A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8.
Cytochrome ba3 is an integral membrane protein that serves as a terminal oxidase of the respiratory chain in some prokaryotes. We have cloned the complete cba operon of Thermus thermophilus HB8 in an Escherichia coli/T. thermophilus shuttle vector. The ba3-encoding operon, cba, was eliminated from the chromosome of T. thermophilus strain MT111 using the pyrE system of Yamagishi and co-workers. Expression of functional cytochrome ba3 occurred in cells grown at reduced dioxygen levels. A hepta-histidine tag was placed at the N-terminus of subunit I, and a purification method for this form of the enzyme was developed. Growth conditions were investigated for moderate sized cultures (2L) with typical yields of approximately 2 mg of highly pure enzyme per liter of culture medium. The physical properties and enzymatic activities of these recombinant enzymes were compared with those of native enzyme. Recombinant enzyme lacking the histidine tag is spectrally identical to wild-type enzyme. Histidine-tagged cytochrome ba3 shows minor differences from wild-type, and it appears be somewhat less active as a cytochrome c552 oxidase. Exemplary mutants were also produced and compared to native protein. Tyrosine I-237, previously found to be covalently bonded to I-His-233, was changed to phenylalanine (I-Y237F) and to histidine (I-Y237H) in the hepta-histidine tagged cytochrome ba3. The Y to F mutant is devoid of enzyme activity whereas the Y to H mutant possesses approximately 5% wild-type oxidase activity; their properties are compared with those of wild-type enzyme. The above versions of the histidine-tagged enzyme have been crystallized, and our analysis of a 2.3 angstrom resolution electron-density map will be discussed elsewhere.
Chen
Ying
Y
Division of Biology, University of California at San Diego, 9500 Gilman Dr., La Jolla CA 92093-0116, USA.
Hunsicker-Wang
Laura
L
Pacoma
Ronald L
RL
Luna
Eugene
E
Fee
James A
JA
D003001:Cloning, Molecular / Q000379:methods*; D003573:Cytochrome b Group / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine; D015336:Molecular Probe Techniques; D020125:Mutation, Missense; D009876:Operon / Q000235:genetics; D010084:Oxidation-Reduction; D010100:Oxygen; D015202:Protein Engineering; D013057:Spectrum Analysis; D016963:Thermus thermophilus / Q000737:chemistry*
D003573:Cytochrome b Group; D006639:Histidine; C058557:cytochrome ba3; D003576:Electron Transport Complex IV; D010100:Oxygen