title: Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking.
abstract: Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M.
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376, USA.
authors.forename: Ablat
authors.initials: A
authors.lastname: Tursun
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376, USA.
authors.forename: Shaotong
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376, USA. Electronic address: svik@smu.edu.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Vik
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine; D030562:Databases, Protein; D042967:Electron Transport Complex I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine; D008301:Maleimides / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D019897:Periplasm / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D000072756:Protein Conformation, alpha-Helical; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011522:Protons; D013329:Structure-Activity Relationship; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008301:Maleimides; D021122:Protein Subunits; D011522:Protons; D025801:Ubiquitin; D009243:NAD; D011092:Polyethylene Glycols; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I; D008239:Lysine; D003545:Cysteine
keywords: Complex I; Cross-linking; Disulfide; PEG-maleimide; Proton channels; Proton translocation