Coordination of nucleotides to metals at the M2 and M3 metal-binding sites of spinach chloroplast F1-ATPase.
Vanadyl (VIV=O)2+ was used as a paramagnetic probe at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase (CF1) in order to detect interaction of the metals with nucleotides. The M2 site can exist in two forms in the presence of ATP. When ATP and VO2+ are added in a 1.5:1 ratio to CF1, the VO2+ EPR spectrum is identical to that of CF1-VO2+ in the absence of ATP. When the M2 site is filled by the addition of ATP and VO2+ in a 3:1 ratio, the VO2+ binds to M2 in a second form with equatorial coordination to a single phosphate. The treatments required to deplete CF1 of the monodentate VO2+(-)nucleotide complex indicate that the VO2+ is coordinated to the ATP at the nonacatalytic N2 site. The presence of uncomplexed nucleotide appears to induce formation of the second form, possibly via ATP binding to the N3 site. This change in coordination at the M2 noncatalytic site may regulate the ATPase activity of CF1. The M3 site also exists in two forms: (i) in latent CF1, no phosphate coordination is evident; and (ii) after the ATPase has been activated, the EPR line shape is consistent with the two phosphates from ADP at N3 coordinated to the VO2+ at M3. This work establishes a connection between the metal- and nucleotide-binding sites as M2-N2 and M3-N3.
Houseman
A L
AL
Department of Botany, Arizona State University, Tempe 85287-1601.
LoBrutto
R
R
Frasch
W D
WD
D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D008670:Metals / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013113:Spin Labels; D014638:Vanadates / Q000378:metabolism
D008670:Metals; D009711:Nucleotides; D013113:Spin Labels; D014638:Vanadates; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol