| 1. |
- 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. |
- Lundgren, Camilla A. K, et al.
(author)
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Scavenging of superoxide by a membrane-bound superoxide oxidase
- 2018
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In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 14, s. 788-793
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Journal article (peer-reviewed)abstract
- Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).
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| 3. |
- von Ballmoos, Christoph, et al.
(author)
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Mimicking respiratory phosphorylation using purified enzymes
- 2016
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In: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1857:4, s. 321-331
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Journal article (peer-reviewed)abstract
- The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP x s(-1) x enzyme(-1). We have also used the novel system to study the phenomenon of mild uncoupling as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.
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