Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala42 mutant B protein were formed, and the ability of such complexes to hydrolyze ATP was investigated. We found that ATP hydrolysis was able to proceed independently only in the wild-type subunit, albeit at a lower rate. With only one ATP molecule hydrolyzed at a time, gyrase could still perform supercoiling, but the limit of this reaction was lower than that observed when both subunits can hydrolyze the nucleotide.