How much iron is good? Revisiting the role of ferrous iron supplementation in cultured cells using genetically encoded NO biosensors

Official URL: https://risc01.sabanciuniv.edu/record=b2708509_(Table of contents)

Although ferrous iron (Fe2+) is an essential cofactor for many metalloproteins, most cell culture media lack or only contain insufficient amounts of this transient metal, resulting in iron (II) deficiency in cultured cells. Well-established techniques permit quantification of Fe2+ in cells with high accuracy; however, these techniques do not provide information about the iron-dependent functionality of metalloprotein. In this study, we demonstrated that genetically encoded nitric oxide probe (geNOps), which comprises a non-heme ironbinding protein (GAF) fused with a fluorescent protein, can be utilized as an indirect model system to study the iron-dependency in cultured cells. In the presence of NO, the GAF domain can be reversibly nitrosylated causing changes in the fluorescence intensity in a concentration-dependent manner. We showed that long-term supplementation of Fe2+ in cultured cells has no effect on the functionality of geNOps. Only short-term provision of freshly prepared Fe2+ combined with vitamin C activated the metalloprotein. The distribution of Fe2+ in cells after short-term treatment was observed with the FeRhoNox1 and the accumulation of total iron was determined using high-resolution light microscopy techniques and Perls/DAB staining. Employing the design of experiment approaches, we optimized the Fe2+ loading procedure regarding Fe2+ and vitamin C concentrations and the duration of cell treatment. This approach yielded a non-toxic Fe2+ supplementation procedure that is essential for cell integrity and the full functionality of metalloprotein. In conclusion, this study exploited a metalloprotein readout to extrapolate the Fe2+ uptake capability of cells under cell culture conditions.