Unraveling the complexities of glycoconjugate vaccines
November 8, 2016

No longer unknown – novel cytochrome c oxidase subunit revealed

A few years ago, researchers solved the structure of the cbb3-1 cytochrome c oxidase from Pseudomonas stutzeri. They were surprised to detect the presence of a fourth protein subunit, consisting of a single transmembrane helix, for which no proteomic or genomic information was available. In a recently published article, they have now described the identification and characterization of that previously unknown subunit.

Cytochrome c oxidases (CcOs) belong to the superfamily of heme-copper oxidases, which are responsible for a key step in aerobic respiration: coupling O2 reduction and transmembrane proton pumping. In Pseudomonas stutzeri, the cbb3-type CcOs are encoded by two cbb3 operons organized in a tandem repeat. Because it is essential to many different pathogenic bacteria, cbb3-CcO has emerged as a promising drug target in the search for new antibiotics. Understanding the enzyme’s structure and function should assist the discovery and development of compounds to target it.

Kohlsteadt and colleagues purified the full CcO complex from Pseudomonas stutzeri, a close relative of the human pathogen Pseudomonas aeruginosa, and obtained a fraction enriched with a 3,986 Da polypeptide. De novo sequencing of this polypeptide on a Bruker ultrafleXtreme MALDI-TOF mass spectrometer followed by an MS_BLAST search identified it as the hypothetical PstZobell_05036 protein.

After matching the amino acid sequence with the electron density of their previously unassigned transmembrane helix and concluding that they had identified the unknown subunit, they renamed the protein “CcoM”. Fitting CcoM into their completed crystal structure indicated a strong interaction with the catalytic subunit CcoN in a ladder-like conformation.

The ccoM gene was found to be located far away from the two cbb3 operons containing the structural genes for the enzyme complex. The predicted promoter contains an arginine nitrate regulator (ANR) binding motif, suggesting upregulation in low oxygen concentrations. A BLAST search indicated that the CcoM protein is almost exclusively present in Pseudomonas species.

In an attempt to assess the physiological function of CcoM in the cbb3-CcO complex, the authors created a DCcoM knockout variant of P. stutzeri ZoBell. They confirmed the absence of CcoM in the complex by MALDI-TOF MS. Under microaerobic conditions, the deletion strain grew similarly to the wild-type strain. However, under anaerobic denitrifying conditions, the deletion strain displayed a significant increase in the lag phase compared to the wild-type strain, indicating that CcoM plays a role in anaerobic respiration. Further studies are needed to determine the detailed role of CcoM in this process.

The biochemical and biophysical properties of the purified oxidases were compared using various methods, including UV-vis spectroscopy, differential scanning calorimetry, and oxygen reductase activity measurements. No differences were observed with respect to heme incorporation. They also displayed similar oxygen consumption rates and catalytic activities. The only significant difference observed between the two oxidases was that the deletion mutant displayed a much lower melting temperature compared to the wild type. Based on these results, the researchers hypothesize that CcoM may play a role in the assembly or stability of the complex.

At several points in this project, MALDI-TOF MS generated detailed data to elucidate the sequence and function of the CcoM protein. Mass spectrometry is an essential tool in the identification and detailed characterization of proteins.


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