Conformational changes in the Escherichia coli ATP synthase (ECF sub(1)F sub(0)) monitored by nucleotide-dependent differences in the reactivity of Cys-87 of the gamma subunit in the mutant beta Glu-381 arrow right Ala

• Published in: The Journal of biological chemistry 1996-01, Vol.271 (30), p.17986-17989
• Main Authors: Feng, Zhaoyang, Aggeler, R, Haughton, MA, Capaldi, R A
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 0021-9258

Cys-87, one of two intrinsic cysteines of the gamma subunit of the Escherichia coli ATP synthase (ECF sub(1)F sub(0)), 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 ECF sub(1) and ECF sub(1)F sub(0) 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 + Mg super(2+) but not with ADP + Mg super(2+) bound, whether P sub(i) 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 ECF sub(1) more than 2-fold by reaction with Cys-87. In ECF sub(1)F sub(0), 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 ECF sub(1), 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 F sub(1)F sub(0)-type ATPase.