Characterization of the recombinant Rieske [2Fe-2S] proteins HcaC and YeaW from E. coli.
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.
Boxhammer
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S
Center for Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, Lubeck, Germany.
Glaser
S
S
Kühl
A
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Wagner
A K
AK
Schmidt
Christian L
CL
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D029968:Escherichia coli Proteins; D005288:Ferredoxins; C532030:HcaC protein, E coli; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C532031:YeaW protein, E coli; D010088:Oxidoreductases