Analysis of chloride-binding domains of the extrinsic 12-kDa protein by proteolysis and directed mutagenesis

Abstract

The extrinsic proteins of 12-kDa and cytochrome c-550 in red algal PSII from Cyanidium caldarium play a role in minimizing the calcium and chloride requirement of oxygen-evolving complex, which resembles closely to the function of the 23- and 17-kDa proteins in higher plant PSII [Enami et al. (1998) Biochemistry 37, 2787-2793]. The 12-kDa protein consists of 93 amino acids with a molecular mass of 10,513 Da [Ohta et al. (1999) B. B. R. C. 260, 245-250]. In order to identify functional domains of the 12-kDa protein, we digested the protein with proteases and then examined its binding and reactivating capabilities by reconstitution of the digested protein with PSII. Treatment of the 12-kDa protein with chymotrypsin resulted in cleavages at Tyr-9 and Phe-83. The digested protein (Leu-10 to Phe-83) was able to bind to PSII completely concomitant with a restoration of oxygen evolution in the presence of NaCl. However, the oxygen evolution was not restored at all in the absence of NaCl. On the other hand, treatment with V8 protease resulted in a peptide fragment from Gly-6 to C-terminus of the 12-kDa protein, which was able to bind to PSII and reactivate oxygen evolution partially even in the absence of NaCl. These results suggest that both the C-terminus and N-terminus of the 12-kDa protein are the functional domain for chloride requirement of oxygen evolution. To confirm this, we are constructing mutants of the 12-kDa protein lacking 10 residues at the C-terminus and 5 or 9 residues at the N-terminus, and will report the binding and reactivation abilities of these mutants.