The ATPase cycle of the AAA-ATPase protein transporter Bcs1 is highly concerted.

Abstract

Bcs1, a homo-heptameric transmembrane AAA-ATPase that belongs to the superfamily of AAA proteins, acts as translocation machinery for the folded Rieske iron-sulfur protein (ISP, a key subunit of the mitochondrial complex-III) across the inner mitochondrial membrane. Cryo-EM structures depicted a ring-shaped transporter with two cavities, a cylindrical one formed by the ATPase domains on the matrix surface and a conical one formed by the transmembrane helices. Structures in different nucleotide states (ATPγS, ADP, APO) provided intuitive snapshots of the functional cycle, but the kinetics and structural transitions of the ATPase cycle remain entirely unknown. Here, using high-speed atomic force microscopy (HS-AFM) and HS-AFM line scanning (HS-AFM-LS), we characterized the ATPase cycle of Bcs1 at the single molecule level with sub-millisecond temporal resolution. We determined, in conditions where the molecules are in a continuous ATPase cycle turnover, that the ATP-bound conformational has a lifetime of ∼3700 ms while the lifetime of ADP/APO conformation is much shorter, ∼400 ms. By monitoring the conformational state of the two Bcs1 protomers on the two opposite sides of the ring at microsecond temporal resolution, we found that ATP-binding and -hydrolysis coupled conformational changes are highly concerted. We propose that the opening of the cone cavity to the intermembrane surface through the straightening of the transmembrane helices in the membrane is energetically costly and membrane elasticity drives the resealing of the gate. Overall, our results establish a working mechanism for the ATPase cycle of Bcs1 at the single molecule level and propose a mechanism for how Bcs1 can translocate a folded protein.