Collin J. Steen*, Jens Niklas, Oleg G. Poluektov, Richard D. Schaller, Graham R. Fleming and Lisa M. Utschig,
{"title":"EPR Spin-Trapping for Monitoring Temporal Dynamics of Singlet Oxygen during Photoprotection in Photosynthesis","authors":"Collin J. Steen*, Jens Niklas, Oleg G. Poluektov, Richard D. Schaller, Graham R. Fleming and Lisa M. Utschig, ","doi":"10.1021/acs.biochem.4c00028","DOIUrl":null,"url":null,"abstract":"<p >A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (<sup>1</sup>O<sub>2</sub>*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of <sup>1</sup>O<sub>2</sub>* in photodamage and cell signaling, few studies directly correlate <sup>1</sup>O<sub>2</sub>* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of <sup>1</sup>O<sub>2</sub>* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of <sup>1</sup>O<sub>2</sub>* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of <sup>1</sup>O<sub>2</sub>* in chlorophyll-containing photosynthetic membranes. We find that the apparent <sup>1</sup>O<sub>2</sub>* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of <sup>1</sup>O<sub>2</sub>* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of <sup>1</sup>O<sub>2</sub>* during high light. Upon saturation of NPQ, the concentration of <sup>1</sup>O<sub>2</sub>* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00028","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry Biochemistry","FirstCategoryId":"1","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.biochem.4c00028","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (1O2*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1O2* in photodamage and cell signaling, few studies directly correlate 1O2* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of 1O2* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1O2* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1O2* in chlorophyll-containing photosynthetic membranes. We find that the apparent 1O2* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1O2* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of 1O2* during high light. Upon saturation of NPQ, the concentration of 1O2* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).
期刊介绍:
Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.