Abstract
Iron- and iron-sulfur cluster (Fe-S)-containing proteins are essential for diverse biological processes, including electron transfer, genome maintenance, metabolism, cellular signaling, and host-pathogen interactions. Despite their broad biological importance and growing links to human disease, Fe-S cluster-dependent proteins remain underexplored as therapeutic targets, largely because it is difficult to define their metal-dependent chemistry using conventional biochemical, spectroscopic, and structural approaches.
This review examines how Mössbauer spectroscopy can be integrated into workflows for metalloprotein characterization, target validation, and drug discovery. Using representative Fe-S cluster-containing proteins, the practical considerations for implementing Mössbauer spectroscopy are outlined, including
Fe-enriched expression, sample preparation, and spectroscopic analysis. Two case studies of experimentally challenging viral Fe-S cluster proteins are then highlighted, the Hepatitis B virus X protein and the Porcine Reproductive and Respiratory Syndrome Virus Nsp1α protease, which demonstrate how direct characterization of metal cofactors can reveal previously unrecognized therapeutic avenues. Relevant literature published through March 2026 was identified using PubMed and Google Scholar with keywords related to Mössbauer spectroscopy, iron-sulfur proteins, viral metalloproteins, and drug discovery.
As drug discovery increasingly seeks to exploit metal-dependent biology, Mössbauer spectroscopy will play an important role in identifying cryptic metalloproteins, defining their native states, and uncovering Fe- and Fe-S cluster-dependent targets. Mössbauer spectroscopy can also be complementary, and integrated with structural and AI-driven approaches to answer emerging challenges in medicinal chemistry.