Structural and Functional Differentiation of Three Groups of Tyrosine Residues by Acetylation of N-Acetylimidazole in Manganese Stabilizing Protein

Abstract

To study its contribution to the assembly of the green plant manganese stabilizing protein (MSP) into photosystem II (PSII), tyrosine residues were specifically acetylated using N-acetylimidazole (NAI). In soluble MSP, three groups of Tyr residues could be differentiated by NAI acetylation: approximately 5 (actually ∼5.2) Tyr residues could be easily acetylated (superficial), 1-2 Tyr residues could be acetylated when the NAI concentration was sufficiently high (superficially buried), and 1-2 Tyr residues could only be acetylated in the presence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did not affect the reconstitution or oxygen-evolving activities of the MSP, and far-UV circular dichroism (CD) analysis showed that the altered MSP retained most of its native secondary structure. These results suggested that the 5.2 Tyr residues are not absolutely essential to the function of MSP. However, further modification of the 1-2 superficially buried Tyr residues (for a total acetylation of ∼6.4 Tyr residues) completely abrogated the MSP rebinding and oxygen evolution activities. Finally, at least one tyrosine residue was inaccessible to NAI until MSP was completely unfolded by 8 M urea. Deacetylation of MSP with 6.4 or 8 acetylated Tyr residues with hydroxylamine restored most of the rebinding and oxygen-evolving activities. A prominent red shift in fluorescence spectra of MSP (excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues, and a further shift could be found after all 8 Tyr residues were modified, indicating a great loss of native secondary structure. Far-UV CD revealed that MSP was mostly unfolded when 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues were modified. Fluorescence and far-UV CD studies revealed that loss of MSP rebinding to PSII membranes following NAI modification correlated well with conformational changes in MSP. Together, these results indicate that different tyrosine residues have different contributions to the binding and assembly of MSP into PSII.In green plants, photosystem II (PSII) 1 catalyzes the lightdriven reduction of plastoquinone and the oxidation of water to molecular oxygen (1). All major components of PSII required for its activity, including chlorophyll a (P680), tyrosine (Y Z ), pheophytin (Pheo), and the primary (Q A ) and secondary (Q B ) quinone acceptors are located within a heterodimeric matrix consisting of polypeptides D1 and D2. Although the essential pathway for photosynthetic water oxidation into molecular oxygen and protons has been established, a detailed comprehensive picture of water oxidation has not been elucidated to date. In particular, the manganese cluster of the water-oxidizing complex (WOC) is not well understood. Site-directed mutagenesis experiments have revealed that several amino acid residues of D1 and D2 are functionally and/or structurally relevant for establishing a competent WOC. However, other extrinsic polypeptides cannot be excluded as being essen...

Structure and Activity of the Photosystem II Manganese-Stabilizing Protein: Role of the Conserved Disulfide Bond

“…The absence of Trp and Tyr signals suggests that the environment around W241 and Y242 in C28A,C51A MSP has become more hydrophilic. This is consistent with the report of Zhang et al (2005) who have shown that one Tyr in MSP is resistant to modification by N-acetylimidazole unless the protein is first unfolded in 8 M urea. Uversky et al (2000) have shown that a defining property of intrinsically disordered proteins is the ratio of charged to hydrophobic residues in their primary amino acid sequences.…”

Structural and functional aspects of the MSP (PsbO) and study of its differences in thermophilic versus mesophilic organisms

“…A huge number of biochemical experiments have been conducted involving site-directed mutagenesis and chemical modification of isolated MSP from several species Eaton-Rye 2005;Han et al 1994;Miura et al 1997;Popelkova et al 2003b;Seidler 1996a;Zhang et al 2005). These studies have identified regions and individual residues of the MSP involved in binding to the intrinsic proteins of PSII.…”

Tyrosine Residues of the Extrinsic 23 kDa Protein Are Important for Its Interaction with Spinach PSII Membranes

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