Application of a YHB1-GFP reporter to detect nitrosative stress in yeast

Yeast flavohemoglobin (Yhb1p) plays a pivotal role in NO(*) detoxification and has also been implicated in oxidative/reductive stress responses. In this study, we have used a YHB1-GFP reporter expressing a full-length chromosome-tagged Yhb1-GFP fusion protein to monitor changes in flavohemoglobin levels after cell treatment with oxidants, antioxidants and nitric oxide donors. Only nitric oxide donors were found to induce a dose-dependent increase in Yhb1-GFP expression while hydrogen peroxide, menadione and diamide caused a moderate diminution of YHB1-GFP fluorescence. Additionally, the levels of endogenous and hydroperoxide-induced ROS production in the Deltayhb1 mutant were comparable to those in the isogenic wild-type strain. Although peroxides increased NO(*) generation while nitrite and nitric oxide donors augmented ROS levels in yeast cells, their effects were generally not more pronounced in Deltayhb1 than in wild-type cells. Taken together, these data suggest that yeast flavohemoglobin does not contribute to cross-protection against oxidative and nitrosative stress.