title: Identification of a histidine-tyrosine cross-link in the active site of the cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides.
abstract: The heme-copper oxidases constitute a superfamily of terminal dioxygen-reducing enzymes located in the inner mitochondrial or in the bacterial cell membrane. The presence of a mechanistically important covalent bond between a histidine ligand of the copper ion (Cu(B)) in the active site and a generally conserved tyrosine residue nearby has been shown to exist in the canonical cytochrome c oxidases. However, according to sequence alignment studies, this critical tyrosine is missing from the subfamily of cbb(3)-type oxidases found in certain bacteria. Recently, homology modeling has suggested that a tyrosine residue located in a different helix might fulfill this role in these enzymes. Here, we show directly by methods of protein chemistry and mass spectrometry that there is indeed a covalent link between this tyrosine and the copper-ligating histidine. The identity of the cross-linked tyrosine was determined by showing that the cross-link is not formed when this residue is replaced by phenylalanine, even though structural integrity is maintained. These results suggest a universal functional importance of the histidine-tyrosine cross-link in the mechanism of O(2) reduction by all heme-copper oxidases.
authors.affiliation: Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P.O. Box 65, Viikinkaari 1, 00014 Helsinki, Finland. virve.rauhamaki@helsinki.fi
authors.forename: Virve
authors.initials: V
authors.lastname: Rauhamäki
authors.forename: Marc
authors.initials: M
authors.lastname: Baumann
authors.forename: Rabah
authors.initials: R
authors.lastname: Soliymani
authors.forename: Anne
authors.initials: A
authors.lastname: Puustinen
authors.forename: Mårten
authors.initials: M
authors.lastname: Wikström
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D006639:Histidine; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV