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 K _m 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 F₁F_O-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 F₁ portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively.

• Acidithiobacillus ferrooxidans
• acidophile
• F₁F_O-ATPase
• atp operon