Primary Structure Characterization of the Photosystem II D1 and D2 Subunits

Abstract

Mass spectrometry techniques have been applied in a protein mapping strategy to elucidate the majority of the primary structures of the D1 and D2 proteins present in the photosystem II reaction center. Evidence verifying the post-translational processing of the initiating methionine residue and acetylation of the free amino group, similar to those reported for other higher plant species, are presented for the two subunits from pea plants (Pisum sativum L.). Further covalent modifications observed on the D1 protein include the COOHterminal processing with a loss of nine amino acids and phosphorylation of Thr 2 . In addition, the studies reported in this paper provide the first definitive characterization of oxidations on specific amino acids of the D1 and D2 proteins. We believe that these oxidations, and to a much lesser extent the phosphorylations, are major contributors to the heterogeneity observed during the electrospray analysis of the intact subunits reported in the accompanying paper (Sharma, J., Panico, M., Barber, J., and Morris, H. R. (1997) J. Biol. Chem. 272, 33153-33157). Significantly, all of the regions that have been identified as those particularly susceptible to oxidation are anticipated (from current models) to be in close proximity to the redox active components of the photosystem II complex.The accurate molecular mass information afforded by techniques such as electrospray ionization (ESI) 1 mass spectrometry (MS) is particularly useful in cases where gene sequences are available; a correlation between experimental and expected measurements can be sufficient in confirming the identity and homogeneity of intact proteins. However, in situations where molecular weight discrepancies are detected or more detailed structural characterization is required, an MS peptide mass mapping strategy is usually applied (1). This procedure uses suitable MS techniques to analyze the peptides generated by enzymatic and/or chemical digestion of the protein under investigation. The molecular weights for the products must match within experimental error those predicted from the protein sequence; those that do not are then presumed to correspond to peptides containing sequence errors or post-translational modifications, or they may provide evidence for contaminating proteins.Detailed structural information of proteins is of fundamental importance in developing an understanding of their biological activities. It therefore follows that analysis of the primary structural features that govern the higher orders of structures of proteins are of considerable interest. The application of a wide range of biochemical and molecular biological studies has identified several post-translational modifications on the D1 and D2 proteins of photosystem II (PSII) reaction centers, for example NH 2 -and COOH-terminal proteolytic processing (2, 3), acetylation (2), lipid attachment and acylation (4, 5), and phosphorylation (2, 6). Most of these covalent processes have been associated with the biosynthesis and assembly of active photosy...

The Thylakoid Lumen Protease Deg1 Is Involved in the Repair of Photosystem II from Photoinhibition in Arabidopsis

“…In this context, it is interesting that three clusters of oxidized residues are found in the D1 protein after exposure to light: at the top of helix B (the pheophytin binding domain), at the bottom of helix D (the P680 domain), and at the lumen-exposed C terminus (Sharma et al, 1997). It is possible that oxidation of residues in the last two domains results in conformational changes nearby, rendering them susceptible to cleavage by Deg1 located on the lumenal surface of the thylakoid membrane and resulting in the 5.2-and 16-kD fragments observed here.…”

“…It is possible that oxidation of residues in the last two domains results in conformational changes nearby, rendering them susceptible to cleavage by Deg1 located on the lumenal surface of the thylakoid membrane and resulting in the 5.2-and 16-kD fragments observed here. Because the D2 protein is less oxidized than D1 (Sharma et al, 1997), it is not surprising that its turnover rate is lower.…”

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

“…Direct experimental evidence for the light-induced oxidation of specific amino acid residues on the D1 and D2 proteins by ROS is lacking. Early studies using electrospray ionization mass spectrometry revealed extensive oxidation of the D1 and D2 proteins in dark-treated PSII reaction centers (18). The use of tandem mass spectrometry recently allowed a more comprehensive identification of oxidized amino acids in PSII (31).…”

“…On the PSII electron donor side, a number of natively oxidized residues were identified in the vicinity of the Mn 4 O 5 Ca cluster on the D1, D2, and CP43 subunits (17). On the PSII electron acceptor side, Sharma et al (18) had identified a peptide of the D1 protein ( 130 E-136 R) that lies in the vicinity of Pheo D1 and contained a single oxidative modification on an unidentified residue. Subsequently, Frankel et al (19) identified a number of natively oxidized residues in the vicinity of Q A and Pheo D1 .…”

Eukaryotic Peptide Deformylases. Nuclear-Encoded and Chloroplast-Targeted Enzymes in Arabidopsis

“…Nevertheless, the N termini of other chloroplast genomeencoded proteins (Michel et al, 1988;Sharma et al, 1997aSharma et al, , 1997bSharma et al, , 1997c, including the large subunit of Rubisco (Houtz et al, 1989), are extensively processed and do not contain an N-formyl group.…”