Biology subjects

Högbom, M.

Publications and source records attributed to Högbom, M..

4 recordsLinked to original sources

bioRxiv2021

Functional design of bacterial superoxide:quinone oxidoreductase

The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions and 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 therefore, cellular defense mechanisms for superoxide neutralization have evolved. 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 potential 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 context of described cytochrome b561 proteins and potential physiological roles of CybB.

biochemistry↗

bioRxiv2021

The respiratory supercomplex from C. glutamicum

Corynebacterium glutamicum is a preferentially aerobic Gram-positive bacterium belonging to the Actinobacteria phylum, which also includes the pathogen Mycobacterium tuberculosis. In the respiratory chain of these bacteria, complexes III (CIII) and IV (CIV) form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. Electron transfer within the CIII2CIV2 supercomplex is linked to transmembrane proton translocation, which maintains an electrochemical proton gradient that drives ATP synthesis and transport processes. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 [A] resolution. The structure shows a central CIII2 dimer flanked by a CIV on each side. One menaquinone is bound in each of the QN and QP sites in each CIII, near the cytoplasmic and periplasmic sides, respectively. In addition, we identified a menaquinone positioned ~14 [A] from heme bL on the periplasmic side. A di-heme cyt. cc subunit provides an electronic connection between each CIII monomer and the adjacent CIV. In CIII2, the Rieske iron-sulfur (FeS) proteins are positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a novel path that conducts protons into CIV.

biochemistry↗

bioRxiv2021

Redetermination of the first unknown protein MicroED structure by high resolution X-ray diffraction

Microcrystal electron diffraction (MicroED) has the potential to considerably impact the field of structural biology. Indeed, the method can solve atomic structures of a wide range of molecules, beyond the reach of single particle cryo-electron microscopy, exploiting crystals too small for X-ray diffraction (XRD) even using X-ray free-electron lasers. However, until the first unknown protein structure - a R2-like ligand-binding oxidase from Sulfolobus acidocaldarius (SaR2lox) - was recently solved at 3.0 [A] resolution, MicroED had only been used to study known protein structures previously obtained by XRD. Here, after adapting sample preparation protocols, the structure of the SaR2lox protein originally solved by MicroED was redetermined by XRD at 2.1 [A] resolution. In light of the higher resolution XRD data and taking into account experimental differences of the methods, the quality of the MicroED structure is examined. The analysis demonstrates that MicroED provided an overall accurate model, revealing biologically relevant information specific to SaR2lox, such as the absence of an ether cross-link, but did not allow to detect the presence of a ligand visible by XRD in the protein binding pocket. Furthermore, strengths and weaknesses of MicroED compared to XRD are discussed in the perspective of this real-life protein example. The study provides fundaments to help MicroED become a method of choice for solving novel protein structures. SynopsisThe first unknown protein structure solved by microcrystal electron diffraction (MicroED) was recently published. The redetermination by X-ray diffraction of this protein structure provides new insights into the strengths and weaknesses of the promising MicroED method.

biochemistry↗

bioRxiv2018

Metal-independent ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

Ribonucleotide reductase (RNR) catalyzes the only known de-novo pathway for production of all four deoxyribonucleotides required for DNA synthesis. In aerobic RNRs, a di-nuclear metal site is viewed as an absolute requirement for generating and stabilizing an essential catalytic radical. Here we describe a new group of RNRs found in Mollicutes, including Mycoplasma pathogens, that possesses a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable DOPA radical species that directly supports ribonucleotide reduction in vitro and in vivo.\n\nThe cofactor synthesis and radical generation processes are fundamentally different from established RNRs and require the flavoprotein NrdI. Several of the pathogens encoding this RNR variant are involved in diseases of the urinary tract and genitalia. Conceivably, this remarkable RNR variant provides an advantage under metal starvation induced by the immune system. We propose that the new RNR subclass is denoted class Ie.

biochemistry↗