Comparative Genomics and Site-Directed Mutagenesis Support the Existence of Only One Input Channel for Protons in the C-Family (cbb₃ Oxidase) of Heme−Copper Oxygen Reductases

Abstract

Oxygen reductase members of the heme-copper superfamily are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling the reduction of molecular oxygen to water to the translocation of protons across the plasma membrane. The protons required for catalysis and pumping in the oxygen reductases are derived from the cytoplasmic side of the membrane, transferred via proton-conducting channels comprised of hydrogen bond chains containing internal water molecules along with polar amino acid side chains. Recent analyses identified eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and H-families of oxygen reductases. Two proton input channels, the K-channel and the D-channel, are well established in the A-family of oxygen reductases (exemplified by the mitochondrial cytochrome c oxidases and by the respiratory oxidases from Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can be identified by the pattern of conserved polar amino acid residues within the protein. The C-family (cbb3 oxidases) is the second most abundant oxygen reductase family after the A-family, making up more than 20% of the sequences of the heme-copper superfamily. In this work, sequence analyses and structural modeling have been used to identify likely proton channels in the C-family. The pattern of conserved polar residues supports the presence of only one proton input channel, which is spatially analogous to the K-channel in the A-family. There is no pattern of conserved residues that could form a D-channel analogue or an alternative proton channel. The functional importance of the residues proposed to be part of the K-channel was tested by site-directed mutagenesis using the cbb3 oxidases from R. sphaeroides and Vibrio cholerae. Several of the residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D, and Y321F. The data strongly suggest that in the C-family only one channel functions for the delivery of both catalytic and pumped protons. In addition, it is also proposed that a pair of acidic residues, which are totally conserved among the C-family, may be part of a proton-conducting exit channel for pumped protons. The residues homologous to these acidic amino acids are highly conserved in the cNOR family of nitric oxide reductases and have previously been implicated as part of a proton-conducting channel delivering protons from the periplasmic side of the membrane to the enzyme active site in the cNOR family. It is possible that the C-family contains a homologous proton-conducting channel that delivers pumped protons in the opposite direction, from the active site to the periplasm.

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

“…It has become increasingly clear, however, that within the large superfamily of heme-copper oxidases some bacterial forms may have solved the proton pumping problem in different ways (4,5), and even those most closely related to eukaryotic oxidases (the aa 3 type) may have different properties with respect to proton pathways (6). This apparent lack of conservation of proton pumping machinery, along with potential involvement of conformational change, makes it a challenging task to identify operative proton paths.…”

Entrance of the proton pathway in cbb 3 -type heme-copper oxidases

“…The HCuO superfamily has previously been divided into subfamilies denoted A, B, and C type (2,3). The A family comprises the enzyme found in mitochondria and other well-studied aa 3 -type (the names aa 3 or cbb 3 refer to the types of hemes found in the complexes) HCuOs from, e.g., Rhodobacter (R.) sphaeroides (4).…”

“…c. Very recently, the three-dimensional structure of the cbb 3 oxidase from P. stutzeri was solved at 3.2-Å resolution (16). The residues constituting the D pathway are missing in cbb 3 s and cbb 3 s presumably have only one pathway (3,16), spatially analogous to the K pathway in the A type, for delivering protons from the cytosol to the catalytic site. This feature is shared between the C-and B-type oxidases (17) and has been proposed to be linked to the lower pumping stoichiometry observed in these HCuOs (n ¼ 2 in 2) (1, 18).…”

“…1A shows the overall structure of the P. stutzeri cbb 3 (16), and Fig. 1B shows a close-up of the suggested proton pathway (3,16). The pathway is composed of residues entirely from the CcoN subunit, except for the suggested pathway entrance, which is at the Glu-49 in CcoP (corresponding to E-25 in the R. sphaeroides CcoP sequence).…”

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

“…8 For a number of bacterial strains, two isoenzymes of cbb 3 -CcO were identified within the genome, 9 and in Pseudomonas aeruginosa it could be shown that its two cbb 3 -CcO isoforms are differentially regulated and possess different respiratory functions. 10 Derived from comparative genomic studies [11][12][13] and the recent X-ray structure, 14 cbb 3 -CcOs exhibit substantial structural differences to the well investigated mitochondrial type cytochrome c oxidase concerning the electron supply system, the proton conducting pathway including two cavities and the coupling region involving the heme b and b 3 propionates as well as two loops linked via a Ca 21 ion. 14 However, they show a close relationship to the nitric oxide reductases (NORs), 15 another distant member of the HCO superfamily, 16 and, interestingly, also display NOR activity in vitro.…”