Research Article: A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

Date Published: January 22, 2014

Publisher: Public Library of Science

Author(s): Sébastien Le Laz, Arlette Kpebe, Marielle Bauzan, Sabrina Lignon, Marc Rousset, Myriam Brugna, Alessandro Giuffrè.


The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa3-type oxidase in S. oneidensis MR-1 are discussed.

Partial Text

In the oxygen respiratory systems, electrons of low-redox potential electron donors are transferred through a series of membrane-bound proteins or complexes and finally, the reduction of molecular oxygen to water is catalyzed by enzymes called terminal oxidases. These oxygen reductases are complicated integral membrane multi-subunit complexes grouped into two major superfamilies. Most of them belong to the well-characterized heme-copper oxidases (HCO) superfamily [1]. HCO have been named cytochrome c oxidases or quinol oxidases, depending on the nature of their electron donor and are able to pump protons across membrane. Additionally, based on biochemical and structural differences in their catalytic subunits and on phylogenetic analysis, a classification of HCO into three families was proposed [2]: i) type A (mitochondrial-like oxidases or aa3-type), ii) type B (ba3-type oxidases) and iii) type C (cbb3-type oxidases) only detected in bacteria [3]. Cytochrome bd-type oxidases, phylogenetically unrelated to the HCO [4], represent the second major oxidases superfamily [5]. Widely distributed among prokaryotes, bd-type oxygen reductases function as quinol-oxidases and are bioenergetically less efficient than HCO since they generate a proton motive force by transmembrane charge separation rather than by pumping protons [6]–[8]. In addition to its role in cell bioenergetics, many studies suggest that cytochrome bd oxidase may be implicated in other important physiological functions. In particular, the enzyme seems to be involved in the bacterial response to a wide variety of stress conditions such as alkalinization of the medium, high temperatures, hydrogen peroxide and nitrosative (NO) stresses [5], [9]–[11]. Cytochrome bd oxidase could also play a determinant role in bacterial pathogenicity by protecting bacteria against the NO-mediated host immune response [12].

In this report, we investigated the aerobic respiratory pathway in S. oneidensis MR-1 to determine the physiological role of the three terminal oxidases in low and in high-O2 environments. Our results suggest that the high-affinity bd-type oxidase is weakly expressed in aerobic conditions and significantly induced under microaerobic conditions which is consistent with previous studies indicating that bd oxidases are typically used by bacteria for aerobic respiration under O2-limited conditions [5], [64], [65]. Taken together, our data also reveal that the cbb3-type oxidase is the most important terminal oxidase under aerobic conditions and has a significant role under microaerobic conditions whereas the low affinity A-type cytochrome c oxidase Cox was not detected in the tested conditions even under aerobic conditions. In addition, characterization of the terminal oxidase deletion mutants confirms that the cbb3-type or the bd-type oxidase is required for aerobic growth and that Cox is unexpectedly not involved in aerobic respiration in these experimental conditions.