Conformational changes in the Escherichia coli ATP synthase (ECF1F0) monitored by nucleotide-dependent differences in the reactivity of Cys-87 of the gamma subunit in the mutant betaGlu-381 --> Ala.
Cys-87, one of two intrinsic cysteines of the gamma subunit of the Escherichia coli ATP synthase (ECF1F0), is in a short segment of this subunit that binds to the bottom domain of a beta subunit close to a glutamate (Glu-381). Cys-87 was unreactive to maleimides under all conditions in wild-type ECF1 and ECF1F0 but became reactive when Glu-381 of beta was replaced by a cysteine or alanine. The reactivity of Cys-87 with maleimides was nucleotide-dependent, occurring with ATP or ADP + EDTA in catalytic sites, in the presence of AMP.PNP + Mg2+ but not with ADP + Mg2+ bound, whether Pi was present or not, and not when nucleotide binding sites were empty. Binding of N-ethylmaleimide had no effect, whereas 7-diethyl-amino-3-(4'-maleimidylphenyl)-4-methylcoumarin increased the ATPase activity of ECF1 more than 2-fold by reaction with Cys-87. In ECF1F0, these reagents inhibited activity. The nucleotide dependence of the reaction of Cys-87 of the gamma subunit depended on the presence of the epsilon subunit. In epsilon subunit-free ECF1, maleimides reacted with Cys-87 under all nucleotide conditions, including when catalytic sites were empty. These results are discussed in terms of nucleotide-dependent movements of the gamma subunit during functioning of the F1F0-type ATPase.
Feng
Z
Z
Institute of Molecular Biology, University of Oregon, Eugene, 97403-1229, USA.
Aggeler
R
R
Haughton
M A
MA
Capaldi
R A
RA
D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D008301:Maleimides; D009711:Nucleotides; D006180:Proton-Translocating ATPases; D003545:Cysteine