Reactivity of a nickel-rubredoxin as a bioanalytical tool for probing nitroxyl and nitric Oxide

Rubredoxins are highly stable iron–sulfur metalloproteins widely used as model systems due to their simple metal-binding site. This study investigates nickel-substituted rubredoxin (Rd-Ni) from Desulfovibrio gigas as a potential platform for detecting reactive nitrogen species, particularly nitric oxide (NO) and nitroxyl (HNO). We hypothesized that NO/HNO binding to the nickel center would induce structural and electronic changes detectable by spectroscopic techniques. Rd-Ni was prepared through in vivo metal substitution and characterized using UV–Vis absorption, fluorescence, circular dichroism, and ICP-AES. Unlike the native iron-containing protein, Rd-Ni exhibited clear spectroscopic responses upon interaction with NO/HNO. ICP-AES analysis further suggested partial demetallation of the nickel center as the underlying reaction mechanism. These findings demonstrate the potential of Rd-Ni as a biomimetic platform for the selective detection of reactive nitrogen species and future biosensor development.