title: Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation.
abstract: Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376.
authors.forename: Shaotong
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376. Electronic address: svik@smu.edu.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Vik
mesh_terms: D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000737:chemistry / Q000201:enzymology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C000589385:NuoN protein, E coli; D011522:Protons; D009243:NAD; D003300:Copper; D009245:NADH Dehydrogenase; C479743:NuoL protein, E coli; C476247:NuoM protein, E coli; D042967:Electron Transport Complex I; D003545:Cysteine
keywords: complex I; cysteine-mediated cross-linking; membrane energetics; membrane protein; methanethiosulfonate; protein cross-linking; proton transport