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
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30481
ISSN1083-351X
AutoresChia-Yuan Hu, Wei Chen, Wayne D. Frasch,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoSite-directed mutations D262C, D262H, D262N, and D262T were made to the β subunit Walker Homology B aspartate of chloroplast F1-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3–14% of wild type as were ATPase activities of purified chloroplast F1 indicating that βD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F1 as VO2+-ATP gave rise to two EPR species designated B and C in wild type and mutants. 51V-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 VO2+, 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 the51V-hyperfine parameters of species B, the predominant conformation of VO2+-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. Site-directed mutations D262C, D262H, D262N, and D262T were made to the β subunit Walker Homology B aspartate of chloroplast F1-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3–14% of wild type as were ATPase activities of purified chloroplast F1 indicating that βD262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F1 as VO2+-ATP gave rise to two EPR species designated B and C in wild type and mutants. 51V-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 VO2+, 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 the51V-hyperfine parameters of species B, the predominant conformation of VO2+-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. the extrinsic membrane portion of the F1F0-ATP synthase chloroplast F1 fluorescence resonance energy transfer phosphate-binding loop also known as the Walker Homology A sequence Walker Homology B sequence 2′(3′)-trinitrophenyl-nucleotides 5′- adenylyl-β,γ-imidodiphosphate F0F1-ATP synthases are found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts where they catalyze ATP synthesis driven by an electrochemical gradient (1Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar). The F0 portion contains membrane-spanning subunits and is responsible for translocating protons across the membrane. The F11portion is an extrinsic membrane complex, composed of five different subunits α, β, γ, δ, and ε, and retains the ability to hydrolyze ATP after purification from F0. In the crystal structure of F1 from bovine heart mitochondria (2Abrahams J.P. Leslie G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar), the catalytic sites are located on each of the three β subunits with some contribution from the proximal α subunit. The enzyme crystallized with one catalytic site that contained Mg2+-AMPPNP, one that contained Mg2+-ADP, and one that was empty. Three noncatalytic sites, each located primarily on an α subunit, contained bound Mg2+-AMPPNP in this structure. Chloroplast F1 has about four metal-nucleotides tightly bound upon purification from F0 (6Shapiro A.B. Huber A.H. McCarty R.E. J. Biol. Chem. 1991; 266: 4194-4200Abstract Full Text PDF PubMed Google Scholar). The metal-nucleotide bound to the site designated Site 3 can be removed by gel filtration chromatography, whereas depletion of Site 2 requires partial unfolding of CF1 by precipitation in ammonium sulfate and EDTA (7Bruist M.F. Hammes G.G. Biochemistry. 1981; 20: 6298-6305Crossref PubMed Scopus (133) Google Scholar), and depletion of Sites 1 and 4 require the removal of the ε subunit (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar). Recent evidence indicates that Site 3 is catalytic (9Chen W. LoBrutto R. Frasch W.D. J. Biol. Chem. 1999; 274: 7089-7094Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Fluorescence resonance energy transfer (FRET) measurements using TNP nucleotides enabled the mapping of the positions of Sites 1–3 relative to each other and to locations of fluorescent groups covalently modified to unique locations on CF1 (10McCarty R.E. Hammes G.G. Trends Biochem. Sci. 1987; 12: 234-237Abstract Full Text PDF Scopus (55) Google Scholar). This FRET map shows a close correspondence to the locations of the metal-nucleotides in the crystal structure of F1 from bovine mitochondria. From this correspondence, Site 2 is a noncatalytic site and Site 1, like Site 3, is catalytic (7Bruist M.F. Hammes G.G. Biochemistry. 1981; 20: 6298-6305Crossref PubMed Scopus (133) Google Scholar). The observation of unique identifiable locations for Sites 1–3 and the correspondence between the FRET map and the crystal structure indicate that each of the metal-nucleotide binding Sites 1–3 can be selectively filled with metal-nucleotide complex (7Bruist M.F. Hammes G.G. Biochemistry. 1981; 20: 6298-6305Crossref PubMed Scopus (133) Google Scholar). The F1 portion binds substrate with high affinity in a manner that allows rapid interconversion of ADP and phosphate with bound ATP in the absence of the proton-motive force (3O'Neal C.C. Boyer P.D. J. Biol. Chem. 1984; 259: 5761-5767Abstract Full Text PDF PubMed Google Scholar). The proton-motive force drives two sequential conformational changes of the catalytic site that decreases the affinity of the enzyme for ATP relative to ADP that facilitates the selective dissociation of ATP. This generates a chemical gradient in which the cellular concentration of ATP is much higher relative to ADP and phosphate than it would be at equilibrium. The conformation of each of three catalytic sites on the enzyme is staggered such that the enzyme contains a catalytic site in each of the three sequential conformations at any instant as supported by the structure of F1 (2Abrahams J.P. Leslie G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar). Nucleotides bind the catalytic sites as a complex with Mg2+(4Frasch W.D. Selman B.R. Biochemistry. 1982; 21: 3636-3643Crossref PubMed Scopus (25) Google Scholar), which serves as a cofactor for the reaction. The decrease in affinity for ATP that results from the sequential conformational changes is directly dependent on the presence of Mg2+ (5Weber J. Bowman C. Senior A.E. J. Biol. Chem. 1996; 271: 18711-18718Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). These differences in affinity, which can be as much as 5 orders of magnitude for the F1 from Escherichia coli, strongly suggest that the selective release of ATP results from changes in the metal ligands that are a consequence of the different conformations of the catalytic site. Thylakoids maintain high rates of photophosphorylation by diverting some of the reducing equivalents derived from the light-driven electron transfer reactions to thioredoxin that in turn keeps a disulfide bond on the γ subunit of CF1 reduced. Formation of this disulfide in darkness converts the enzyme from the activated to the latent state that has very low ATPase activity. Addition of ADP accelerates the dark decay of ATPase activity, suggesting that tightly bound ADP in a catalytic site serves a regulatory function. This is required as part of the mechanism to maintain the enzyme in its latent state in the dark (14Dunham K.R. Selman B.R. J. Biol. Chem. 1981; 256: 212-218Abstract Full Text PDF PubMed Google Scholar, 15Zhou J.M. Xue Z. Du Z. Melese T. Boyer P.D. Biochemistry. 1988; 27: 5129-5135Crossref PubMed Scopus (71) Google Scholar). Conversion of this ADP from loosely to tightly bound correlates with formation of latent CF1 (14Dunham K.R. Selman B.R. J. Biol. Chem. 1981; 256: 212-218Abstract Full Text PDF PubMed Google Scholar). This regulatory interconversion only occurs upon subsequent addition of Mg2+ (15Zhou J.M. Xue Z. Du Z. Melese T. Boyer P.D. Biochemistry. 1988; 27: 5129-5135Crossref PubMed Scopus (71) Google Scholar, 16Feldman R.I. Boyer P.D. J. Biol. Chem. 1985; 260: 13088-13094Abstract Full Text PDF PubMed Google Scholar). Thus, the metal that serves as a cofactor by binding as a complex with the nucleotide can also serve in a regulatory role by forming a nonfunctional conformation. Vanadyl (VIV=O)2+ has been used as a direct probe to identify the types of groups that serve as metal ligands at Sites 2 and 3 of CF1 (11Houseman A.L. LoBrutto R. Frasch W.D. Biochemistry. 1994; 33: 10000-10006Crossref PubMed Scopus (22) Google Scholar, 13Houseman A.L.P. LoBrutto R. Frasch W.D. Biochemistry. 1994; 33: 4910-4917Crossref PubMed Scopus (41) Google Scholar, 27Houseman A.L. LoBrutto R. Frasch W.D. Biochemistry. 1995; 34: 3277-3285Crossref PubMed Scopus (36) Google Scholar). The A and g tensors of51V hyperfine couplings from the EPR spectrum of the bound VO2+ are a direct measure of the nature of the equatorial metal binding ligands (28Chasteen N.D. Berliner L. Reuben J. Biological Magnetic Resonance. Plenum Press, New York1981: 53-119Crossref Google Scholar). In a mixed ligand environment, each type of ligand contributes independently to the observed51V-hyperfine coupling (28Chasteen N.D. Berliner L. Reuben J. Biological Magnetic Resonance. Plenum Press, New York1981: 53-119Crossref Google Scholar, 29Holyk N. An Electron Paramagnetic Resonance Study of Model Oxovanadium (IV) Complexes in Aqueous Solution: Correlation of Magnetic Properties with Ligand Type and Metal Chelate Structure. M.Sc. thesis. University of New Hampshire, Durham, NH1979Google Scholar). As a result, the51V-hyperfine parameters can provide information concerning the type of groups coordinated to the enzyme-bound metal. When Site 2 of latent CF1 is filled with VO2+-nucleotide, the bound VO2+-ATP gives rise to EPR species A (11Houseman A.L. LoBrutto R. Frasch W.D. Biochemistry. 1994; 33: 10000-10006Crossref PubMed Scopus (22) Google Scholar). At Site 3, the majority of VO2+-nucleotide binds in a ligand environment that gives rise to EPR species B, whereas a smaller fraction binds to Site 3 in a form that gives rise to EPR species C (11Houseman A.L. LoBrutto R. Frasch W.D. Biochemistry. 1994; 33: 10000-10006Crossref PubMed Scopus (22) Google Scholar, 12Nilges M.J. Electron Paramagnetic Resonance Studies of Low Symmetry Nickel (I) and Molybenum (V) Complexes. Ph.D. thesis. University of Illinois, Urbana, IL1979Google Scholar). Upon activation of the enzyme, all of the signal intensity of species B converts to species C, suggesting that the latter results from the metal ligands when the enzyme is catalytically active. Titration of VO2+-nucleotide to CF1 that had been depleted of metal-nucleotide only from Site 3 showed that the VO2+binds selectively to a single site (11Houseman A.L. LoBrutto R. Frasch W.D. Biochemistry. 1994; 33: 10000-10006Crossref PubMed Scopus (22) Google Scholar, 13Houseman A.L.P. LoBrutto R. Frasch W.D. Biochemistry. 1994; 33: 4910-4917Crossref PubMed Scopus (41) Google Scholar). It is possible for the Mg2+ bound at each catalytic site to have up to six ligands. However, in the two catalytic sites in the crystal structure of F1 from bovine mitochondria that contain Mg2+-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 also conserved among several Mg2+-nucleotide binding proteins including adenylate kinase, phosphofructokinase, human mdrI protein, ATP/ADP translocase, elongation factor Tu, as well as the α and β subunits of the F1-ATPases (17Walker J.E. Saraste M. Runswick M., J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4254) Google Scholar, 18Higgins C.F. Hiles I.D. Salmond G.P.C. Gill D.R. Downie J.A. Evans I.J. Holland I.B. Gray L. Buckel S.D. Bell A.W. Hermodson M.A. Nature. 1986; 323: 448-450Crossref PubMed Scopus (497) Google Scholar, 19Fry D.C. Kuby S.A. Mildvan A.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 907-911Crossref PubMed Scopus (388) Google Scholar, 20Fry D.C. Kuby S.A. Mildvan A.S. Biochemistry. 1985; 24: 4680-4694Crossref PubMed Scopus (118) Google Scholar, 21Chen C.-J. Chin J.-E. Ueda K. Pastan I. Gottesman M.M. Roninson I.B. Cell. 1986; 41: 381-389Abstract Full Text PDF Scopus (1717) Google Scholar, 22Duncan T.M. Parsonage D. Senior A.E. FEBS Lett. 1986; 208: 1-6Crossref PubMed Scopus (63) Google Scholar). This motif, with the consensus sequence of four hydrophobic residues followed by an aspartate, terminates a β-strand with the carboxyl group facing the binding pocket for metal-nucleotide (2Abrahams J.P. Leslie G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar, 20Fry D.C. Kuby S.A. Mildvan A.S. Biochemistry. 1985; 24: 4680-4694Crossref PubMed Scopus (118) Google Scholar). The aspartate carboxyl of WHB has been suggested to hydrogen bond to a water that is coordinated to the metal, or to coordinate to a metal directly in several proteins including the β subunit of the F1-ATPase (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar, 23Yohda M. Ohta S. Hisabori T. Kagawa Y. Biochim. Biophys. Acta. 1988; 933: 156-164Crossref PubMed Scopus (55) Google Scholar, 24Story R.M. Steitz T.A. Nature. 1992; 355: 374-376Crossref PubMed Scopus (559) Google Scholar, 25Al-Shawi M.K. Senior A.E. Biochemistry. 1992; 31: 878-885Crossref PubMed Scopus (39) Google Scholar, 26Weber J. Hammond S.T. Wilke-Mounts S. Senior A.E. Biochemistry. 1998; 37: 608-614Crossref PubMed Scopus (80) Google Scholar). The crystal structure of the bovine mitochondrial F1-ATPase shows the closest carboxyl-oxygen of this residue to be 3.9 Å to 4.3 Å from the metal at catalytic sites that contain bound Mg2+-AMPPNP and Mg2+-ADP, respectively (2Abrahams J.P. Leslie G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar). Recently, site-directed mutations of the P-loop threonine of the β subunit in CF1 from Chlamydomonas (βT168) were compared to determine whether changes in the EPR spectra of VO2+ bound to catalytic Site 3 could be detected (9Chen W. LoBrutto R. Frasch W.D. J. Biol. Chem. 1999; 274: 7089-7094Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The mutations were found to cause changes in both the signal intensity and51V hyperfine parameters of the bound VO2+ that gave rise to EPR species C in a manner that indicated that this residue was a metal ligand in the activated conformation. The lack of changes in EPR species B in the mutant CF1 indicated that the P-loop threonine is not a ligand in the form that predominates in the latent state of the enzyme. We now report an analysis of site-directed mutants of the WHB-aspartate (βD262) of Chlamydomonas CF1 by EPR spectroscopy of VO2+ bound to catalytic Site 3. The results presented here indicate that βD262 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. Chlamydomonas reinhardtii strains (CC-125 and CC-373) were obtained from the C. reinhardtii Culture Collection at Duke University. The plasmid pWT-373 (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar) was used as a template for double-stranded, oligonucleotide-mediated, site-directed mutagenesis following the protocol described in Stratagene Chameleon double-stranded site-directed mutagenesis manual. Each mutagenesis reaction requires a selection primer and a mutagenic primer. The sequence of the selection primer is the same for every mutagenesis reaction, 5′-CGC CCC GAA GAA CGG ATC CCA ATG ATG AGC AC-3′. The sequences of the mutagenic primers for different mutated plasmids are listed as follows. 1) pD262C, 5′-TTA TTC TTC ATT TGT AAC ATT TTC CGG TTC GTA CAA GCT G; 2) pD262H, 5′-TTA TTC TTC ATT CAT AAC ATT TTC CGT TTC; 3) pD262N, 5′-TTA TTC TTC ATT AAC AAC ATT TTC CGT TTC; 4) pD262T, 5′-TAT TCT TCA TTA CAA ACA TTT TCC GGT TCG TAC AAG CTG G. Mutated plasmid DNA was transformed into the C. reinhardtiichloroplast genome using biolistic transformation following procedures as described previously (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar, 30Boynton J.E. Gillham N.W. Harris E.H. Hosler J.P. Johnson A.M. Jones A.R. Randolph-Anderson B.L. Robertson D. Klein T.M. Shark K.B. Samford J.C. Science. 1988; 240: 1534-1538Crossref PubMed Scopus (712) Google Scholar, 31Webber A.N. Gibbs P.B. Ward J.B. Bingham S.E. J. Biol. Chem. 1993; 268: 12990-12995Abstract Full Text PDF PubMed Google Scholar). Southern blot analyses and double-stranded DNA sequencing (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 9.31-9.55Google Scholar) were used to verify the presence of homoplasmic cell lines with desired mutations. Cell cultures and photoautotrophic growth curves of each C. reinhardtii strain were maintained and measured as per Hu et al. (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar). Thylakoid membranes in which electron transfer and ATP synthesis were tightly coupled were prepared, and photophosphorylation assays were measured as per Hu et al. (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar). The ability of purified thylakoids to generate a light-driven proton gradient was monitored using 9-aminoacridine fluorescence quenching as per Chenet al. (9Chen W. LoBrutto R. Frasch W.D. J. Biol. Chem. 1999; 274: 7089-7094Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Isolation of soluble CF1-ATPase from C. reinhardtii and the selective filling of VO2+-ATP into Site 3 were carried out as per Chen et al. (9Chen W. LoBrutto R. Frasch W.D. J. Biol. Chem. 1999; 274: 7089-7094Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The ATPase activity was determined using the coupled ATPase assay including lactic dehydrogenase, and pyruvate kinase as described by Harris and Bashford (33Harris D.H. Bashford C.L. Spectrophotometry and Spectrofluorimetry. IRL Press Ltd., Oxford, UK1987Google Scholar). To activate the CF1, final concentrations of 50 mm dithiothreitol and 20% ethanol were incubated with purified CF1 for more than one h at room temperature. The reaction rates were determined from the initial slopes, typically in the first 20–30 s after adding the protein into the reaction mixture. EPR experiments were carried out at X-band (9 GHz) using a Bruker 580E spectrometer with a TE102 standard cavity and a liquid nitrogen flow cryostat operating at 125 K. Simulations of the CW-EPR spectra employed the program QPOWA (12Nilges M.J. Electron Paramagnetic Resonance Studies of Low Symmetry Nickel (I) and Molybenum (V) Complexes. Ph.D. thesis. University of Illinois, Urbana, IL1979Google Scholar,34Maurice A.M. Acquisition of Anisotropic Information by Computational Analysis of Isotropic EPR Spectra Ph.D. thesis. University of Illinois, Urbana, IL1980Google Scholar). Growth curves of wild type and mutantChlamydomonas cultures were obtained under photoautotrophic conditions at 25 °C at a light intensity of 80 microEinstein·m−2 s−1. As shown in Table I, all of the mutations of βD262 caused dramatic decreases in the ability to grow photoautotrophically compared with wild type. The D262N, C, and T mutants grew at 5–10% of the wild type rate. The D262H mutant was completely incapable of photoautotrophic growth.Table IFunctional comparison of wild-type and D262 mutantsGenotypePhotoautotrophic growth rateaMeasured as the rate of increase in optical density (cell scattering) of the liquid culture at 720 nm in log phase at 25 °C with a light intensity of 80 microEinstein ·m−2 s−1.ATP synthase activitybBased on the rate of 250 μmol of ATP (mg of chlorophyll · h)−1 using Mg2+-ADP and phosphate concentrations of 2 and 3 mm, respectively, with thylakoids from wild type.ATPase activitycBased on the rate of 9.6 μmol of ATP hydrolyzed (mg of CF1 · min)−1 using 10 mmMg2+-ATP with CF1 purified from wild-typeChlamydomonas.%Wild-type100100100βD262H030βD262N9104βD262C5145βD262T41010a Measured as the rate of increase in optical density (cell scattering) of the liquid culture at 720 nm in log phase at 25 °C with a light intensity of 80 microEinstein ·m−2 s−1.b Based on the rate of 250 μmol of ATP (mg of chlorophyll · h)−1 using Mg2+-ADP and phosphate concentrations of 2 and 3 mm, respectively, with thylakoids from wild type.c Based on the rate of 9.6 μmol of ATP hydrolyzed (mg of CF1 · min)−1 using 10 mmMg2+-ATP with CF1 purified from wild-typeChlamydomonas. Open table in a new tab The effects of these mutations on rates of phenazine methylsulfate-dependent photophosphorylation of isolated thylakoids, and ATPase activity of purified CF1 preparations are summarized in Table I. The results are consistent with the relative ability of the mutants to grow photoautotrophically. In all cases, the activities of the D262N, C, and T mutants were about 10% of wild type, whereas those of the D262H mutant were negligible. The subunit composition of CF1 isolated from wild type and mutants are compared by SDS-polyacrylamide gel electrophoresis in Fig.1. All mutants were found to contain α, β, γ, and ε subunits as does the wild type. The abundance of the δ subunit relative to the other subunits was variable among preparations. This subunit is known to be weakly associated with theChlamydomonas CF1 and is easily lost when the enzyme preparation is stored (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar, 35Merchant S. Selman B.R. Eur. J. Biochem. 1983; 137: 373-376Crossref PubMed Scopus (7) Google Scholar). The variability of the abundance of the δ subunit among preparations did not differ from that of wild-type CF1. Other bands visible in these preparations are polypeptides that have been reported previously to copurify with CF1 from Chlamydomonas (36Selman-Reimer S. Merchant S. Selman B. Biochemistry. 1981; 20: 5476-5482Crossref PubMed Scopus (40) Google Scholar). It is also noteworthy that no significant differences in the yields of purified CF1 were observed between preparations from wild type and mutants. The low rates of photophosphorylation were not the result of the inability of the thylakoids to form a light-driven proton gradient. Fig. 2 shows the relative fluorescence quenching of 9-aminoacridine in the wild type and mutant thylakoids upon illumination. The rate and extent of fluorescence quenching was about the same in each of the mutants as in the wild type. Thus, none of the mutations caused the membranes to become uncoupled. Combined with the observations that the yield and subunit composition of the mutant proteins are the same as wild type, it is unlikely that any of the mutations has caused a large conformational change that interferes with folding and assembly of the CF1F0complex. Fig. 3shows parallel transitions of EPR spectra of VO2+ bound to Site 3 of CF1 from wild type Chlamydomonas. Because of the anisotropy that results from the oxo group of VO2+, the single unpaired electron and the nuclear spin I = 7/2 result in an EPR spectrum that consists of 8 transitions from that fraction of molecules where the molecular axis (defined by the V=O bond) is aligned parallel with the magnetic field of the spectrometer, and 8 transitions from the perpendicular alignment. The center of each group of 8 transitions and the spacing between them is determined by the values of g and A, respectively. The magnitude of these values depends on the strength of the hyperfine coupling between the unpaired electron and the51V nucleus. The 51V-hyperfine coupling of VO2+ is sensitive to the types of group coordinated at the equatorial positions. Of these parameters, the coupling constant for the equatorial ligand donor group, A ∥, shows the largest and most easily discerned changes as a function of the types of groups that serve as equatorial ligands. These changes will be evident as differences in the spacing between the −7/2, −5/2, +3/2, +5/2, and +7/2 transitions shown in Fig. 3, which do not overlap with perpendicular transitions. Addition of an equivalent of VO2+-ATP to CF1under conditions in which all other higher affinity binding sites for metal-nucleotides were filled with Mg2+-nucleotide complexes resulted in two sets of parallel transitions for VO2+. The simulated spectrum for each set (Fig. 3, spectrab and c) and the values ofA ∥ and g ∥ used to generate these spectra are given in TableII. Spectra b and c correspond to EPR species B and C that result from the two specific binding environments for VO2+ as a complex with nucleotide in Site 3 each with its own set of equatorial ligands. In the latent form of the enzyme, species B predominates, but activation induces the conversion of species B into species C. The ratios of the amplitudes of the simulated spectra for EPR species B and C from each mutant that, when summed, reproduced the experimental spectra are also shown in Table II. These data provide the ratio of the amount of vanadyl bound in the form that gives rise to species B versus species C.Table IIExperimental 51V-hyperfine parameters derived from VO2+bound as a complex with ATP at Site 3 in CF1 from wild-type and βD262 mutantsStrainRatio of C:BSpecies BSpecies CA∥g∥A∥g∥Wild-type0.87498.21.948456.51.957βD262H0.28493.81.949458.01.957βD262T0.51502.21.950456.51.957βD262N0.68498.21.948456.51.957βD262C0.17498.21.948457.51.957Experimental parameters were determined by simulation of entire spectrum using QPOWA. Open table in a new tab Experimental parameters were determined by simulation of entire spectrum using QPOWA. The parallel transitions of the EPR spectrum from VO2+-ATP bound to Site 3 of CF1 with the D262H mutation is shown in Fig. 4 along with the simulations of EPR species B and C that best fit the experimental data. The value ofA ∥ for EPR species B in this mutant decreased 4.4 MHz from that of the wild type enzyme to a value of 493.8 MHz, whereas the 51V-hyperfine components of species C remained unchanged (Table II). This mutation also caused an increase in the species C:B ratio by more than 3-fold of that observed with the wild type enzyme. No significant differences in A ∥ could be discerned for species B in the spectrum of VO2+ bound to the D262N mutant (Fig. 5)versus those of wild type enzyme. However, much higher signal-to-noise in the EPR spectrum is required to resolve the small difference in A ∥ that would be anticipated if the asparagine side chain were to become a ligand (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar) such that this experiment serves as a negative control. This conservative change in the side chain did increase the signal intensity of species B relative to species C by about 20% such that the ratio of the C:B signal intensity decreased to 0.68. The EPR spectrum that resulted from VO2+-ATP bound to Site 3 of CF1 that contained the D262T mutation is shown in Fig.6. A value of 502.2 MHz forA ∥ was derived from the simulation of EPR species B, which represents a 4 MHz increase inA ∥ of species B from that of wild type. This mutation decreased the ratio of species C:B by 1.7-fold. In the D262C mutant, if there were a single substitution of the sulfhydryl for the carboxyl in the equatorial ligands of the bound VO2+, a decrease in A ∥ of 31.6 MHz from that of the wild type is expected. However,A ∥ of species B remained unchanged (Fig.7). This mutation did have the largest effect on the C:B ratio of any of the mutants, causing a decrease of more than 5-fold from that of wild type. None of the mutations was found to change the 51V-hyperfine parameters of EPR species C. This indicates that the WHB-aspartate is not an equatorial ligand to VO2+ bound at Site 3 in the species C conformation. This also serves as a negative control that indicates that the changes in 51V-hyperfine parameters observed in species B are specific for that binding environment. All of the mutations were found to change the ratio of EPR species C:B from that observed in wild type CF1. BecauseA ∥ and g ∥ are not affected by the mutations, these changes are indicative of changes in the affinity of the VO2+-ATP complex for the conformation of Site 3 that gives rise to species B. The results presented here indicate that βD262 serves as an essential residue of the chloroplast F1F0-ATP synthase in both a catalytic and regulatory capacity. Catalytic function of the enzyme was significantly affected by every mutation examined. In addition, every mutation changed the ratio of EPR species C:B and/or the 51V-hyperfine parameters of EPR species B, the predominant conformation of VO2+-nucleotide bound to Site 3 of latent CF1. None of the crystal structures of F1 determined to date (2Abrahams J.P. Leslie G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar, 38Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar, 39Shirakihara Y. Leslie A.G. Abrahams J.P. Walker J.E. Ueda T. Sekimoto Y. Kambara M. Saika K. Kagawa Y. Yoshida M. Structure ( Lond. ). 1997; 5: 825-836Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) has provided any information concerning the conformation of the metal-nucleotide bound to CF1 in the latent state. Weber et al. (5Weber J. Bowman C. Senior A.E. J. Biol. Chem. 1996; 271: 18711-18718Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) measured the binding affinities of nucleotides to the catalytic sites of the βY331W mutant of E. coli F1 by the fluorescence quenching that results from a direct interaction between the adenine ring of the nucleotide and the tryptophan residue. These studies revealed that Mg2+ was responsible for the large differences in affinities of nucleotide among the three catalytic sites. When the WHB-aspartate was mutated to create a double mutant βY331W/βD242N of E. coli F1, the nucleotide binding affinities of the three catalytic sites were not increased by Mg2+ but became closely similar to the lower affinity observed when nucleotide binds alone (26Weber J. Hammond S.T. Wilke-Mounts S. Senior A.E. Biochemistry. 1998; 37: 608-614Crossref PubMed Scopus (80) Google Scholar). Based on these results Weber et al. (26Weber J. Hammond S.T. Wilke-Mounts S. Senior A.E. Biochemistry. 1998; 37: 608-614Crossref PubMed Scopus (80) Google Scholar) concluded that there must be a water molecule not visible in the crystal structure that is both hydrogen-bonded to the WHB carboxyl and coordinated to the metal in all three catalytic sites. The results presented here for EPR species C show that, in this conformation, the WHB carboxyl is neither an equatorial ligand to VO2+ nor is it hydrogen-bonded to a water molecule coordinated at an equatorial position at Site 3. The51V-hyperfine parameters of EPR species C, the species present exclusively in the activated enzyme, were not changed significantly by any of the mutations examined. These mutations did inhibit catalytic function as observed for the D242N mutant in E. coli F1 (26Weber J. Hammond S.T. Wilke-Mounts S. Senior A.E. Biochemistry. 1998; 37: 608-614Crossref PubMed Scopus (80) Google Scholar). Therefore, it is possible that the WHB carboxyl has hydrogen bonded to the vanadyl-oxo or to a water molecule coordinated at the axial position of the VO2+ in Site 3. The loss of activity in these mutants could also be explained if this residue were to serve as a direct ligand in one of the other catalytic sites. This will be resolved as more of the residues that serve as metal ligands at each catalytic site are identified. The EPR data that result from VO2+ bound to Site 3 of mutant CF1 provide insight into the metal ligation responsible for the changes in EPR species B in each of these mutants. Based on the measured coupling constants of A ∥from model studies (28Chasteen N.D. Berliner L. Reuben J. Biological Magnetic Resonance. Plenum Press, New York1981: 53-119Crossref Google Scholar, 37Hamstra B.J. Houseman A.L. Colpas G.J. Kampf J.W. LoBrutto R. Frasch W.D. Pecoraro V.L. Inorg. Chem. 1997; 36: 4866-4874Crossref PubMed Scopus (118) Google Scholar), the hyperfine coupling for a given group of equatorial ligands can be calculated from Eq. 1A∥calc=∑niA∥i/4Equation 1 where i counts the different types of equatorial ligand donor groups, n i(= 1–4) is the number of ligands of type i, and A ∥i is the measured coupling constant for equatorial ligand donor group of typei (28Chasteen N.D. Berliner L. Reuben J. Biological Magnetic Resonance. Plenum Press, New York1981: 53-119Crossref Google Scholar). Similar equations can be written forg ∥ and for A iso, though the changes in A ∥ are the largest and most easily discerned. Table III shows the values ofA ∥ and g ∥ calculated from Eq. 1 that give the closest fit to the experimental data derived from simulation of the spectra (Table II) and summarizes the equatorial ligands used for these calculations. The best fit of the data for species B at Site 3 of wild type CF1 includes a water, as well as carboxyl, hydroxyl, and phosphate groups as equatorial ligands to VO2+ (9Chen W. LoBrutto R. Frasch W.D. J. Biol. Chem. 1999; 274: 7089-7094Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). One interpretation of the data presented here is that the putative carboxyl ligand is D262. In this case, we expect to observe a change in 51V-hyperfine parameters consistent with displacement of the carboxyl by the type of group substituted in the D262 mutation. Alternatively, D262 may be hydrogen-bonded to the putative water ligand derived from the best fit to EPR species B. No changes 51V-hyperfine parameters are typically expected except in the rare event that the mutation causes a ligand to be displaced. Instead, the differences in the ability of the mutated groups to hydrogen bond to water are anticipated to change the ratio of EPR species C:B.Table IIIBest fits of the 51V-hyperfine parameters of VO2+ bound as a complex with ATP at Site 3 in CF1 from wild-type and βD262 mutants to the groups of equatorial ligands based on Eq. 1StrainCalculatedaFrom Eq. 1 based on the ligands shown.Most probable equatorial ligandsA∥g∥Wild-type498.81.946RCOOROHH2OPiβD262H492.71.948R = NR′ROHH2OPiβD262T501.51.946RNH2ArOHH2OPiβD262N498.81.946RCONROHH2OPiβD262C497.41.946RSHH2OH2OPia From Eq. 1 based on the ligands shown. Open table in a new tab If D262 acts as a direct metal ligand, the 4.4 MHz change inA ∥ of species B observed with D262H can be easily explained as a simple substitution of an imidazole nitrogen for a carboxyl oxygen as an equatorial ligand to the bound VO2+. This change in A ∥ is more difficult to explain if D262 is only hydrogen-bonded to the coordinated water because this requires a double substitution of imidazole nitrogen for water and water for carboxyl. The EPR data shown for the D262N mutation does not provide any information to distinguish between the possible roles of the carboxyl as a direct ligand, or as an indirect ligand hydrogen bonded to a coordinated water. An asparagine side chain coordinated to VO2+ shows changes in A ∥ that can be resolved only at much higher signal-to-noise than that reported here (8Hu C.-Y. Houseman A.L.P. Morgan L. Webber A.N. Frasch W.D. Biochemistry. 1996; 35: 12201-12211Crossref PubMed Scopus (16) Google Scholar). The ability of this side chain to hydrogen bond to water also allows for the latter possibility. It is noteworthy that the D262N mutation causes a small change in the ratio of EPR species C:B signal intensities. If the βD262T mutation were to result solely in the substitution of a carboxyl for a hydroxyl oxygen, a decrease inA ∥ of about 22 MHz is expected. However, an increase in A ∥ of 4 MHz was observed in this mutant to a value of 502.2 MHz. These data are best fit to a set of equatorial ligands where the carboxyl group and the hydroxyl group have been substituted for an amino group and a tyrosine hydroxyl. Although these are somewhat unexpected substitutions, both the P-loop lysine (βK157) and the catch-loop tyrosine (βY311) are in close proximity to the Mg2+ in the catalytic sites of F1 from bovine mitochondria (2Abrahams J.P. Leslie G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar). Clearly, the D262T mutation caused profound changes in the equatorial ligands of VO2+ bound at catalytic Site 3. Because a hydroxyl group is capable of hydrogen bonding, the changes observed here are inconsistent with D262 serving only to hydrogen bond to a coordinated water. The D262C mutation did not change the 51V-hyperfine parameters. Although this appears to favor hydrogen bonding over direct ligation, sulfhydryl groups are relatively poor in forming hydrogen bonds. Consequently, if the group at this position is needed to hydrogen bond to the coordinated water, this mutant should increase the ratio of EPR species C:B. However, a large decrease in the ratio of EPR species C:B was observed. Furthermore, a double substitution of equatorial ligands of sulfhydryl sulfur for carboxyl oxygen and water oxygen for hydroxyl oxygen will also result in51V-hyperfine parameters closely similar to those of wild type (Table III). The results presented here strongly favor the conclusion that the WHB carboxyl serves as an equatorial ligand to the VO2+ bound in Site 3 in the form that gives rise to EPR species B, the form of Site 3 that predominates in latent CF1. Upon activation of the enzyme with dithiothreitol, the signal intensity of species B converts into species C. The lack of effects of the mutations to the WHB carboxyl on EPR species C indicates that activation of the enzyme causes the loss of this carboxyl group as an equatorial ligand. Chenet al. (9Chen W. LoBrutto R. Frasch W.D. J. Biol. Chem. 1999; 274: 7089-7094Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) showed that the P-loop threonine (βT168 inChlamydomonas) serves as an equatorial ligand to VO2+ at Site 3 under conditions that give rise to species C but not to that which results in species B. When combined with the results presented here, it appears that activation changes the conformation of the enzyme to cause the substitution of the WHB carboxyl for the P-loop threonine as a metal ligand at Site 3. We thank David Lowry for excellent technical support.
Referência(s)