Biophysical Society Conference | Tahoe 2023

Proton Reactions: From Basic Science to Biomedical Applications

Tuesday Speaker Abstracts

FROM PROTON REACTIONS TO ADVANCED BIOPHYSICAL VISUALIZATION METHODS FOR BIOMEDICAL APPLICATION: CYTOCHROME C OXIDASE PROTON PATHWAYS, ADVERSE EFFECTS OF DRUG ACTION, AND REACTIVE OXYGEN SPECIES Jens Balke 1 ; Victor Manuel Loyo Cruz 1 ; Ulrike Alexiev 1 ; 1 Freie Universität Berlin, Physics, Berlin, Germany In recent years, my group has specialized in developing techniques from time-resolved fluorescence spectroscopy into advanced spectromicroscopic methods to bridge the gap between the molecular and the cellular level [1-3]. Here, we are using cytochrome c oxidase (CcO) as an example for this approach. As part of the respiratory chain, CcO contributes to the proton motive force (pmf) driving ATP synthesis. On the molecular level a clear picture of the proton exit pathway(s) remains elusive. On the cellular level, CcO dysfunction seems to be associated with increased reactive oxygen species production and cellular toxicity, although a molecular picture is missing. Using CcO from P. denitrificans we address these open questions with the help of selected CcO variants harboring surface mutations [4, 5] including proton collecting antennae and newly discovered surface switch residues. A voltage-sensitive fluorescent protein sensor [3] for pmf detection at the single enzyme level was developed. Surface switch residues influence proton exit and thereby the build-up of a pmf. Along the same lines, the molecular action of the cancer drug doxorubicin (DOX) has been studied, the mechanistic details of its adverse cytotoxic effects being controversial. We show that DOX directly affects electron-coupled proton transfer. To translate this insight to the cellular level, we developed Cluster-Fluorescence Lifetime Imaging (FLIM) [1] and FLIM-ROX [2] to quantify minute amounts of possible oxidative stress induced by DOX.[1] Brodwolf, R. et al., Theranostics 10, 6322 (2020)[2] Balke, J. et al., Small 14, 1800310 (2018) [3] Silapetere, A. et al., Nat. Communications 13, 5501 (2022) [4] Wolf, A. et al., FEBS J. 287, 1232 (2019)[5] Wolf, A. et al., Chemical Science 11, 3804 (2020)

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