SITE-DIRECTED MUTANTS OF THE CYTOCHROME-BO UBIQUINOL OXIDASE OF ESCHERICHIA-COLI - AMINO-ACID SUBSTITUTIONS FOR 2 HISTIDINES THAT ARE PUTATIVE CU(B) LIGANDS

The bo-type ubiquinol oxidase of Escherichia coli is a member of the s uperfamily of structurally related heme-copper respiratory oxidases. T he members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordi nate low-spin heme, and a high-spin heme. The high-spin heme is magnet ically coupled to a copper, Cu(B), 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 t his 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-coppe r oxidases are ligands to Cu(B). Previously reported mutagenesis studi es of the E. coli bo-type oxidase and the aa3-type oxidase of Rhodobac ter sphaeroides supported this model, as substitutions at these two po sitions produced nonfunctional enzymes but did not perturb the visible spectra of the two heme groups. In this work, six different amino aci ds, including potential copper-liganding residues, were substituted fo r H333 and H334 of the E. coli oxidase. All of the mutations resulted in inactive, but assembled, oxidase with both of the heme components p resent. However, cryogenic Fourier transform infrared (FTIR) spectrosc opy of the CO adducts revealed that dramatic changes occur at the binu clear center as a result of each mutation and that Cu(B) appears to be absent. To varying degrees, the protein environment monitored by CO b ound to heme o in the mutants is perturbed by the H333 and H334 mutant s. Electron paramagnetic resonance (EPR) spectroscopy of oxidation-red uction potentiometric titrations of the mutant in which H333 is replac ed by Leu demonstrate that the high-spin heme is no longer magneticall y coupled to Cu(B). The results confirm that both H333 and H334 are es sential for the assembly and/or maintenance of Cu(B) in the oxidase.