|PhD student U173/U178
Characterisation of the Escherichia coli cytochrome b561 superoxide:ubiquinone oxidoreductase
All aerobic species have to deal with Reactive Oxygen Species (ROS) that are derived by incomplete reduction of molecular oxygen (O2). ROS are known to cause damage to important cellular components like proteins, lipids and nucleic acids and are therefore involved in development of different diseases and aging. However, they also play an important role in cell signalling processes –. In aerobic respiration O2 therefore plays a dual role. While it acts as terminal electron acceptor of the respiratory chain, it also can undergo a one-electron reduction by electron leakage from one of the respiratory enzymes forming a superoxide anion (O2·–). O2·– is not as reactive as the name implies, but it is able to reduce cytochrome c, oxidize and destabilize iron-sulfur clusters and acts as a precursor for other more dangerous ROS like hydroperoxyl radicals (HO2·), hydrogen peroxide (H2O2) and hydroxyl radicals (OH·). In combination with nitric oxide (NO·) it can react to peroxynitrite (ONOO–) which is a strong oxidant ,.
Nature has evolved different mechanisms to keep the cellular ROS concentrations low. While higher organisms are using different low-molecular weight antioxidants (e.g. ascorbate, glutathione, tocopherol and carotene) in combination with highly effective enzymes, bacteria often only use enzymes. An effective way to keep the ROS concentration low is to remove the precursor O2·–. In all living systems this process is fulfilled by superoxide dismutase (SOD) that convert O2·– to O2 and H2O2 which is finally detoxified by catalase ,. Recently, an enzyme from E. coli which was first described in 1984, was structurally and biochemically characterised ,. The data indicate, that cytochrome b561 (CybB) acts as a superoxide oxidase (SOO), reflecting a third and so far undescribed type of superoxide reacting enzyme.
Fig. 1: 3D structure of CybB. CybB is an integral membrane protein composed of four transmembrane helices containing two b-type hemes as prosthetic groups. In the proposed mechanism, O2·– is oxidised to O2 and the released electron is transferred to ubiquinone. From this mechanism CybB can be classified as superoxide:ubiquinone oxidoreductase, a previously undescribed enzyme class. It is able to keep the O2·– concentration and therefore the ROS concentration low and feed the electrons back into the respiratory chain 
The aim of this project is the mechanistic and biochemical characterisation of CybB in detergent solubilized form but also in cell membrane mimicking systems like liposomes and nanodiscs. The exact mechanism of the electron transfer as well as the mode of superoxide binding is unknown and its determination therefore the priority of our work.
- Hancock, J. T., Desikan, R. & Neill, S. J. Role of reactive oxygen species in cell signalling pathways. Biochem. Soc. Trans. 29, 345–350 (2001)
- Dröge, W. Free Radicals in the Physiological Control of Cell Function. Physiol. Rev. 82, 47–95 (2002)
- Sabharwal, S. S. & Schumacker, P. T. Mitochondrial ROS in cancer: Initiators, amplifiers or an Achilles’ heel? Nat. Rev. Cancer 14, 709–721 (2014)
- Riera, C. E., Merkwirth, C., De Magalhaes Filho, C. D. & Dillin, A. Signaling Networks Determining Life Span. Annu. Rev. Biochem. 85, 35–64 (2016)
- Barnham, K. J., Masters, C. L. & Bush, A. I. Neurodegenerative diseases and oxidative stress. Biomed. Pharmacother. 58, 39–46 (2004)
- Turrens, J. F. Mitochondrial formation of reactive oxygen species. J. Physiol. 552, 335–344 (2003)
- Beckman, J. S. & Koppenol, W. H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. Physiol. 271, C1424–C1437 (2017)
- SIES, H. Strategies of antioxidant defense. Eur. J. Biochem. 215, 213–219 (1993)
- Sheng, Y. et al. Superoxide dismutases and superoxide reductases. Chem. Rev. 114, 3854–3918 (2014)
- Murakami, H., Kita, K. & Anraku, Y. Cloning of cybB, the gene for cytochrome b561 of Escherichia coli K12. MGG Mol. Gen. Genet. 198, 1–6 (1984)
- Lundgren, C. A. K. et al. Scavenging of superoxide by a membrane-bound superoxide oxidase. Nat. Chem. Biol. 14, 788–793 (2018)
Abou-Hamdan A., Mahler R., Grossenbacher P., Biner O., Sjöstrand D., Lochner M., Högbom M., von Ballmoos C. (2022) Functional design of bacterial superoxide:quinone oxidoreductase. BBA. 2022 Jun, doi: 10.1016/j.bbabio.2022.148583. Epub ahead of print. PMID: 35671795.
Previous work in the group:
2018 – 2019 Lab assistant
2016 – 2018 Master student:
Project: Towards a synthetic respiratory chain: “Functional co-reconstitution of bc1 complex and cytochrome c oxidase from Rhodobacter sphaeroides”