Coupling Scheme of the Rotary Motor Thermophilic F1

Abstract

Thermophilic F1 (TF1) is an ATP-driven rotary molecular motor driven by sequential hydrolysis of ATP in three catalytic sites. Rotation occurs in steps of 120° per ATP, and the 120° step is further resolved into 80-90° and 40-30° substeps. In the standard coupling scheme, ATP binding starts rotation from an ATP-waiting angle at 0°, and at ∼200° the ATP is cleaved into ADP and Pi, and the ADP is released around 240° after a third ATP is bound. Pi release is at 200° or 320°, yet unsettled. With human mitochondrial F1 (HF1), Suzuki (2014) has indicated different angle dependence: cleavage occurs at 210° and Pi release at 305°. A peculiar finding in HF1 was that supposedly slowly hydrolyzed ATPγS not only lengthened the dwell at 210° but to much greater extent the 305° dwell, implying that the thio-Pi release is much slower than the ATPγS cleavage. We thus re-examined under a microscope how ATPγS affects TF1. With fluorescently (Cy3) labeled ATPγS we observed a remarkably long dwell at only 200° after its binding. With unlabeled ATPγS mixed in ATP, we observed only one long dwell per ATPγS binding, compared to two consecutive long dwells in HF1. The long dwell with unlabeled ATPγS, presumably at 200°, comprised two reactions with rates 460 s−1 and 30 s−1, compared to 2400 s−1 and 820 s−1 with regular ATP. We have yet to decide which corresponds to cleavage and (thio-)Pi release, but kinetic difference from HF1 is obvious: either (thio-)Pi release occurs at 200° in TF1, or thio-Pi release is not slow in TF1. Nonhydrolyzable Cy3-AMPPNP halted rotation at 200° after binding, implying that ATP cleavage occurs at 200° or before, a conclusion previously drawn on the assumption of slow cleavage of ATPγS and a mutant.