Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

Abstract

Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N’-bis (2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.

Genetically encoded protein sensors for metal ion detection in biological systems: a review and bibliometric analysis

“…Its recognition motif is the bacterially derived GAF structural domain, which is Fe 2+ -dependent and needs to be activated by Fe 2+ to function accordingly. 115 In addition, the discovery of lanmodulin opened up the possibility of using genetically encoded fluorescent protein sensors to detect lanthanides. Lanmodulin is the first natural, highly selective chelator for lanthanide (LnIII) ions.…”