Abstract
The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba 3-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix “subunit IIa”, which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA2) of 59000 Da, subunit II (encoded by coxB2) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba 3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.
Highlights
In aerobic respiration of prokaryotic and eukaryotic organisms, the reduction of molecular oxygen to water is catalyzed by terminal oxidases which are integral membrane multi-subunit enzymatic complexes belonging to the heme-copper oxidases superfamily [1]
A BLAST search using the corrected protein sequence indicates high similarity with proteins, annotated cytochrome c oxidase subunit II, from bacteria belonging to Aquificales order (Hydrogenivirga sp., Thermocrinis albus, and Hydrogenobacter thermophilus, 74%, 61% and 56% identity respectively) (Supplementary Figure S1)
Some similarities were found with other bacterial and archaeal proteins, including the characterized ba3 cytochrome c oxidases from T. thermophilus (31% identity) [7], Rhodothermus (R.) marinus (36% identity) [9] and Geobacillus (G.) stearothermophilus (36% identity) [10,11]
Summary
In aerobic respiration of prokaryotic and eukaryotic organisms, the reduction of molecular oxygen to water is catalyzed by terminal oxidases which are integral membrane multi-subunit enzymatic complexes belonging to the heme-copper oxidases superfamily [1]. These respiratory enzymes, which pump protons across membrane, have been named cytochrome c oxidases or quinol oxidases, depending on the electron donor that they oxidize. Subunit II of oxidases A and B is made of a transmembrane domain and a peripheral one in which the copper CuA centre is located This subunit binds the electron donor (reduced cytochrome c, and other metalloproteins) and transfers electrons to subunit I [2]
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