Preliminary Estimation of Rotary Torque Produced by Proton-Motive Force in Fully Functional F0F1-ATPase

摘要

F0F1-ATPase is a rotary molecular motor. It is well known that the rotary torque is generated by ATP hydrolysis in F1 but little is known about how it produces the proton-motive force (PMF) in F0. Here a cross-linking approach was used to estimate the rotary torque produced by PMF. Three mutant E. coli strains were used in this study: SWM92 (δW28L F0F1, as control), MM10 (αP280C γA285C F0F1) and PP2 (αA334C/ δL262C F0F1). The oxidized inner membranes from mutant MM10 having a disulfide bridge in the top of γ subunit exhibited good ATP synthesis activity, while the oxidized PP2 inner membranes having a disulfide bridge in the middle of δ subunit synthesized ATP very poorly. We conclude that the rotary torque generated by PMF is sufficient to uncoil the α-helix in the top of δ subunit (MM10) and to overcome the Ramachandran activation barriers (25-30kJ/mol, i.e. about 40-50pN·nm), but cannot cleave the disulfide bond in the middle of the δ subunit (200 kJ/mol, i.e. 330pN·nm) (PP2). Consequently a preliminary estimation is that the rotary torque generated by PMF in the fully functional F0F1 motor is greater than 40-50pN·nm but less than 330pN·nm.