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 other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the 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-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.
Backgren
C
C
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, University of Helsinki, P.O. Box 8, 00014 Helsinki, Finland.
Hummer
G
G
Wikström
M
M
Puustinen
A
A
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry*; D006860:Hydrogen Bonding; D017136:Ion Transport; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010231:Paracoccus denitrificans / Q000201:enzymology; D011522:Protons
D011522:Protons; D018698:Glutamic Acid; D003576:Electron Transport Complex IV