Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N.
ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.
Gumbiowski
Karin
K
Universität Osnabrück, FB Biologie, Abt. Biophysik, Barbarastrasse 11, 49076 Osnabrück, Germany.
Pänke
Oliver
O
Junge
Wolfgang
W
Engelbrecht
Siegfried
S
D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D021122:Protein Subunits; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases