N-Formylkynurenine as a Marker of High Light Stress in Photosynthesis

Abstract

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganesecalcium active site. Light-dependent damage and repair occur in PSII under conditions of high light stress. The core reaction center complex is composed of the D1, D2, CP43, and CP47 intrinsic polypeptides. In this study, a new chromophore formed from the oxidative post-translational modification of tryptophan is identified in the CP43 subunit. Tandem mass spectrometry peptide sequencing is consistent with the oxidation of the CP43 tryptophan side chain, Trp-365, to produce N-formylkynurenine (NFK). Characterization with ultraviolet visible absorption and ultraviolet resonance Raman spectroscopy supports this assignment. An optical assay suggests that the yield of NFK increases 2-fold (2.2 ؎ 0.5) under high light illumination. A concomitant 2.4 ؎ 0.5-fold decrease is observed in the steady-state rate of oxygen evolution under the high light conditions. NFK is the product formed from reaction of tryptophan with singlet oxygen, which can be produced under high light stress in PSII. Reactive oxygen species reactions lead to oxidative damage of the reaction center, D1 protein turnover, and inhibition of electron transfer. Our results are consistent with a role for the CP43 NFK modification in photoinhibition.Oxygenic photosynthesis is the enzyme-catalyzed conversion of light energy to biochemical energy, and this process occurs in the membranes of plants, algae, and cyanobacteria. In oxygenic photosynthesis, photosystem II (PSII) 4 catalyzes the light-driven oxidation of water and reduction of plastoquinone. On the acceptor side of PSII, electrons are transferred sequentially to two quinone molecules, Q A and Q B (1). On the donor side, a Mn 4 Ca active site is the binding site for water and the site of oxygen production. Each monomer is composed of 20 protein subunits, chlorophylls, carotenoids, and redox-active plastoquinones (2, 3). Calcium and chloride are required for activity under physiological conditions (4). The chloride binding site has been assigned near the active site (2, 3).The D1, D2, CP43, and CP47 polypeptides form the intrinsic core complex of PSII. The D1 and D2 membrane spanning proteins bind the electron transfer cofactors active in water oxidation (2, 3). This central heterodimeric core is symmetrically flanked by the CP43 and CP47 proteins, which bind light-harvesting antennae chlorophyll (Chl) molecules (5). Each of these core polypeptides is composed of intrinsic membrane-spanning helices, as well as several hydrophilic loops that protrude into the interior lumen of the thylakoid membrane (2, 3). The lumenal loop regions of CP43 have been implicated as important in assembly and protection from photoinhibition (see Ref. and references therein).The active site of water oxidation, the Mn 4 Ca cluster, is located on the lumenal surface and is protected by three extrinsic polypeptides (6). In plants, these extrinsic proteins, the 18-kDa, 24-kDa, and psbO (or the 33-kDa, manganes...

Light-Induced Oxidative Stress, N-Formylkynurenine, and Oxygenic Photosynthesis

“…This corrects for the yield of tryptic peptides [19]. These data are presented in the bar graph shown in Figure 5.…”

“…HPLC separation, isolation of NFK-containing peptides, and quantitative NFK assay were carried out as previously described [19]. Retention times for Fractions A–D were 25, 26, 28, and 34 minutes and were reproducible to ±0.6 min (Table S1).…”

Release of ecologically relevant metabolites by the cyanobacterium Synechococcus elongatus CCMP 1631

“…Anderson et al, (2002) detected kynurenine and other tryptophan oxidation products in the place of tryptophan residues in one of the PSII proteins (CP43) suggesting that these modifications are a result of reactive oxygen species generated during the light reactions. Oxidation of tryptophan residues to N-formylkynurenine in the D1 protein by ROS generated during photosynthesis was also recently observed (Dreaden et al, 2011;. The PSII proteins, particularly D1, are known to turn over rapidly relative to other cellular proteins (Ohad et al, 1984) and the presence of these tryptophan oxidation products may be an indicator of ROS-damaged proteins (Anderson et al, 2002).…”

Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences

“…One aspect of the sensing and repair mechanism seems to rely on the oxidation of Trp residues to NFK in core proteins of PSII, a reaction occurring readily in the presence of 1 O 2 . In PSII, increasing oxidation of Trp365 to NFK in the CP43 subunit correlates with increasing light stress and increasing photoinhibition in thylakoid membranes [91, 93]. While a small increase in ionic strength results in a decrease in NFK365 and leads instead to a Trp to NFK conversion in a different intrinsic polypeptide (D1), both the CP43 and the D1 Trp oxidations to NFK appear to be linked to D1 degradation and subsequent replacement of the damaged D1 proteins.…”