Nickel‐Substituted Rubredoxin Undergoes Demetallation Upon Reaction With Nitric Oxide and Nitroxyl

Nitric oxide (NO) and nitroxyl (HNO) are closely related reactive nitrogen species with distinct biological functions, yet differentiating their chemistry remains challenging because of their high reactivity and overlapping reaction pathways. Developing simple protein‐based platforms to study their reactivity is therefore of considerable interest. Rubredoxins are small, stable iron–thiolate proteins widely used as model systems for investigating metal–thiolate coordination and electron transfer. Here, we investigated the reactivity of native iron‐rubredoxin (Fe‐Rd) and its nickel‐substituted analog (Ni‐Rd) toward NO and HNO to determine whether cysteine ligand modification could disrupt metal–thiolate coordination and induce demetallation. Electronic absorption and fluorescence spectroscopy showed that Fe‐Rd is highly resistant to both species, whereas Ni‐Rd exhibited pronounced spectral changes consistent with metal release, including loss of the Ni─S ligand‐to‐metal charge‐transfer band and increased intrinsic fluorescence. Inductively coupled plasma atomic emission spectroscopy confirmed partial nickel release following treatment with both NO and HNO donors. Despite demetallation, circular dichroism, thermal denaturation, and size‐exclusion chromatography demonstrated that Ni‐Rd largely retains its secondary structure, thermal stability, and monomeric state. These findings establish Ni‐Rd as a robust minimal metalloprotein platform for probing reactive nitrogen species‐induced thiolate chemistry.