Abstract
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.
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Acknowledgements
The authors wish to thank A. Petersen, Research Center Borstel, for the N-terminal sequencing of HcaC, E. Bill, Max Planck Institute for Bioinorganic Chemistry, for recording the EPR spectrum of HcaC, L. Böttger, Institute of Physics, University of Luebeck, for assistance with recording the YeaW EPR spectra, D. Mutschall and W. Verheyen Institute of Biochemistry, University of Luebeck, for skilful experimental assistance.
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Boxhammer, S., Glaser, S., Kühl, A. et al. Characterization of the recombinant Rieske [2Fe–2S] proteins HcaC and YeaW from E. coli . Biometals 21, 459–467 (2008). https://doi.org/10.1007/s10534-008-9134-y
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DOI: https://doi.org/10.1007/s10534-008-9134-y