Virve Rauhamäki

Cytochrome cbb ₃ belongs to the superfamily of respiratory heme-copper oxidases that couple the reduction of molecular oxygen to proton translocation across the bacterial or mitochondrial membrane. The cbb ₃ -type enzymes are found only in bacteria, and are both structurally and functionally the most distant from their mitochondrial counterparts. The mechanistic H ⁺ /e ⁻ stoichiometry of proton translocation in these cbb ₃ -type cytochrome c oxidases has remained controversial. A stoichiometric efficiency of only one-half that of the mitochondrial aa ₃ -type enzyme was recently proposed to be related to adaptation of the organism to microaerobic environments. Here, proton translocation by the Rhodobacter sphaeroides enzyme was studied using purified cytochrome cbb ₃ reconstituted into liposomes. An H ⁺ /e ⁻ stoichiometry of proton translocation close to unity was observed using the oxygen pulse method, but solely in conditions in which the vast majority of the enzyme was fully reduced in the anaerobic state before the O ₂ pulse. These data were compared with results using whole cells or spheroplasts, and the discrepancies in the literature data were discussed. Our results suggest that a proton-pumping efficiency of 1 H ⁺ /e ⁻ may be achieved using the single-proton uptake pathway identified in the structure of cytochrome cbb ₃ . The mechanism of proton pumping thus differs from that of the aa ₃ -type oxidases of mitochondria and bacteria.

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.