Abstract
Bcs1 is a AAA-ATPase that transports the Rieske iron-sulfur protein (ISP) across the inner mitochondrial membrane. Bcs1 is a particular molecular machine in many regards: In contrast to canonical, hexameric, soluble AAA-ATPases (i) Bcs1 is heptameric, (ii) Bcs1 is a transmembrane protein with each subunit featuring a transmembrane helix, (iii) Bcs1 transports ISP in its folded state, and (iv) Bcs1 works using a concerted mechanism and not a hand-over-hand or stochastic mechanism. How Bcs1 binds and transports folded ISP is unknown. Here we used high-speed atomic force microscopy (HS-AFM) single-molecule analysis and report that Bcs1 subunits are conformationally fully coupled: When Bcs1 is exposed to a mixture of AMP-PNP and ADP where the probability to be in the AMP-PNP or the ADP conformation is equal, all Bcs1 ring complexes are either in AMP-PNP or ADP state, and none forms a hetero-conformer ring. When Bcs1 is exposed to a mixture of AMP-PNP and ATP, traces of AMP-PNP inhibit Bcs1 action showing that all subunits in the ring must be compatible to hydrolyze ATP and undergo a conformational change for function. Furthermore, ISP binds exclusively to the matrix cavity of apo-conformation AAA-domains. Finally, ISP-Bcs1 binding is long enough to outlast the apo-conformation lifetime in ATP-turnover conditions, assuring high transport efficiency. Our single-molecule structural and kinetic data reveals that Bcs1 works according to a unique mechanism so far unknown for any AAA-ATPase.