Cholate Extraction of a Heme-Protein from Spinach Thylakoids and its Possible Involvement in PS-II Oxygen Evolution

A heme-protein has been obtained from spinach thylakoids using an extraction procedure similar to that described by Spector and Winget [1] for the isolation of a manganese protein claimed to be involved in photosynthetic oxygen evolution. The protein isolated in this study displays a Soret peak near 400 nm, similar to the absorption spectrum for other heme-proteins, e.g., peroxidase and catalase. The heme nature of this protein was confirmed by its reduced pyridine hemochromagen spectrum with a peak at 557 nm, indicating also that the prosthetic group is a protoporphyrin. In addition, the iron exhibits an extremely temperature-sensitive high-spin EPR signal in the g = 6.0 region at liquid helium temperatures. The pl of the protein was determined by isoelectric focusing to be about 6.0 and the apparent polypeptide molecular weight by SDS-gel electrophoresis was found to be about 58,000, contrary to the earlier estimate reported in the Proceedings of the 5th International Congress on Photosynthesis, Halkidiki, Greece. Sedimentation studies indicate that the native molecular weight of the protein is approximately four times greater than that determined by SDS electrophoresis. It was shown that the isolated heme-protein could bring about enhancement of oxygen evolution when incorporated into liposomes containing cholate- extracted chloroplast material.