Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+.
Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.
Houseman
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AL
Department of Botany, Arizona State University, Tempe 85287-1601.
Morgan
L
L
LoBrutto
R
R
Frasch
W D
WD
D000255:Adenosine Triphosphate / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006868:Hydrolysis; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D014638:Vanadates / Q000378:metabolism; D014675:Vegetables
D008024:Ligands; D008670:Metals; D014638:Vanadates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium