부산대학교 도서관

F-ATP synthase synthesizes ATP at the expense of ion motive force by a rotary coupling mechanism. A central shaft, subunitγ, functionally connects the ion-driven rotary motor, FO, with the rotary chemical reactor, F1. Using polarized spectrophotometry we have demonstrated previously the functional rotation of the C-terminalα-helical portion ofγ in the supposed‘hydrophobic bearing’ formed by the (αβ)3 hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between theα-helix ofγ and subunitα did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a‘functional unwinding’ of theα-helix to form a swivel joint. Furthermore, they suggested a firm clamping of that part ofγ even without the engineered cross-link, i.e. in the wild-type enzyme. Here, we rechecked the rotational mobility of the C-terminal portion ofγ relative to (αβ)3. Non-fluorescent, engineered F1 (αP280C/γA285C) was oxidized to form a (nonfluorescent)αγ heterodimer. In a second mutant, containing just the point mutation withinα, all subunits were labelled with a fluorescent dye. Following disassembly and reassembly of the combined preparations and cystine reduction, the enzyme was exposed to ATP or 5′-adenylyl-imidodiphosphate (AMP-PNP). After reoxidation, we found fluorescentαγ dimers in all cases in accordance with rotary motion of the entireγ subunit under these conditions. Molecular dynamics simulations covering a time range of nanoseconds therefore do not necessarily account for motional freedom in microseconds. The rotation ofγ within hours is compatible with the spectroscopically detected blockade of rotation in the AMP-PNP-inhibited enzyme in the time-range of seconds. [ABSTRACT FROM AUTHOR]