Intra-molecular rotation of FoF1 ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of FoF1 with the epsilon subunit connected to a globular protein [cytochrome b(562) (epsilon-Cyt) or flavodoxin reductase (epsilon-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing epsilon-Cyt and epsilon-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with epsilon-Cyt or epsilon-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction. (C) 2014 Elsevier Inc. All rights reserved.