Characterization of two isoforms of cbb3 cytochrome oxidase from Pseudomonas stutzeri ZoBell

The cbb₃ cytochrome oxidase is a member of the heme-copper oxidase (HCO) superfamily, which couples the reduction of dioxygen to transmembrane proton pumping. This reaction generates an electrochemical proton potential, which is used for ATP synthesis. The cbb₃ oxidases are characterized by a higher catalytic activity at low oxygen concentrations. This feature is considered to be essential for the pathogenicity of many bacterial species. They are also an evolutionary close relative to the bacterial nitric oxide reductase (NOR), which catalyzes the reduction of NO to N₂O.

Pseudomonas stutzeri ZoBell contains two independent operons encoding cbb₃-isoforms, Cbb₃-1 and Cbb₃-2, respectively. The two isoforms differ from each other in their respiratory and regulatory functions under different oxygen tensions. The x-ray structure of Cbb₃-1 at 3.2 angstrom resolution showed an electron transfer pathway that differs from the mitochondria-like oxidase (subfamily A) and ba₃-like oxidase (subfamily B). Moreover, structural differences around heme b and heme b₃-CuB binuclear center and the existence of one proton pathway indicate a different redox-driven proton pumping mechanism. To further investigate structural and functional details of both isoforms, each of the two operons has been deleted by homologous recombination. The absence of each of the operons was confirmed by PCR and subsequent sequence analysis. These deletion strains are used for the expression of recombinant tagged Cbb₃-isoforms. Both homologously produced isoforms were isolated and identified by in-gel detection using TMBZ/H₂O₂ assay and electrospray ionization mass spectrometry (ESI-MS). Compared with Cbb₃-1, Cbb₃-2 showed a low level of expression under both aerobic and micro-aerobic growth conditions. Further studies are in progress focusing on functional and structural analysis of both Cbb₃-isoforms by combining site-directed mutagenesis and x-ray crystallography.