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- Abou-Hamdan, Abbas, et al.
(author)
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Functional design of bacterial superoxide : quinone oxidoreductase
- 2022
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In: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1863:7
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Journal article (peer-reviewed)abstract
- The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions. It can perform a range of cellular functions, including bacterial warfare and iron uptake, signalling and host immune response in eukaryotes. However, it also serves as precursor for more deleterious species such as the hydroxyl anion or peroxynitrite and defense mechanisms to neutralize superoxide are important for cellular health. In addition to the soluble proteins superoxide dismutase and superoxide reductase, recently the membrane embedded diheme cytochrome b561 (CybB) from E. coli has been proposed to act as a superoxide:quinone oxidoreductase. Here, we confirm superoxide and cellular ubiquinones or menaquinones as natural substrates and show that quinone binding to the enzyme accelerates the reaction with superoxide. The reactivity of the substrates is in accordance with the here determined midpoint potentials of the two b hemes (+48 and -23 mV / NHE). Our data suggest that the enzyme can work near the diffusion limit in the forward direction and can also catalyse the reverse reaction efficiently under physiological conditions. The data is discussed in the context of described cytochrome b561 proteins and potential physiological roles of CybB.
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| 2. |
- Riepl, Daniel, et al.
(author)
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Molecular principles of proton-coupled quinone reduction in the membrane-bound superoxide oxidase
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Other publication (other academic/artistic)abstract
- Reactive oxygen species (ROS) are physiologically harmful radicals generated as biproducts of aerobic respiration. To detoxify ROS, most cells employ superoxide scavenging enzymes that disproportionate superoxide (O2•-) to oxygen (O2) and hydrogen peroxide (H2O2). However, the recently discovered membrane-bound superoxide oxidase (SOO) (Nature Chemical Biol 2018) is a minimal 4-helical bundle protein that catalyzes the direct oxidation of O2•- to O2 and drives quinone reduction by mechanistic principles that remain unknown. Here we combine multiscale hybrid quantum/classical (QM/MM) free energy calculations and microsecond molecular dynamics simulations with functional assays and site-directed mutagenesis experiments to probe the energetics and dynamics underlying the charge transfer reactions of the superoxide (O2•-)-driven quinone reduction. We identify a cluster of charged residues at the periplasmic side of the membrane that functions as a O2•- collecting antenna, which shuttles the electrons to the active site for quinone reduction. Based on multidimensional QM/MM string simulations, we suggest that a proton-coupled electron transfer (PCET) reaction from the active site heme b and nearby histidine residues (H87, H158) catalyzes the quinol (QH2) formation, followed by proton uptake from the cytoplasmic side of the membrane. The functional relevance of the identified residues is supported by site-directed mutagenesis and activity assays, with mutations leading to inhibition of the O2•--driven quinone reduction activity. We suggest that the coupled electron and proton transfer reactions build up a proton motive force that support the bacterial energy transduction machinery, with the PCET reactions providing unique design principles of a minimal oxidoreductase.
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