Proton translocation by cytochrome c oxidase can take place without the conserved glutamic acid in subunit I

A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O-2 reduction and proton- pumping mechanisms of these respiratory enzymes. Its mutation to residues o ther than aspartic acid dramatically inhibits activity, and proton transloc ation is lost. However, this glutamic acid is replaced by a nonacidic resid ue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase f rom Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of t he wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fol d and proton translocation is restored. Molecular dynamics simulations sugg est that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisi ngly, the glutamic acid is thus not indispensable, which puts important con straints on the catalytic mechanism of these enzymes.