Rotary F1-ATPase. Is the C-terminus of subunit gamma fixed or mobile?
F-ATP synthase synthesizes ATP at the expense of ion motive force by a rotary coupling mechanism. A central shaft, subunit gamma, functionally connects the ion-driven rotary motor, F(O), with the rotary chemical reactor, F(1). Using polarized spectrophotometry we have demonstrated previously the functional rotation of the C-terminal alpha-helical portion of gamma in the supposed 'hydrophobic bearing' formed by the (alpha beta)(3) hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between the alpha-helix of gamma and subunit alpha did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a 'functional unwinding' of the alpha-helix to form a swivel joint. Furthermore, they suggested a firm clamping of that part of gamma even without the engineered cross-link, i.e. in the wild-type enzyme. Here, we rechecked the rotational mobility of the C-terminal portion of gamma relative to (alpha beta)(3). Non-fluorescent, engineered F(1) (alpha P280C/gamma A285C) was oxidized to form a (nonfluorescent) alpha gamma heterodimer. In a second mutant, containing just the point mutation within alpha, 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 fluorescent alpha gamma dimers in all cases in accordance with rotary motion of the entire gamma 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 of gamma within hours is compatible with the spectroscopically detected blockade of rotation in the AMP-PNP-inhibited enzyme in the time-range of seconds.
Müller
Martin
M
Universität Osnabrück, FB Biologie/Chemie, Abt. Biophysik, D-49076 Osnabrück, Germany.
Gumbiowski
Karin
K
Cherepanov
Dmitry A
DA
Winkler
Stephanie
S
Junge
Wolfgang
W
Engelbrecht
Siegfried
S
Pänke
Oliver
O
D000255:Adenosine Triphosphate / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D012399:Rotation
D003432:Cross-Linking Reagents; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases