Glutamate-89 in subunit II of cytochrome bo(3) from Escherichia coli is required for the function of the heme-copper oxidase

Recent electrostatics calculations on the cytochrome c oxidase from Paracoc cus denitrificans revealed an unexpected coupling between the redox stare o f the heme-copper center and the state of protonation of a glutamic acid (E 78(II)) that is 25 Angstrom away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper o xidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The f unctional importance of several residues in this cluster (E89(II), W93(II), T94(II), and P96(II)) was examined by site-directed mutagenesis of the cor responding region of the cytochrome bo(3) quinol oxidase from Escherichia c oli (where E89(II) is the equivalent of residue E78(II) of the P. denitrifi cans oxidase), Substitution of E89(II) with either alanine or glutamine res ulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase, The quinol oxidase activity of the W93V mutant is also re duced to about 30% of that of the wild-type oxidase, Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the hem e-copper center, The data suggest that E89(II) (E. coli numbering) is criti cal for the function of the heme copper oxidases, The proximity to K362 sug gests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mut ant, Thus, it appears that the inhibition resulting from the E89(II) mutati on is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.