Abstract
Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme.
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Acknowledgments
This work was supported by grant R15GM099014 from the NIH. We thank the following students for technical assistance: April Wiseman, Caitlyn Le, Sarah Bruyere, Kayla Wilson, Emily Helm, Shivani Sharma, and Sarah Simmons. We also thank Alan J. Wolfe (Loyola University, Chicago IL USA) for providing plasmids.
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Zhu, S., Canales, A., Bedair, M. et al. Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L. J Bioenerg Biomembr 48, 325–333 (2016). https://doi.org/10.1007/s10863-016-9655-y
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DOI: https://doi.org/10.1007/s10863-016-9655-y