Effects of Nucleotides on the Protein Ligands to Metals at the M2 and M3 Metal-Binding Sites of the Spinach Chloroplast F1-ATPase

Andrew L P Houseman, Russell LoBrutto, Wayne Frasch

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

We have identified the most probable protein ligands at the catalytic M3 and noncatalytic M2 metal-binding sites in the spinach chloroplast F1-ATPase (CF1) and here propose possible residues in the protein sequence for these ligands in latent CFi in the absence of nucleotide. The changes in the metal ligands at these sites upon binding of nucleotide to the N2 and N3 sites and upon activation of latent CF1 provide a possible molecular basis for inhibition of ATPase activity by free metal, for the lack of activity in the latent state, and for the gating mechanism of the ATPase H+ pump. To these ends, the Mg2+ analogue, vanadyl (VIV=O)2+, was used as a paramagnetic probe at the M2 and M3 metal-binding sites. EPR and ESEEM spectra of VO2+ were obtained, and simulations of the full EPR spectra imply the ligand sets at the different metal-binding sites. When VO2+ is added to CF1 in the absence of ATP, the most likely set of ligands at the M2 site are 1 ROH (±T176), 2 H2O, and 1 RCOO- (±D269 or ±D270), where the suggested amino acid designations of the residues are given in parentheses according to the mitochondrial sequence. Evidence suggests a possible axial nitrogen ligand at this site (±K175). When the M2 site is filled by addition of VO2+ and ATP, the metal binds as a second species in which N2-bound ATP and M2-bound VO2+ form a monodentate complex with concomitant exchange of the equatorial protein ligands by 3 H2O. The M3 sites also exists in two forms: (i) in latent CF1, the data are best fit with 1 ROH (βT163), 1 RCOO-(β256 or βE188), and 2 H2O as ligands; and (ii) after the ATPase has been activated, the two phosphates of the ADP bound to the N3 site may coordinate to the metal bound to the M3 site. The best fit of the coordination sphere for this second form of M3-bound VO2+ is 2 ROH (β163 and aS344) and 2 phosphates (from ADP). Evidence suggests that there is an axial nitrogen ligand at this site (βK162) and/or the M2 site (αK175). The determination of metal ligands provided by this and earlier studies with vanadyl permits a correlation between the sites of the crystal structure and the order of filling: N1, N2, N3, etc.

Original languageEnglish (US)
Pages (from-to)3277-3285
Number of pages9
JournalBiochemistry
Volume34
Issue number10
DOIs
StatePublished - 1995

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Effects of Nucleotides on the Protein Ligands to Metals at the M2 and M3 Metal-Binding Sites of the Spinach Chloroplast F<sub>1</sub>-ATPase'. Together they form a unique fingerprint.

Cite this