The C subunit of Ideonella dechloratans chlorate reductase: expression, purification, refolding, and heme reconstitution.
The C subunit of Ideonella dechloratans chlorate reductase has been expressed in Escherichia coli as a GST fusion protein. Purification from inclusion bodies, followed by refolding and reconstitution with heme, produced a protein with a heme/protein ratio of 0.4, and with UV-vis spectral characteristics similar to those of native chlorate reductase. Wavelength maxima for the alpha and beta bands in the reduced state were 559 and 529 nm for both native chlorate reductase and the reconstituted recombinant subunit, whereas the reduced Soret bands were found at 426 and 424 nm, respectively. These results support the notion of the C subunit as the cytochrome b moiety of I. dechloratans chlorate reductase. Moreover, the availability of a recombinant version of the C subunit is expected to facilitate further studies of electron transfer and protein interaction included in the reaction catalyzed by chlorate reductase.
Karlsson
Jan
J
Karlstad University, Department of Chemistry, SE 65188 Karlstad, Sweden.
Nilsson
Thomas
T
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020562:Betaproteobacteria / Q000201:enzymology*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006418:Heme / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013917:Thrombin / Q000737:chemistry
D001426:Bacterial Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006418:Heme; D010088:Oxidoreductases; C021777:chlorate reductase; D013917:Thrombin