F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane.
Incubation of right-side-out oriented membrane vesicles of Escherichia coli with tetranitromethane resulted in the nitration of tyrosine residues (Tyr-10 and Tyr-73) of subunit c from the ATP synthase. Cleavage of the protein with cyanogen bromide and separation of the resulting fragments, especially of the tyrosine-containing peptides, clearly demonstrated that the distribution of the nitro groups is similar at any time and at any pH value chosen for the analysis. Furthermore, the percentage of 3-nitrotyrosine present in the two peptide fragments was in good agreement with that obtained for the intact polypeptide chain. While the modification of the tyrosine residues in subunit c with the lipophilic tetranitromethane is independent of the orientation of the membrane vesicles, the subsequent partial conversion of the 3-nitrotyrosine to the amino form only occurred when membrane vesicles with right-side-out orientation were treated with the ionic, water-soluble sodium dithionite, which at certain concentrations cannot penetrate biological membranes. Cleavage of subunit c isolated from nitrated and subsequently reduced membrane vesicles and separation of the resulting fragments by high-pressure liquid chromatography showed that the 3-nitrotyrosine in the Tyr-73-containing peptides has been completely reduced, while the nitro group in peptides containing Tyr-10 remained nearly unaffected.
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014443:Tyrosine; D006180:Proton-Translocating ATPases; D013774:Tetranitromethane; D003488:Cyanogen Bromide