Site-directed mutants of the cytochrome bo ubiquinol oxidase of Escherichia coli: amino acid substitutions for two histidines that are putative CuB ligands.
The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of structurally related heme-copper respiratory oxidases. The members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordinate low-spin heme, and a high-spin heme. The high-spin heme is magnetically coupled to a copper, CuB, forming a binuclear center which is the site of oxygen reduction to water. Vectorial proton translocation across the membrane bilayer appears to be another common feature of this superfamily of oxidases. It has been proposed previously that the two adjacent histidines in putative transmembrane helix VII (H333 and H334 in the E. coli sequence) of the largest subunit of the heme-copper oxidases are ligands to CuB. Previously reported mutagenesis studies of the E. coli bo-type oxidase and the aa3-type oxidase of Rhodobacter sphaeroides supported this model, as substitutions at these two positions produced nonfunctional enzymes but did not perturb the visible spectra of the two heme groups. In this work, six different amino acids, including potential copper-liganding residues, were substituted for H333 and H334 of the E. coli oxidase. All of the mutations resulted in inactive, but assembled, oxidase with both of the heme components present. However, cryogenic Fourier transform infrared (FTIR) spectroscopy of the CO adducts revealed that dramatic changes occur at the binuclear center as a result of each mutation and that CuB appears to be absent.(ABSTRACT TRUNCATED AT 250 WORDS)
Calhoun
M W
MW
School of Chemical Sciences, University of Illinois, Urbana 61801.
Hill
J J
JJ
Lemieux
L J
LJ
Ingledew
W J
WJ
Alben
J O
JO
Gennis
R B
RB
D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared
D008024:Ligands; D006639:Histidine; D003300:Copper; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV