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
Abstract:Site-directed mutations D262C, D262H, D262N, and D262T were made to the  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 D262 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 p… Show more
“…Although the calculated value of A ∥ for this ligand set from eq 1 deviates from the experimental value by 2.7 MHz, it is the only ligand set that fits with the ligands available in the catalytic site. It is noteworthy that when changes in the 51 V hyperfine tensors of VO 2+ bound to catalytic site 3 were induced by site-directed mutations to βT168 and βD262, the cysteine mutation induced a second ligand substitution in addition to the coordination by the sulfhydryl ( , ). 2 Parallel regions of VO 2+ EPR spectra of the VO 2+ −ATP complex bound to site 3 of CF 1 βY317C (a).…”
Section: Resultsmentioning
confidence: 99%
“…Biochemical Characterization of C. reinhardtii Mutants. Cell cultures were maintained for each C. reinhardtii strain as per Hu et al (21). The growth curves of the mutants were determined by the change in optical density (cell scattering) at 720 nm under photoautotrophic conditions as per Hu et al (21).…”
Section: Methodsmentioning
confidence: 99%
“…For PMS-dependent photophosphorylation measurements, the thylakoid particles were prepared as per Hu et al (21). Samples were assayed in 1 mL of reaction mixture that contained 10 µg of chlorophyll of dark-adapted thylakoid membranes, 50 mM Tricine-NaOH (pH 8.0), 10 mM NaCl, 0.3 mM MgCl 2 , 2 mM NaH 2 PO 4 , 0.3 mM ADP, 2.0 × 10 5 cpm [R-32 P]ADP, 50 mM dithiothreitol, and 0.08 mM phenazine methyl sulfate.…”
Section: Methodsmentioning
confidence: 99%
“…In the latent form, the predominant EPR species observed when VO 2+ -ADP complex is tightly bound to site 3 is known as species B (12,13). Under these conditions, the Walker homology B-aspartate (βD262 in Chlamydomonas CF 1 ) was identified as a metal ligand at site 3 (21). The P-loop threonine (βT168 in Chlamydomonas CF 1 ) is not a metal ligand in site 3 of latent CF 1 but becomes a ligand in lieu of the Walker homology B-aspartate upon activation when the EPR signal intensity from species B is converted to species C (15).…”
Site-directed mutants Y317C, Y317E, Y317F, Y317G, and Y317K were made to the catch-loop tyrosine on the beta subunit of the chloroplast F(1)-ATPase in Chlamydomonas. EPR spectra of VO(2+)-ATP bound to site 3 of CF(1) from wild type and mutants were obtained. Every mutant changed the (51)V hyperfine parameters of the VO(2+) bound at this site in the catalytically active conformation of the enzyme but had no effect on these parameters in the form that predominates when the enzyme activity is latent. These results indicate that this residue is a ligand to the metal of the Mg(2+)-nucleotide complex that binds to the empty catalytic site. The mutations also decreased the k(cat) of the ATPase activity to a much greater extent than k(cat)/K(M). Thus, these mutations limit the rate of product (Mg(2+)-ADP and phosphate) release in the ATPase direction or, conversely, the initial binding of substrates in the ATP synthesis direction. On the basis of these observations, coordination of betaY317 by Mg(2+)-ADP that binds to the empty catalytic site provides a means by which substrate binding could trigger gamma subunit rotation and consequent conformation changes of beta subunits during ATP synthesis.
“…Although the calculated value of A ∥ for this ligand set from eq 1 deviates from the experimental value by 2.7 MHz, it is the only ligand set that fits with the ligands available in the catalytic site. It is noteworthy that when changes in the 51 V hyperfine tensors of VO 2+ bound to catalytic site 3 were induced by site-directed mutations to βT168 and βD262, the cysteine mutation induced a second ligand substitution in addition to the coordination by the sulfhydryl ( , ). 2 Parallel regions of VO 2+ EPR spectra of the VO 2+ −ATP complex bound to site 3 of CF 1 βY317C (a).…”
Section: Resultsmentioning
confidence: 99%
“…Biochemical Characterization of C. reinhardtii Mutants. Cell cultures were maintained for each C. reinhardtii strain as per Hu et al (21). The growth curves of the mutants were determined by the change in optical density (cell scattering) at 720 nm under photoautotrophic conditions as per Hu et al (21).…”
Section: Methodsmentioning
confidence: 99%
“…For PMS-dependent photophosphorylation measurements, the thylakoid particles were prepared as per Hu et al (21). Samples were assayed in 1 mL of reaction mixture that contained 10 µg of chlorophyll of dark-adapted thylakoid membranes, 50 mM Tricine-NaOH (pH 8.0), 10 mM NaCl, 0.3 mM MgCl 2 , 2 mM NaH 2 PO 4 , 0.3 mM ADP, 2.0 × 10 5 cpm [R-32 P]ADP, 50 mM dithiothreitol, and 0.08 mM phenazine methyl sulfate.…”
Section: Methodsmentioning
confidence: 99%
“…In the latent form, the predominant EPR species observed when VO 2+ -ADP complex is tightly bound to site 3 is known as species B (12,13). Under these conditions, the Walker homology B-aspartate (βD262 in Chlamydomonas CF 1 ) was identified as a metal ligand at site 3 (21). The P-loop threonine (βT168 in Chlamydomonas CF 1 ) is not a metal ligand in site 3 of latent CF 1 but becomes a ligand in lieu of the Walker homology B-aspartate upon activation when the EPR signal intensity from species B is converted to species C (15).…”
Site-directed mutants Y317C, Y317E, Y317F, Y317G, and Y317K were made to the catch-loop tyrosine on the beta subunit of the chloroplast F(1)-ATPase in Chlamydomonas. EPR spectra of VO(2+)-ATP bound to site 3 of CF(1) from wild type and mutants were obtained. Every mutant changed the (51)V hyperfine parameters of the VO(2+) bound at this site in the catalytically active conformation of the enzyme but had no effect on these parameters in the form that predominates when the enzyme activity is latent. These results indicate that this residue is a ligand to the metal of the Mg(2+)-nucleotide complex that binds to the empty catalytic site. The mutations also decreased the k(cat) of the ATPase activity to a much greater extent than k(cat)/K(M). Thus, these mutations limit the rate of product (Mg(2+)-ADP and phosphate) release in the ATPase direction or, conversely, the initial binding of substrates in the ATP synthesis direction. On the basis of these observations, coordination of betaY317 by Mg(2+)-ADP that binds to the empty catalytic site provides a means by which substrate binding could trigger gamma subunit rotation and consequent conformation changes of beta subunits during ATP synthesis.
“…The similar dose-response patterns GTP exhibited in UV-crosslinking PrrC ( Figure 5A ) or ACNase activation ( 18 ) suggest that PrrC harbors the activating GTPase, although such an activity remains to be demonstrated. It is noteworthy that Asp 222 of PrrC's Walker B motif could be important for hydrolysis of the bound nucleotide as in well-characterized ATPases ( 42 ). Thus, the Asp 222 mutants used here could be devoid of the anticipated GTPase activity.…”
The tRNALys anticodon nuclease PrrC is associated in latent form with the type Ic DNA restriction endonuclease EcoprrI and activated by a phage T4-encoded inhibitor of EcoprrI. The activation also requires the hydrolysis of GTP and presence of dTTP and is inhibited by ATP. The N-proximal NTPase domain of PrrC has been implicated in relaying the activating signal to a C-proximal anticodon nuclease site by interacting with the requisite nucleotide cofactors [Amitsur et al. (2003) Mol. Microbiol., 50, 129–143]. Means described here to bypass PrrC's self-limiting translation and thermal instability allowed purifying an active mutant form of the protein, demonstrating its oligomeric structure and confirming its anticipated interactions with the nucleotide cofactors of the activation reaction. Mutagenesis and chemical rescue data shown implicate the C-proximal Arg320, Glu324 and, possibly, His356 in anticodon nuclease catalysis. This triad exists in all the known PrrC homologs but only some of them feature residues needed for tRNALys recognition by the Escherichia coli prototype. The differential conservation and consistent genetic linkage of the PrrC proteins with EcoprrI homologs portray them as a family of restriction RNases of diverse substrate specificities that are mobilized when an associated DNA restriction nuclease is compromised.
The Mg(2+) cofactor of the F(1)F(0) ATP synthase is required for the asymmetry of the catalytic sites that leads to the differences in affinity for nucleotides. Vanadyl (V(IV)=O)(2+) is a functional surrogate for Mg(2+) in the F(1)-ATPase. The (51)V-hyperfine parameters derived from EPR spectra of VO(2+) bound to specific sites on the enzyme provide a direct probe of the metal ligands at each site. Site-directed mutations of residues that serve as metal ligands were found to cause measurable changes in the (51)V-hyperfine parameters of the bound VO(2+), thereby providing a means by which metal ligands were identified in the functional enzyme in several conformations. At the low-affinity catalytic site comparable to beta(E) in mitochondrial F(1), activation of the chloroplast F(1)-ATPase activity induces a conformational change that inserts the P-loop threonine and catch-loop tyrosine hydroxyl groups into the metal coordination sphere thereby displacing an amino group and the Walker homology B aspartate. Kinetic evidence suggests that coordination of this tyrosine by the metal when the empty site binds substrate may provide an escapement mechanism that allows the gamma subunit to rotate and the conformation of the catalytic sites to change, thereby allowing rotation only when the catalytic sites are filled. In the high-affinity conformation analogous to the beta(DP) site of mitochondrial F(1), the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from betaE197 in Chlamydomonas CF(1). Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity.
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