Three genes within the genome of E. coli K12 are predicted to encode proteins containing the typical Rieske iron–sulfur cluster-binding motifs. Two of these, hcaC and yeaW, were overexpressed in E. coli BL21 and Tuner (DE3) pLacI. The recombinant proteins were purified and analyzed by UV/Vis- and EPR-spectroscopy. HcaC and YeaW display the typical redox-dependent UV/Vis-spectra of iron–sulfur proteins. The EPR spectrum of reduced HcaC shows characteristic g-values of a Rieske cluster whereas the g-values for YeaW are close to the upper limit for this type of iron–sulfur cluster. Both iron–sulfur clusters could be reduced by dithionite, but not by ascorbate, confirming their classification as low-potential Rieske proteins as derived from the amino acid sequences. A phylogenetic analysis of the two proteins reveals that HcaC clearly segregates with the Rieske ferredoxins of class IIB oxygenases whereas the classification of YeaW remains doubtful.