Characterization of the Metal Binding Environment of Catalytic Site 1 of Chloroplast F₁-ATPase from Chlamydomonas

Abstract

Metal ligands of the VO(2+)-adenosine diphosphate (ADP) complex bound to high-affinity catalytic site 1 of chloroplast F(1) adenosine triphosphatase (CF(1) ATPase) were characterized by electron paramagnetic resonance (EPR) spectroscopy. This EPR spectrum contains two EPR species designated E and F not observed when VO(2+)-nucleotide is bound to site 3 of CF(1). Site-directed mutations betaE197C, betaE197D, and betaE197S in Chlamydomonas CF(1) impair ATP synthase and ATPase activity catalyzed by CF(1)F(o) and soluble CF(1), respectively, indicating that this residue is important for enzyme function. These mutations caused large changes in the (51)V hyperfine tensors of VO(2+)-nucleotide bound to site 1 but not to site 3. Mutations to the Walker homology B aspartate betaD262C, betaD262H, and betaD262T of Chlamydomonas CF(1) caused similar effects on the EPR spectrum of VO(2+)-ADP bound to site 1. These results indicate that the conversion of the low-affinity site 3 conformation to high-affinity site 1 involves the incorporation betaE197 and betaD262 as metal ligands.

Interactions of γT273 and γE275 with the β Subunit PSAV Segment that Links the γ Subunit to the Catalytic Site Walker Homology B Aspartate Are Important to the Function ofEscherichia coliF1FoATP Synthase

“…In the β subunit, this sleeve encompasses residues V254 (MF 1 V268)-T270 (MF 1 T284). The loop and R helix containing βD242 (MF 1 D256) and βR246 (MF 1 R260) that coordinate the Mg 2+ -ATP complex (13) and bind the ATP γ-phosphate (14), respectively, end with residues V254 (MF 1 V268)-I261 (MF 1 I275) (Figure 1C). First, as these residues exit the R helix, several backbone hydrogen bonds reinforce the V254-I261 structure.…”

High-Affinity Metal-Binding Site in Beef Heart Mitochondrial F1ATPase: An EPR Spectroscopy Study

“…Among divalent cations that could replace the natural Mg 2+ in the ATP hydrolysis reaction, Mn 2+ and VO 2+ are of special interest because of their paramagnetic properties that can be investigated by continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR techniques, such as electron spin-echo envelope modulation (ESEEM). EPR techniques have been used to study the binding site(s) of the metal cation in F 1 from different sources and in particular to investigate the specific metal−nucleotide interaction in enzymes from thermophilic bacteria PS3 (TF1) and from chloroplast (CF1) ( − ).…”

Identification of the P-Loop Lysine as a Metal Ligand in the Absence of Nucleotide at Catalytic Site 3 of Chloroplast F1-ATPase from Chlamydomonas reinhardtii

“…These results suggest that an important step in the conversion of species B to C that takes place upon activation of CF 1 -ATPase is the replacement of βD262 as an equatorial ligand by βT168. Consequently, βD262, a residue known to be important for catalysis as a metal ligand in site 1 (20,29,46), also contributes to the nonfunctional, regulatory metal-binding conformation in site 3. In a similar manner, the contribution of the P-loop lysine at catalytic site 3 as a metal ligand in the absence of nucleotide allows this residue to serve a regulatory role via the stabilization of entrapped Mg 2+ -ADP at the high-affinity catalytic sites.…”

“…The catalytic sites of CF 1 -ATPase are designated sites 4, 1, and 3 in order of decreasing affinity for Mg 2+ -nucleotide ( , ). Vanadyl has been used as a direct probe to identify the groups that serve as metal ligands at sites 1 and 3 of the latent and activated forms of the chloroplast F 1 -ATPase ( − ). Vanadyl (V IV O) 2+ is a cation composed of vanadium(IV) double-bonded to oxygen which results in a net charge of 2+.…”

“…The catalytic sites of CF 1 -ATPase are designated sites 4, 1, and 3 in order of decreasing affinity for Mg 2+ -nucleotide (25,26). Vanadyl has been used as a direct probe to identify the groups that serve as metal ligands at sites 1 and 3 of the latent and activated forms of the chloroplast F 1 -ATPase (27)(28)(29). Vanadyl (V IV dO) 2+ is a cation composed of vanadium-(IV) double-bonded to oxygen which results in a net charge of 2+.…”