Reconstitution of cytochrome b-560 (QPs1) of bovine heart mitochondrial succinate-ubiquinone reductase.
The QPs1 subunit of bovine heart mitochondrial succinate-ubiquinone reductase was overexpressed in Escherichia coli DH5 alpha cells as a glutathione S-transferase fusion protein (GST-QPs1) using the expression vector, pGEX/QPs1. The yield of soluble active recombinant GST-QPs1 fusion protein depends on the IPTG concentration, induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth with 0.5 mM IPTG at 27 degrees C in an enriched medium containing betaine and sorbitol. QPs1 is released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs1 shows one protein band in SDS-polyacrylamide gel electrophoresis corresponding to subunit III of mitochondrial succinate-ubiquinone reductase. However, partial N-terminal amino acid sequence analysis of recombinant QPs1 shows two extra amino acid residues, glycine and serine, at the N-terminus of mature QPs1, resulting from the recombinant manipulation. When isolated recombinant QPs1 is dispersed in 0.01% dodecyl maltoside, it is in a highly aggregated form with an apparent molecular mass of over 1 million. Recombinant GST-QPs1 contains little cytochrome b-560 heme. However, addition of hemin chloride restores the spectral characteristics of cytochrome b-560. Cytochrome b-560 restoration varies with the amount of hemin used. Maximum reconstitution is obtained when the molar ratio of heme to fusion protein used in the system is 0.6. Reconstituted cytochrome b-560 shows a EPR signal at g = 2.91 which corresponds to one of the EPR signals of cytochrome b-560 in a QPs preparation. When GST-QPs1 with reconstituted cytochrome b-560 is treated with thrombin to cleave GST from QPs1, no change in the absorption and EPR characteristics of cytochrome b-560 is observed, indicating that the bis-histidine ligands of reconstituted cytochrome b-560 are provided by QPs1.
Lee
G Y
GY
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078, USA.
Zhu
J
J
Yu
L
L
Yu
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D042963:Electron Transport Complex II; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013385:Succinate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
D003573:Cytochrome b Group; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; C025355:cytochrome b560; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D005982:Glutathione Transferase; D013917:Thrombin