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Primary Structure Characterization of the Photosystem II D1 and D2 Subunits

1997; Elsevier BV; Volume: 272; Issue: 52 Linguagem: Inglês

10.1074/jbc.272.52.33158

1083-351X

Jyoti Sharma, Maria Panico, Catherine A. Shipton, Fredrick Nilsson, Howard R. Morris, James Barber,

Enzyme Structure and Function

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 COOH-terminal processing with a loss of nine amino acids and phosphorylation of Thr2. 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. 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 COOH-terminal processing with a loss of nine amino acids and phosphorylation of Thr2. 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) 1The abbreviations used are: ESI, electrospray ionization; MS, mass spectrometry; PSII, photosystem II; CNBr, cyanogen bromide; FAB, fast atom bombardment; HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; Hse, homoserine. 1The abbreviations used are: ESI, electrospray ionization; MS, mass spectrometry; PSII, photosystem II; CNBr, cyanogen bromide; FAB, fast atom bombardment; HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; Hse, homoserine. 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 (1Morris H.R. Panico M. Taylor G.W. Biochem. Biophys. Res. Commun. 1983; 30: 299-305Crossref Scopus (149) Google Scholar). 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 NH2- and COOH-terminal proteolytic processing (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar, 3Takahashi M. Shiraishi T. Asada K. FEBS Lett. 1988; 420: 6-8Crossref Scopus (66) Google Scholar), acetylation (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar), lipid attachment and acylation (4Mattoo A.K. Edelman M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1497-1501Crossref PubMed Scopus (207) Google Scholar, 5Kruse O. Radunz A. Schmid G.H. Z. Naturforsch. 1994; 49c: 9651-9654Google Scholar), and phosphorylation (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar, 6Owen G.C. Ohad I. J. Cell Biol. 1982; 93: 712-718Crossref PubMed Scopus (69) Google Scholar). Most of these covalent processes have been associated with the biosynthesis and assembly of active photosynthetic complexes. By contrast, details of the structural modifications that influence other aspects of PSII function, in particular the triggering and subsequent degradation of damaged PSII reaction center proteins that are observed during illumination conditions, remain to be resolved (7Barber J. Andersson B. Trends Biochem. Sci. 1992; 17: 61-66Abstract Full Text PDF PubMed Scopus (829) Google Scholar). The majority of experimental evidence obtained to date favors the possibility of oxidative damage resulting in conformational changes and eventually the degradation of the D1 subunit and to a lesser extent the D2 subunit (8Aro E.-M. Virgin I. Andersson B. Biochim. Biophys. Acta. 1993; 1143: 113-134Crossref PubMed Scopus (1879) Google Scholar). In the accompanying paper (34Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) we described how we utilized the powerful ESI-MS technique to examine the D1 and D2 proteins that had been isolated from dark treated PSII reaction center preparations. The data obtained indicated the presence of molecular heterogeneity within the populations of both subunits. In the studies reported in this paper we have employed a protein mapping strategy to characterize the nature and sites of the structural modifications that result in the observed diversity. These procedures are detailed in the accompanying paper (34Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Separation of the D1 and D2 protein bands to enable isolation of these subunits was performed by preparative polyacrylamide gel electrophoresis using the LKB 2001 vertical electrophoresis apparatus. Linear 2-mm-thick 15% non–SDS-polyacrylamide gels containing 6 m urea were used for this procedure. The buffer system was 0.2 m glycine, 25 mmTris-HCl, and 0.1% SDS. The reaction center samples were solubilized in an equal volume of 25 mm Tris-HCl, 4% SDS, and 2% β-mercaptoethanol (pH 8.9) before application to the gel (a maximum of 200 μg of chlorophyll was loaded on each gel). After the resolution of the D1 and D2 proteins by preparative polyacrylamide gel electrophoresis, the proteins were identified by negative staining using a 5% (w/v) solution of copper chloride in 0.2 mglycine, 25 mm Tris-HCl. The relevant bands were cut out and washed in a solution of 0.2 m glycine, 25 mm Tris-HCl (elution buffer) to remove any excess copper solution. The gel slices were then reduced by incubation in a solution of 10% β-mercaptoethanol in elution buffer. Finally, the proteins were eluted from the gel slices into elution buffer using a Hoefer Scientific Instruments GT114 tube gel unit, running at 4 °C, 10 mA/tube for 4 h. The D1 and D2 proteins purified by the gel elution technique were dialyzed against 2 × 2000 ml of 20% aqueous formic acid at 4 °C for 6 h. These samples were then made up to a concentration of 70% aqueous formic acid before the digestion. A few crystals of CNBr were added to the sample solutions, and the reaction mixture was incubated in the dark for 5 h at room temperature. The reaction mixture was diluted with 5 volumes of water and dialyzed against 2 × 2000 ml of 50 mm ammonium bicarbonate (pH 8.5) at 4 °C for 6 h. The appropriate amount of trypsin (1:50 w/w, enzyme:substrate) was added and incubated for 6 h at 37 °C. Digestion was terminated by freezing followed by lyophilization. Partial separation of the polypeptides produced by enzymatic digestion was accomplished using an Aquapore OD-300 (220 × 4.6 mm) column fitted onto a Kontron HPLC system (Datasystem 450, HPLC pump 420, detector 430, and mixer M800). The samples were dissolved in 0.1% trifluoroacetic acid (buffer A) for injection on the column. The column was held for 5 min at 0% B followed by a linear increase to 100% B (90% acetonitrile in aqueous 0.1% trifluoroacetic acid) over 60 min at a flow rate of 1.0 ml min−1. Elution was monitored at 214 and 280 nm, and fractions were collected at 1-min intervals. The samples for manual sequencing were dried down in a glass tube and redissolved in 100 μl of Milli-Q water (purified by the Millipore system). 100 μl of a 5% solution of phenylisothiocynate in pyridine was added to each sample, and the coupling reaction was flushed with nitrogen before its incubation at 45 °C. After 1 h this reaction was terminated by removing excess reagents under a stream of nitrogen. To each of the dried samples 100 μl of trifluoroacetic acid was added, and the mixtures were incubated for a further 10 min at 45 °C, causing the cleavage of the phenylthiocarbamyl-labeled terminal amino acids to form the thiohydantoin derivative. The products of the remaining truncated peptides were subsequently dried under nitrogen and analyzed by MS without the removal of phenylthiohydantoin derivatives. FAB mass spectra were acquired using a Fisons VG ZAB2 SEFPD mass spectrometer fitted with a cesium ion gun operating at 30 kV. The instrument was calibrated using cesium iodide clusters. Data acquisition and processing were performed using VG Analytical Opus software. The samples were dissolved either in aqueous 5% acetic acid or in 40% propan-1-ol in aqueous 5% acetic acid (for the more hydrophobic peptides), and 1-μl aliquots were loaded onto the probe that had been treated previously with a 1:1 mixture of glycerol:monothioglycerol. These procedures are detailed in the accompanying paper (34Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Matrix-assisted laser desorption ionization (MALDI) mass spectra were acquired using a Fisons VG ZAB2 SEFPD mass spectrometer fitted with a UV laser (337 nm). The instrument was calibrated using cesium iodide clusters. Data acquisition and processing were performed using VG Analytical Opus software. The samples were dissolved in 60% propan-1-ol in 5% aqueous, and 1-μl aliquots were loaded onto the probe that had been treated with a saturated solution of 2,5-dihydroxybenzoic acid made up in a 70:30 v:v solution of 90% acetonitrile in aqueous 0.1% trifluoroacetic acid and aqueous 0.1% trifluoroacetic acid, respectively, followed by air-drying. The MS mapping strategy used in these studies to analyze the primary structure of the D1 and D2 proteins is facilitated greatly by the availability of pure protein. Thus an alternative purification protocol enabling better separation of these PSII subunits than that reported by our HPLC purification system (described in Ref.34Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) was required. The D1 and D2 proteins share sequence homology and are of approximately the same mass, consequently distinct separation of these subunits is an extremely difficult task to achieve. One technique by which it has been possible to segregate these proteins is SDS-polyacrylamide gel electrophoresis; 15% acrylamide gels containing 6 m urea have been found to give a good separation of the two subunits. We have developed an isolation procedure based on this electrophoretic system followed by electroelution of proteins from the gel. This purification protocol was found to be far more efficient than the electroblotting procedure that we had originally developed for use in these mapping studies. The main problem encountered during the development of our purification technique was that the use of SDS had to be minimized to enable MS analysis. Fortunately, the resolution of the D1 and D2 proteins on polyacrylamide gels that did not contain SDS (in the gel matrix) was found to be similar to those with SDS (Fig.1), with the only obvious difference being that the former had a larger proportion of aggregated products. By contrast, analysis of the products after the gel elution procedure indicated that the presence of SDS had a marked effect on this step; in fact it was found that protein elution from gels that did not utilize SDS in the gel matrix was less efficient than that of the standard SDS-containing system. However, by incubating the non–SDS-containing gel slices in a buffer containing mercaptoethanol before electroelution we were able to increase the purification yields significantly. The pure forms of the D1 and D2 proteins, isolated using the gel elution technique, were extremely hydrophobic, and even slight changes in the procedure led to their precipitation. Moreover, even the propan-1-ol and 5% acetic acid solvent system, which had enabled partial purification of the D1 and D2 for ESI-MS analysis (reported in Ref. 34Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), failed to elute these gel-purified proteins from the reverse phase HPLC column. Thus, special care has to be taken in handling these proteins after their isolation. We found that an effective method for mapping the D1 and D2 proteins avoided any drying or concentrating steps and instead utilized a dialysis strategy to transfer buffers. Application of two consecutive digestions, CNBr followed by trypsin digestion leading to the cleavage of all accessible methionine, arginine, and lysine residues, was found to give the greatest peptide yields. The spectrum presented in Fig. 2 was obtained by direct FAB-MS analysis of an aliquot taken from the successive CNBr and tryptic digestion mixture of the D1 protein purified by the gel elution technique. Most of the observed quaismolecular ions can be assigned to expected D1 peptides (calculated from the protein sequence derived from pea psbA gene; see Ref. 9Shapiro D.R. Tewari K.K. Plant Mol. Biol. 1986; 6: 1-12Crossref PubMed Scopus (18) Google Scholar), and some important information on the primary structure can also be obtained from this spectrum. For example, as can be seen, the quasimolecular ions detected at m/z 744.4, 824.4, 900.4, and 980.4 have all been assigned to NH2-terminal tryptic peptides. The quasimolecular ions m/z 744.4 and 900.4 differ by a mass equivalent to an arginine residue (156 atomic mass units), and their molecular weights correspond to that expected for the NH2-terminal processed (des-Met) and acetylated peptides (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar), acetyl-Thr2-Arg7 and acetyl-Thr2-Arg8, respectively. By analogy, the quasimolecular ions m/z 824.4 and 980.4 also differ by 156 atomic mass units; these signals are both 80 atomic mass units larger than the 744 and 900 components, respectively. The 80-atomic mass unit increment can be ascribed to a phosphoryl group (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar), and these components are likely to represent the NH2-terminal phosphorylated peptides, acetyl/phosphoryl-Thr2-Arg7 and acetyl/phosphoryl-Thr2-Arg8, respectively. Furthermore, although the spectrum is only semiquantitative, it is clear that in these samples the nonphosphorylated form predominates. Verification of these assignments was obtained after purification of the individual components. Direct FAB-MS analysis of complicated mixtures, such as the one under investigation here, often leads to suppression effects (10Morris H.R. Panico M. Barber M. Sedwick R.D. Tyler A.N. Biochim. Biophys. Res. Commun. 1981; 101: 623-631Crossref PubMed Scopus (215) Google Scholar). This phenomenon is usually overcome by partial separation of the digestion products; in this study we used reverse phase HPLC. All fractions correlating with regions of UV absorbance were screened by FAB-MS and ESI-MS. A large majority of the molecular ions detected during this process could be assigned to expected peptides (TableI; note that the methionine cleavage by CNBr results in the formation of homoserine, which is detected as the lactone in these experiments). These assignments have been verified by manual Edman reaction followed by MS analysis of the truncated peptides. The mass shift observed on all of these assigned components corresponds to the molecular weight of the expected NH2-terminal residue (from the nucleotide-derived protein sequence) except on the NH2-terminal tryptic peptides (Thr2-Arg7 and Thr2-Arg8) where no mass shift was detected because the D1 protein is blocked at its NH2 terminus.Table IMS mapping of the CNBr/tryptic peptides from D1 proteinPeptideFractionExpected [M+H]+Measured [M+H]+Total + 16 atomic mass units observedAcThr2-Arg728744.4744.4AcThr2-Arg828–29900.5900.4Ac/PThr2-Arg727824.4824.4Ac/PThr2-Arg827–28980.4980.4Asp9-Arg1626976.4976.5Ile38-Arg64ppt2826.92827.3Gly128-Arg136291179.61179.61Glu130-Arg13625966.4966.51Arg140-Hse172413380.93380.7Pro173-Hse18332–341135.41135.4Ile184-Hse194301266.61266.6Ile184-Hse199311916.91916.93Leu200-Hse21436–401364.71364.6His215-Arg22528–291169.61169.6Glu226-Arg23822–231499.61499.6Phe239-Arg257312188.52188.4Leu258-Arg269311459.71459.6Ala294-Arg312322114.22114.2Val313-Arg323321314.81314.92Ala324-Arg334241286.61286.62His332-Ala344301533.61533.42Asn335-Ala34431–321111.61111.6 Open table in a new tab In fractions 27–29 the four molecular ions attributed to the processed NH2-terminal peptides, which had been detected during examination of the digestion mixture, were identified. By contrast, the molecular ions representing the unprocessed NH2-terminal peptide of the D1 protein were not observed in any of the UV-absorbing fractions. The NH2-terminal processing event characterized for the pea D1 protein entails removal of the initiating formylmethionine residue followed by N-acetylation of the second residue, Thr2. This process is therefore similar to that reported for the D1 protein of spinach (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar). In addition, our studies have also provided direct evidence for the presence of an NH2-terminal phosphorylation site on the D1 subunits of pea plants, which is also analogous to that observed in its spinach counterpart (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar). Even though it is difficult to quantitate exactly, careful analysis of these samples indicates that the phosphorylated form occurs in approximately one-third of the D1 population. This modification would thus contribute to the heterogeneity observed by the ESI-MS analysis of the intact protein (34Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The COOH terminus of the D1 protein in most photosynthetic organisms (except Euglena) is thought to be processed to the mature form (11Keller M. Stutz E. FEBS Lett. 1986; 175: 173-177Crossref Scopus (46) Google Scholar). In spinach plants this event was found to result in the removal of nine amino acid residues 335–344 (3Takahashi M. Shiraishi T. Asada K. FEBS Lett. 1988; 420: 6-8Crossref Scopus (66) Google Scholar); however, as yet there has been no direct evidence confirming the occurrence of this modification in pea plants (the organism under investigation in these studies). During our analyses of the D1 protein we did not detect the quasimolecular ion corresponding to the unprocessed COOH-terminal peptide, m/z 1679.8, Asp335-Gly353, although the quasimolecular ion at m/z 1111.6 which corresponds to the processed COOH-terminal component, Asn335-Ala344, was observed readily (Fig. 2). These findings not only provide direct evidence verifying that the D1 protein in pea plants is processed proteolytically in a way similar to that reported for spinach and other plants, but it also indicates that at least in the granal regions of thylakoid membranes (the regions from which these PSII reaction centers were isolated) there is no heterogeneity at this modification site. During the screening of the isolated products we observed that several molecular ions that corresponded to peptides of the D1 protein had additional satellite components at multiples of 16-atomic mass unit increments associated with them. The 16-atomic mass unit increment can be ascribed to an oxygen atom; thus it seems that the molecular ions with these mass increments are oxidized products. The D1 peptides found to carry this modification are presented in Table I. In some cases, for example, Glu130-Arg136 (966.4), Gly128-Arg136 (1179.6), Ala324-Arg334 (1286.6), and Ile184-Hse199 (1916.9), the quasimolecular ions corresponding to the oxidized peptides were quite prominent. On peptides Glu130-Arg136 and Gly128-Arg136, the quasimolecular ions m/z 982.4 and 1195.6, which are both 16 atomic mass units larger than the signals expected for these molecules, are the principal components detected. Similarly, for the peptide Ala324-Arg334 the main quasimolecular ion observed, at m/z 1318.6, is 32 atomic mass units heavier (addition of two oxygen atoms) than that of its calculated mass, whereas on the peptide Ile184-Hse199 quasimolecular ions at m/z 1933.1, 1949.1, and 1965.2 corresponding to additions of 16, 32, and 48 atomic mass units (one, two, and three oxygen atoms, respectively) were detected. Collectively, these results, which show the absence of oxidations on some peptides while others have up to three oxygen additions, indicate that there is a degree of specificity of this modification. According to the folding model of the D1 protein (Fig.3), which has been developed by analogy to the l-subunit of the corresponding bacterial photosynthetic unit (28Michel H. Deisenhofer J. Biochemistry. 1988; 27: 1-7Crossref Scopus (644) Google Scholar), the observed oxidation sites appear to be located primarily in two lumenal regions, the COOH-terminal region and the beginning of the fourth transmembrane segment, and one stromal region, the end of the II transmembrane segment. This group of modifications exemplifies a further source of heterogeneity which may also explain the ESI-MS data obtained for the intact D1 protein presented in the accompanying paper (34Sharma J. Panico M. Barber J. Morris H.R. J. Biol. Chem. 1997; 272: 33153-33157Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). At the time of transferring the CNBr digestion products into the buffer required for tryptic digestion we noted a slight precipitate. This product, which could probably be attributed to the poor solubility of some extremely hydrophobic peptides, remained insoluble in the solvent conditions used to initiate the HPLC separation. To complete the analysis of the D1 protein we analyzed this precipitate by MALDI-MS, a technique that is anticipated to be more amenable to hydrophobic molecules. The D1 protein digestion mixture was centrifuged and subsequently separated into soluble and precipitated phases. Direct MALDI-MS analysis of the precipitate identified (among other signals) the quasimolecular ion, m/z 2827.3 (Fig.4). This component can be assigned to peptide Ile38-Arg64, which had not been observed previously. The quasimolecular ion at m/z 2615.3, also present in this spectrum, is yet to be assigned. It is quite clear from the above observations that the extreme hydrophobic properties coupled with the structural complexity of the D1 protein have made it a particularly difficult molecule to study; nevertheless the application of several complementary techniques has enabled a significant portion of this subunit to be mapped successfully and has also allowed characterization of several post-translational modifications. The overall map of proven MS assignments is shown in Fig. 3. Direct FAB-MS analysis of an aliquot from the successive CNBr and tryptic digestion experiment on the gel-purified D2 (Fig. 5) depicts a host of quasimolecular ions, most of which can be attributed to expected D2 peptides (derived from the pea psbD sequence; see Ref. 12Rasmussen O.F. Bookjans G. Stutmann B.M. Henningsen K.W. Plant Mol. Biol. 1984; 3: 191-199Crossref PubMed Scopus (74) Google Scholar). Verification of these assignments has been carried out after purification of the individual components. One of the regions mapped by this process is the NH2-terminal tryptic peptide, acetyl-Thr2-Lys7 (m/z644.4); however, unlike the D1 protein, the quasimolecular ion with an 80-atomic mass unit increment (m/z 724.4), corresponding to the phosphorylated form of this peptide, was not immediately apparent in the FAB map spectrum. Subsequently, a more rigorous analysis of the D2 protein was carried out on the partially purified digestion products obtained by reverse phase HPLC. All fractions correlating with regions of UV absorbance on the HPLC chromatogram were screened by FAB-MS and ESI-MS. A significant number of the molecular ions observed during this process could be assigned to expected D2 peptides (Table II). These assignments have been confirmed by manual Edman degradation followed by MS analysis. Collectively, the data obtained in this study have enabled a large proportion of the D2 protein to be mapped and have also revealed interesting structural information.Table IIMS mapping of the CNBr/tryptic peptides from D2 proteinPeptideFractionExpected [M+H]+Measured [M+H]+Total +16 atomic mass units observedAcThr2-Lys722644.4644.4AcThr2-Lys10261020.61020.5Asp11-Arg24341818.51818.6Trp105-Arg135403540.43539.8Leu128-Arg135241032.61032.6Gln130-Arg13523763.4763.4Ser136-Hse199ppt7241.67242.02Phe182-Hse200352380.72380.72Gly201-Arg23437–393361.43361.4Ala235-Hse247231440.61440.6Phe253-Lys265321530.71530.7Arg266-Hse28234–352133.32133.21Leu273-Arg295382428.02428.4Ser283-Arg295331282.71282.7Ser283-Arg30538–392492.52492.8Ala296-Arg305251227.61227.5Ala306-Lys31826–271547.51547.6Asn319-Arg327251041.61041.6Ala328-Arg349292622.92623.13Ala331-Arg349282234.12234.1Ala331-Leu353ppt2589.72589.4 Open table in a new tab The D2 proteins of spinach plants have been shown previously to be NH2-terminally processed (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar). This modification is similar to that reported for the D1 protein and entails the removal of the initiating methionine residue followed by N-acetylation. Michel et al. (2Michel H. Hunt D.F. Shabanowitz J. Bennett J. J. Biol. Chem. 1988; 263: 1123-1130Abstract Full Text PDF PubMed Google Scholar) also found that the NH2-terminal regions of both the D1 and D2 were phosphorylated. In this study on the D2 protein isolated from pea plants, we did not detect the quasimolecular ion corresponding to the unprocessed NH2-terminal peptide, m/z733.4 (Met1-Lys7), although the quasimolecular ion at m/z 644.4, which corresponds to the NH2-terminal processed tryptic peptide acetyl-Thr2-Lys7, was observed in fraction 22. Careful analysis of the spectra obtained from the other fractions in this region did not reveal the presence of an NH2-terminal quasimolecular ion with an 80-atomic mass unit increment which would correspond to the phosphorylated form. The anticipated quasimolecular ion of the NH2-terminal phosphorylated peptide was also not present in the direct FAB analysis of the digestion mixture. There is no reason to suppose that the quasimolecular ion from the phosphorylated peptide would not have been detected, if present, since it was clearly seen in the D1 protein. Thus, it seems that the D2 protein that we purified from pea PSII reaction center preparations is NH2-terminally processed but not phosphorylated. Several of the molecular ions that have been assigned to particular D2 peptides appear to have associated satellite signals at 16-atomic mass unit mass increments. The quasimolecular ion at m/z 2133.2 attributed to the peptide Arg266-Hse282 has only a single 16-atomic mass unit (one oxygen)