Methods in Enzymology, Volume 599 is the second of two volumes focused on Fe-S cluster enzymes. Topics of interest in this new release include steps towards understanding mitochondrial Fe/S cluster biogenesis, iron sulfur clusters in zinc finger proteins, electrochemistry of Iron-sulfur enzymes, NRVS for Fe in biology and its experiment and basic interpretation, methods for studying iron regulatory protein 1, an important protein in human iron metabolism, the characterization of glutaredoxin Fe-S cluster binding interactions using circular dichroism spectroscopy, fluorescent reporters to track Fe-S cluster assembly and transfer reactions, methods for studying the Fe-S cluster containing base excision repair glycosylase MUTYH, and more.