title: 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.
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.
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX, 75275-0376, USA.|Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
authors.forename: Shaotong
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX, 75275-0376, USA.|Department of Biochemistry, University of Wisconsin, Madison, WI, 53706, USA.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Canales
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX, 75275-0376, USA.|University of Texas Southwestern Medical School, Dallas, TX, 75390, USA.
authors.forename: Mai
authors.initials: M
authors.lastname: Bedair
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX, 75275-0376, USA. svik@smu.edu.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Vik
mesh_terms: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D021122:Protein Subunits; D042967:Electron Transport Complex I; D003545:Cysteine
keywords: Complex I; Cross-linking; Methanethiosulfonate; Subunit interactions; nuoK