Vanadyl as a probe of the function of the F1-ATPase-Mg2+ cofactor.
The Mg2+ dependent asymmetry of the F(1)-ATPase catalytic sites leads to the differences in affinity for nucleotides and is an essential component of the binding-change mechanism. Changes in metal ligands during the catalytic cycle responsible for this asymmetry were characterized by vanadyl (V(IV) + O)2+, a functional surrogate for Mg2+. The (51)V-hyperfine parameters derived from EPR spectra of VO2+ bound to specific sites on F(1) provide a direct probe of the metal ligands. Site-directed mutations of metal ligand residues cause measurable changes in the (51)V-hyperfine parameters of the bound VO2+, thereby providing a means to identification. Initial binding of the metal-nucleotide to the low-affinity catalytic site conformation results in metal coordination by hydroxyl groups from the P-loop threonine and catch-loop threonine. Upon conversion to the high-affinity conformation, carboxyl groups from the Walker homology B aspartate and MF(1)betaE197 become ligands in lieu of the hydroxyl groups.
Frasch
W D
WD
Center for the Study of Early Events in Photosynthesis, Department of Plant Biology, Arizona State University, Tempe, Arizona 85287-1601, USA. frasch@asu.edu
D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021122:Protein Subunits; D014638:Vanadates / Q000378:metabolism*
D008024:Ligands; D021122:Protein Subunits; D014638:Vanadates; D025261:Mitochondrial Proton-Translocating ATPases; D008274:Magnesium