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Pseudomonas stutzeri; Cytochrome c oxidase; Cbb3; Subunit CcoQ; Knockout mutant; Assembly
Abstract:
The Cbb3-type cytochrome c oxidases (Cbb3-CcOs), the second most abundant CcOs, catalyze the reduction of molecular oxygen to water, even at micromolar oxygen concentrations. In Pseudomonas stutzeri ZoBell, two tandemly organized cbb3-operons encode the isoforms Cbb3-1 and Cbb3-2 both possessing subunits CcoN, CcoO and CcoP. However, only the cbb3-2 operon contains an additional ccoQ gene. CcoQ consists of 62 amino acids and is predicted to possess one transmembrane spanning helix. The physiological role of CcoQ was investigated based on a CcoQ-deletion mutant and wild-type Cbb3-2 crystals not containing subunit CcoQ. Cbb3-2 isolated from the deletion mutant is inactive and appears as a dispersed band on blue native-PAGE gels. Surprisingly, in the absence of ccoQ, Cbb3-1 also shows a strongly reduced activity. Our data suggest that CcoQ primarily functions as an assembly factor for Cbb3-2 but is also required for correct assembly of Cbb3-1. In contrast, once correctly assembled, Cbb3-1 and Cbb3-2 possess a full enzymatic activity even in the absence of CcoQ.