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

Tina M Dreaden,Jun Chen,Sascha Rexroth,Bridgette A Barry

doi:10.1074/jbc.m110.212928

Tina M Dreaden, Jun Chen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.212928

Copy DOI

Abstract

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganese-calcium 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.

Highlights

Photoinhibition and High Light Stress—Examination of CP43 protein sequences in other organisms, both prokaryotes and eukaryotes, indicates strict sequence conservation of Trp-365
Summary—In this paper, we provide evidence that PSII contains a modified form of tryptophan, NFK
Mass spectrometry on purified peptides shows a mass shift of ϩ32 m/z for the 363AP(W*)LEPLRGPNGLDLSR379 peptide from CP43

Summary

EXPERIMENTAL PROCEDURES

Removal of psbO and the Mn4Ca cluster (supplemental Fig. S1, step 1) was performed by incubation with 800 mM Tris-NaOH, pH 8.0, for 45 min at room temperature in the light [37] These Tris-washed PSII membranes were washed three times with a buffer of 400 mM sucrose, 50 mM HEPESNaOH, pH 7.5, and resuspended in the same buffer to yield a chlorophyll concentration of 2– 4 mg/ml. A 220 ␮W 229-nm probe beam from a frequency-doubled argon-ion laser (Cambridge LEXEL 95, Fremont, CA) was used to record Raman spectra of the model compounds, kynurenine and NFK, which were dissolved in water In spinach PSII membranes, at 25 °C, and a light intensity of 4,000 ␮mol of photons/(m21⁄7s), the half-time for oxygen evolution was reported as ϳ30 min [46].

RESULTS

DISCUSSION

Methods