; Intracellular Membranes - metabolism; Ion-Selective Electrodes; Kinetics; Manganese - metabolism; PHOTOSYNTHESIS; Photosynthetic Reaction Center Complex Proteins - metabolism; Photosystem II Protein Complex; Proteins - metabolism; Spinacia oleracea
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971356z

The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2+-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 μM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2+-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2+-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 μM. This number of very high-affinity Ca2+-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721−724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 μM. The loss of 2 very high-affinity Ca2+-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2−3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 μM. These sites are assigned to Ca2+ binding to the vacant Mn sites.