Characterization of chimeric heme-copper respiratory oxidases using subunits I of Escherichia coli cytochrome b o and Halobacterium salinarium cytochrome aa3.
We constructed chimeric enzymes with the Escherichia coli cytochrome bo and the Halobacterium salinarium cytochrome aa3 through recombinant DNA techniques and investigated their spectroscopic and biochemical properties. Although most of the chimeras could not retain hemes in the molecule, the chimeric enzyme containing helix VII of subunit I of the H. salinarium cytochrome aa3 showed the spectral properties similar to those of the native E. coli oxidase, suggesting that both the low-spin heme b and the high-spin heme o are associated with the chimeric subunit I. However, CuB was absent in the chimera. Helix VII of subunit I of the H. salinarium cytochrome aa3 is 70% similar to the counterpart of the E. coli cytochrome bo and further contains two invariant histidines which serve as the CuB ligands. These results indicate that helix VII must be arranged properly relative to helix VI which provides the third CuB ligand.
Denda
K
K
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Yokohama, Japan.
Mogi
T
T
Anraku
Y
Y
Yamanaka
T
T
Fukumori
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D003300:Copper / Q000378:metabolism*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006217:Halobacterium / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D003580:Cytochromes; D017931:DNA Primers; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008667:Metalloproteins; D011993:Recombinant Fusion Proteins; D003300:Copper; C017168:cytochrome bo, E coli; D003576:Electron Transport Complex IV