Biochemical consequences of two clinically relevant ND-gene mutations in Escherichia coli respiratory complex I

Abstract

NADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in energy metabolism by coupling electron transfer from NADH to quinone with proton translocation across the membrane. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction that manifest in a wide variety of neurodegenerative diseases. Seven subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations discovered in mitochondria from patients’ tissues. However, whether or how these genetic aberrations affect complex I at a molecular level is unknown. Here, we used Escherichia coli as a model system to biochemically characterize two mutations that were found in mtDNA of patients. The V253AMT-ND5 mutation completely disturbed the assembly of complex I, while the mutation D199GMT-ND1 led to the assembly of a stable complex capable to catalyze redox-driven proton translocation. However, the latter mutation perturbs quinone reduction leading to a diminished activity. D199MT-ND1 is part of a cluster of charged amino acid residues that are suggested to be important for efficient coupling of quinone reduction and proton translocation. A mechanism considering the role of D199MT-ND1 for energy conservation in complex I is discussed.

The clinically relevant triple mutation in the mtND1 gene inactivates Escherichia coli complex I

“…To determine an effect of the mutation on complex I, strains were grown aerobically in minimal medium with 25 m m acetate as sole carbon source [25] at 37 °C while agitating at 180 rpm. Expression of the nuo operon was induced by an addition of 0.02% (w/v) l ‐arabinose.…”

“…Expression of the nuo operon was induced by an addition of 0.02% (w/v) l ‐arabinose. For protein preparation, cells were grown in a rich autoinduction medium containing 34 µg·mL −1 chloramphenicol as described [25]. Cells were harvested by centrifugation, suspended in a fivefold volume buffer A (50 m m MES/NaOH, 50 m m NaCl, pH 6.0) containing 0.1 m m PMSF and a few grains of DNAseI and disrupted by three passages through an HPL‐6 (Maximator, 1000‐1500 bar) [25].…”

“…For protein preparation, cells were grown in a rich autoinduction medium containing 34 µg·mL −1 chloramphenicol as described [25]. Cells were harvested by centrifugation, suspended in a fivefold volume buffer A (50 m m MES/NaOH, 50 m m NaCl, pH 6.0) containing 0.1 m m PMSF and a few grains of DNAseI and disrupted by three passages through an HPL‐6 (Maximator, 1000‐1500 bar) [25]. Cytoplasmic membranes were obtained by differential centrifugation [25] and suspended in an equal volume (1 : 1, w/v) of buffer A* (buffer A with 5 m m MgCl 2 ) containing 0.1 m m PMSF.…”

“…This problem can be circumvented by using bacterial model systems such as Escherichia coli that have been developed to identify the effect of specific mutations on biochemical properties of the mutated complex [21–25]. Without question, eukaryotic model systems such as Yarrowia lipolytica are evolutionarily much closer related to the human enzyme, but the bacterial systems have the invaluable advantage that mutations, even multiple mutations, found in mtDNA can be integrated into their genome without technical problems.…”

“…They are encoded by the nuo ‐genes and add up to a molecular mass of approximately 530 kDa [30]. We work with a chromosomal nuo null strain, in which all nuo ‐genes are expressed from one plasmid, thus facilitating their mutation in our experimental approach [25]. An efficient and straightforward purification strategy was developed that allows a detailed analysis of the enzyme variants with respect to their composition, stability and catalytic activity [25].…”