Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon

Abstract

ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP. The Km, value for ATP was 1.8 mM. ATPase activity was optimum at pH 8.5 at 45°C, and was activated by sulfite. Azide strongly inhibited the enzyme activity, whereas the enzyme was relatively resistant to vanadate, nitrate, and N,N′- dicyclohexylcarbodiimide. The genes encoding the subunits for the F 1FO-ATPase from A. ferrooxidans NASF-1 were cloned as three overlapping fragments by PCR cloning and sequenced. The molecular masses of the α, β, γ, δ, and ε subunits of the F 1 portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively.