Proton-translocating carboxyl of subunit c of F1Fo H(+)-ATP synthase: the unique environment suggested by the pKa determined by 1H NMR.
Subunit c of the H(+)-transporting F1Fo ATP synthase (EC 3.6.1.34) is thought to fold across the membrane as a hairpin of two alpha helices with a conserved Asp/Glu residue, centered in the second membrane-spanning helix, which is thought to function in H+ translocation. NMR studies indicate that the purified subunit c from Escherichia coli is also folded as a hairpin in a chloroform/methanol/H2O (4:4:1) solvent mixture [Girvin, M. E., & Fillingame, R. H. (1993) Biochemistry 32, 12167-12177] and that the conserved Asp remains uniquely reactive in this solvent mixture [Girvin, M. E., & Fillingame, R. H. (1994) Biochemistry 33, 665-674]. The pKa of Asp61 is of interest because of its unique reactivity and because it is thought to protonate and deprotonate during each proton translocation cycle. We have determined the pKa value of the carboxyl group of the functional Asp in wild type and two functional, mutant subunit c proteins, i.e. the Ala24-->Asp (D24D61) and the Ala24-->Asp/Asp61-->Asn (D24N61) mutant proteins. The pKa values were determined by 1H NMR spectroscopy by measuring changes in the alpha and beta proton chemical shifts by constant time two-dimensional (2D) correlated spectroscopy. The pKa of Asp61 in the purified wild type protein was 7.1. This pKa was significantly higher than the pKa of the other two Asp residues, i.e. Asp7 and Asp44 which were 5.4 and 5.6, respectively. The pKa of the two Glu residues in the protein were determined by 2D total correlation spectroscopy and found to be approximately 5.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706, USA.
F M
FM
Assadi-Porter
R H
RH
Fillingame
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid; D006859:Hydrogen; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D016296:Mutagenesis; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*
D046911:Macromolecular Substances; D001224:Aspartic Acid; D018698:Glutamic Acid; D006859:Hydrogen; D006180:Proton-Translocating ATPases