71 - The Genetically-encoded Photosensitizer mini Singlet Oxygen Generator produces superoxide

Abstract

While reactive oxygen species (ROS) are known to have a harmful role within a cell, ROS can also have a protective signaling role. One limitation to differentiating these roles is the inability to spatially and temporally control ROS production. ROS-generating proteins (RGPs) overcome this limitation by using light to control ROS production. RGPs have been used in a variety of applications, from cell ablation to mutagenesis; however, the type of ROS produced is often not well characterized. One RGP, mini “Singlet Oxygen Generator” was reported to generate large quantities of singlet oxygen. However, the contribution of superoxide remains unclear due to indirect measures with confounding interpretations. We measured the light-dependent superoxide production of purified miniSOG using HPLC separation of dihydroethidium oxidation products. We demonstrate that miniSOG generates superoxide in an SOD-dependent and azide/catalase-independent manner. miniSOG’s superoxide production rate was lower than its free chromophore, flavin mononucleotide. miniSOG produced superoxide at a rate of ~4.0 µmol superoxide/min/µmol photosensitizer for an excitation fluence rate of 5.9 mW/mm2 at 470±20 nm, and the rate remained consistent across fluences. This contrasts miniSOG’s singlet oxygen production which has been shown to be fluence-dependent. Understanding the type and amount of biologically available ROS produced by miniSOG can improve the experimental design and interpretation of the results. Our results may aid in the design of new RGPs that can be more selective in their photosensitization mechanism.