Metal Ligation by Walker Homology B Aspartate βD262 at Site 3 of the Latent but Not Activated Form of the Chloroplast F1-ATPase from Chlamydomonas reinhardtii

Chia-Yuan Hu,Wei Chen,Wayne D Frasch

doi:10.1074/jbc.274.43.30481

Abstract

Site-directed mutations D262C, D262H, D262N, and D262T were made to the beta subunit Walker Homology B aspartate of chloroplast F(1)-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3-14% of wild type as were ATPase activities of purified chloroplast F(1) indicating that betaD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F(1) as VO(2+)-ATP gave rise to two EPR species designated B and C in wild type and mutants. (51)V-hyperfine parameters of species C, present exclusively in the activated enzyme state, did not change significantly by the mutations examined indicating that it is not an equatorial ligand to VO(2+), nor is it hydrogen-bonded to a coordinated water at an equatorial position. Every mutation changed the ratio of EPR species C/B and/or the (51)V-hyperfine parameters of species B, the predominant conformation of VO(2+)-nucleotide bound to Site 3 in the latent (down-regulated) state. The results indicate that the Walker Homology B aspartate coordinates the metal of the predominant metal-nucleotide conformation at Site 3 in the latent state but not in the conformation present exclusively upon activation and elucidates one of the specific changes in metal ligation involved with activation.

Highlights

Site-directed mutations D262C, D262H, D262N, and D262T were made to the ␤ subunit Walker Homology B aspartate of chloroplast F1-ATPase in Chlamydomonas
The results presented here indicate that ␤D262 serves as an essential residue of the chloroplast F1F0-ATP synthase in both a catalytic and regulatory capacity
Every mutation changed the ratio of EPR species C:B and/or the 51V-hyperfine parameters of EPR species B, the predominant conformation of VO2ϩ-nucleotide bound to Site 3 of latent CF1

Summary

The abbreviations used are

F1, the extrinsic membrane portion of the F1F0-ATP synthase; CF1, chloroplast F1; FRET, fluorescence resonance energy transfer; P-loop, phosphate-binding loop known as the Walker Homology A sequence; WHB, Walker Homology B sequence; TNP-nucleotides, 2Ј(3Ј)-trinitrophenyl-nucleotides; AMPPNP, 5Јadenylyl-␤,␥-imidodiphosphate. In the two catalytic sites in the crystal structure of F1 from bovine mitochondria that contain Mg2ϩ-nucleotide, only the oxygens of the phosphates and the hydroxyl of Thr-156 were within the 2.5 Å distance that would suggest that they were ligands This threonine is a residue in a motif composed of GXXXXGKT known as Walker Homology A or phosphate-binding loop (P-loop) conserved among several enzymes that catalyze ATP hydrolysis. The Walker Homology B (WHB) motif is conserved among several Mg2ϩ-nucleotide binding proteins including adenylate kinase, phosphofructokinase, human mdrI protein, ATP/ADP translocase, elongation factor Tu, as well as the ␣ and ␤ subunits of the F1-ATPases [17,18,19,20,21,22] This motif, with the consensus sequence of four hydrophobic residues followed by an aspartate, terminates a ␤-strand with the carboxyl group facing the binding pocket for metal-nucleotide [2, 20]. The results presented here indicate that ␤D262 participates in metal binding at Site 3 in the metal-nucleotide complex that predominates in the latent form, but not in the complex that occurs in the activated form of the enzyme

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION