Variant G129D of NuoEF from Aquifex aeolicus bound to NAD+

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH‐OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X‐ray crystallographic analysis, we report the structure of NADH‐OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH‐OH. Furthermore, the structure sheds light on the specificity of NADH‐OH towards the unique Rossmann‐fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH‐OH acts as a lead‐structure for the synthesis of a novel class of ROS suppressors.

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Structural Basis for Inhibition of ROS‐Producing Respiratory Complex I by NADH‐OH

Vranas

Wohlwend

Qiu

et al. 2021

Angew Chem Int Ed

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Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry

Sandberg

Bunkenborg²,

Thyssen³

et al. 2021

Amino Acids

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.

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Structure of respiratory complex I – An emerging blueprint for the mechanism

Kampjut

Sazanov

2022

Current Opinion in Structural Biology

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Variant G129D of NuoEF from Aquifex aeolicus bound to NAD+

Cited by 11 publications

References 44 publications

Structural Basis for Inhibition of ROS‐Producing Respiratory Complex I by NADH‐OH

Structural Basis for Inhibition of ROS‐Producing Respiratory Complex I by NADH‐OH

Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry

Structure of respiratory complex I – An emerging blueprint for the mechanism

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