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Removal of the ability to form a salt bridge or hydrogen bonds between the beta subunit catch loop (beta Y297-D305) and the gamma subunit of Escherichia coli F1Fo-ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues beta T304, beta D305, beta D302, gamma Q269, and gamma R268 were found to be very important for ATP hydrolysis catalyzed by soluble F1-ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue beta D305 would not tolerate substitution with Val or Ser and had extremely low activity as beta D305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the gamma subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which alpha beta gamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.

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.

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.

Vanadyl (VIV=O)2+ was used as a paramagnetic probe at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase (CF1) in order to detect interaction of the metals with nucleotides. The M2 site can exist in two forms in the presence of ATP. When ATP and VO2+ are added in a 1.5:1 ratio to CF1, the VO2+ EPR spectrum is identical to that of CF1-VO2+ in the absence of ATP. When the M2 site is filled by the addition of ATP and VO2+ in a 3:1 ratio, the VO2+ binds to M2 in a second form with equatorial coordination to a single phosphate. The treatments required to deplete CF1 of the monodentate VO2+(-)nucleotide complex indicate that the VO2+ is coordinated to the ATP at the nonacatalytic N2 site. The presence of uncomplexed nucleotide appears to induce formation of the second form, possibly via ATP binding to the N3 site. This change in coordination at the M2 noncatalytic site may regulate the ATPase activity of CF1. The M3 site also exists in two forms: (i) in latent CF1, no phosphate coordination is evident; and (ii) after the ATPase has been activated, the EPR line shape is consistent with the two phosphates from ADP at N3 coordinated to the VO2+ at M3. This work establishes a connection between the metal- and nucleotide-binding sites as M2-N2 and M3-N3.

Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.

The involvement of H2O2 generated by photosystem II (PSII) in the process of photoinhibition of thylakoids with a functional oxygen-evolving complex (OEC) was investigated. The rate of photoinhibition was decreased to the rate of loss of activity in the dark when bovine Fe-catalase was present during the photoinhibitory illumination. Photoinhibition was accelerated for both Cl−-depleted and Cl−-sufficient thylakoids when KCN was present to inhibit the thylakoid-bound Fe-catalase. We propose that these preparations become photoinhibed by reactions with H2O2 produced via oxidation of water by the Cl−-depleted OEC and by reduction of O2 at the QB site when PSII is illuminated without an electron acceptor.

The five polypeptides of chloroplast coupling factor one (CF1) have a subunit stoichiometry of 3α, 3β, γ, δ, e designated in order of decreasing molecular weight (1–3). Nucleotide binding and ATPsynthase/ATPase activity have been associated with the β subunit per CF1 and evidence suggests that the three active sites are functionally linked (4). Purified CF1 is a latent ATPase which can be activated to show specificity for Mg2+(5–8) or Ca2+(9–12).

The correlation of the extent to which Tris inactivates photosynthetic O2 evolution with the loss of 2/3 of the Mn bound to thylakoid membranes provided the first evidence that the oxygen-evolving complex (OEC) contained four bound Mn (1). Due to the linear dependence of activity and bound Mn, Tris was hypothesized to cause the concerted release of all four Mn from each reaction center. Later experiments which quantitated functional Mn after the removal of adventitious Mn by washing thylakoids with 50 mM CaCl2 confirmed the ratio of 4 Mn/OEC (2). However, in these experiments the amount of Mn released during inactivation by Tris or NH2OH was variable with the conditions of the extraction. This partial extraction of Mn was interpreted to result from a loss of one to three Mn/OEC in all of the reaction centers rather than the loss of 4 Mn/OEC in 1/4 to 3/4 of the reaction centers, respectively. Tris and NH2OH are now known to remove the extrinsic membrane proteins associated with the OEC (3,4). In the absence of these proteins, the mM concentrations of Ca2+ used to remove adventitious Mn from thylakoids also activate the OEC and inhibit the loss of Mn from the enzyme (5–7).

Washing PSII preparations in 1 M NaCl removes about 1 of the 2–3 bound Ca2+ per Photosystem II reaction center and causes the loss of water oxidation (1). Partial recovery of this activity is specific to the addition of Ca2+ (2) or, less effectively, to Sr2+ (3,4). This salt wash also removes the P17 and P23 proteins from the preparation and it has been hypothesized (4,5) that the loss of Ca2+ results from a decreased affinity of the cation for the P23-less reaction centers. However, the presence of a high affinity (10 µM) and a low affinity (1 mM) Ca2+ binding sites have been observed in P17, P23-depleted PSII preparations (6,7). Alternatively, it has been proposed that the relative proportion of Ca2+ removed from these sites depends on the presence of light or EGTA during the salt wash (1,7). Experiments show that the high affinity Ca2+ binding site is required to form a functional S3 state (8,9) while the low affinity site is involved with photoactivation (10), H2O2-driven S1/S-1 cycling (11) and for electron transfer to Z+ (12).

The integration of microfluidic devices with single molecule motor detection techniques allows chip based devices to reach sensitivity levels previously unattainable.

The influence of the binding of ADP and ATP on the high-affinity Mn(II) binding site of chloroplast coupling factor 1 (CF{sub 1}) was studied by analysis of field-dependent solvent proton and deuteron spin-lattice relaxation data. In order to characterize metal-nucleotide complexes of CF{sub 1} under conditions similar to those of the NMR experiments, the enzyme was analyzed for bound nucleotides and Mn(II) after incubation with AdN and MnCl{sub 2} and removal of labile ligands by extensive gel filtration chromatography. In the field-dependent NMR experiments, the Mn(II) binding site of CF{sub 1} was studied for three mole ratios of added Mn(II) to CF{sub 1}, 0.5, 1.0, and 1.5, in the presence of an excess of either ADP or ATP. The results were extrapolated to zero Mn(II) concentration to characterize the environment of the first Mn(II) binding site of Cf{sub 1}. In the presence of both adenine nucleotides, pronounced changes in the Mn(II) environment relative to that in Mn(II)-CF{sub 1} were evident; the local relaxation rate maxima were more pronounced and shifted to higher field strengths, and the relaxation rate per bound Mn(II) increased at all field strengths. Analysis of the data revealed that the number of exchangeable water molecules liganded tomore » bound Mn(II) increased from one in the binary Mn(II)-CF{sub 1} complex to three and two in the ternary Mn(II)-ADP-CF{sub 1} and Mn(II)-ATP-CF{sub 1} complexes, respectively; these results suggest that a water ligand to bound Mn(II) in the Mn(II)-ADP-CF{sub 1} complex is replaced by the {gamma}-phosphate of ATP in the Mn(II)-ATP-CF{sub 1} complex. A binding model is presented to account for these observations.« less

Purified 125I-labeled 33-kDa protein binds to calcium-washed photosystem II preparations at high-affinity and low-affinity binding sites. Filling 70% of the high-affinity site with 33-kDa protein induces 63% of the maximum achievable reconstitution of O2-evolving activity. When N-succinimidyl [(4-azidophenyl)dithio]propionate modified 33-kDa protein was reconstituted into Ca(II)-washed membranes under conditions that primarily filled the high-affinity site and then cross-linked to adjacent proteins by illumination of the photoaffinity label, a cross-linked protein complex was formed that could be solubilized from the membranes with sodium dodecyl sulfate. The protein complex consisted of 22-, 24-, 26-, 28-, 29-, and 31-kDa proteins cross-linked to the 33-kDa protein and contained about 3-4 mol of Mn/mol of protein.

A method for the quantitative determination of small amounts of protein samples was developed employing neutron activation analysis. Current methods of protein concentration determination are severely limited as a result of differences in the specific characteristics of each protein. Silver binding has been used as a sensitive colorimetric method to indicate the presence of protein. However, silver-protein complexes can have a variety of absorbance spectra unique to each protein, which complicate the analysis. Various amounts of specific proteins were equilibrated in an excess of silver nitrate prior to the reduction of the silver by the addition of NaBH4, HCHO, and NaOH. The protein-silver complex was rapidly separated from the unbound silver by centrifugation chromatography and the amount of bound silver was determined by INAA. The amount of silver was proportional to the amount of protein present in each sample. When the silver was not reduced prior to removal of the unbound silver by chromatography, only negligible amounts of silver remained bound to the protein. The stoichiometry of bound silver to protein on a molar basis showed relatively small differences for the proteins that were examined. This ratio was found to depend on the conditions of the binding and reduction of the silver. The results suggest that the binding of silver is not specific to any charged or polar groups on these proteins and may, therefore, provide a means of determination of the concentration of protein that has general application for all proteins.

Washing thylakoid membranes with 1 M LiCl causes the release of the beta subunit from the chloroplast energy transducing complex (CF1.CF0) in spinach chloroplasts. This protein purifies by size exclusion chromatography as a 180-kDa aggregate and, thus, is probably composed of a trimer of beta polypeptides. The purified aggregate binds ADP to a high and a low affinity site with dissociation constants of 15 and 202 microM, respectively. Mg2+ is required for ADP to bind to both sites. Manganese binds to the protein in a cooperative manner to at least two sites with high affinity. The beta subunit preparation catalyzes Mg2+-dependent ATP hydrolysis at rates which are comparable to other subunit-deficient CF1 preparations and is increased by treatments known to activate the Mg2+-ATPase activity of CF1. However, Ca2+ is not an effective cofactor for this reaction and treatments which activate the Ca2+-ATPase of CF1 are either ineffective or inhibitory.

The evolution of O2 from H2O2 catalyzed by the oxygen-evolving complex (OEC) in darkness was examined with photosystem II reaction center complex preparations from spinach. Flash illumination of dark-adapted reaction centers was used to make S0-enriched or S1-enriched complexes. The membranes catalyzed O2 evolution from H2O2 when preset to either the S0 or S1 state. However, only the S0-state reaction was inhibited by carbonyl cyanide m-chlorophenylhydrazone and dependent on chloride. These results indicate that (1) the S0-dependent and S1-dependent catalytic cycles can be separated by flash illumination, (2) the S0-dependent reaction involves the formation of the S2 state, and (3) the S1-dependent reaction does not involve the formation of the S2 or S3 states. A kinetic study of the S1-dependent reaction revealed a rapid equilibrium ordered mechanism in which (1) the binding of Ca(II) must precede the binding of H2O2 to the OEC and (2) the reaction of Ca(II) with the free enzyme is at thermodynamic equilibrium such that Ca(II) does not necessarily dissociate after each catalytic cycle.

The reaction mechanism and substrate specificity of soluble chloroplast coupling factor 1 (CF1) from spinach were determined by using the purified isomers of chromium-nucleotide complexes either as substrates for the enzyme or as inhibitors of the Ca2+-dependent ATPase activity. The isolation of CrADP( [32P]Pi) formed upon the addition of the enzyme to [32P]Pi and lambda-bidentate CrADP and the observation that the lambda-bidentate CrADP epimer was 20-fold more effective in inhibiting the Ca2+-dependent ATPase activity than was the delta epimer suggest that the substrate of phosphorylation catalyzed by CF1 is the lambda-bidentate metal ADP epimer. Tridentate CrATP was hydrolyzed by soluble CF1 to CrADP(Pi) at an initial rate of 3.2 mumol (mg of CF1)-1 min-1, indicating that the tridentate metal ATP is the substrate for ATP hydrolysis. From these results a mechanism for the phosphorylation of ADP catalyzed by coupling factor 1 is proposed whereby the bidentate metal ADP isomer associates with the enzyme, phosphate inserts into the coordination sphere of the metal, and the oxygen of the beta-phosphate of ADP attacks the inorganic phosphate by an SN2 type reaction. The resulting product is the tridentate ATP ligand.

The metal binding sites of chloroplast coupling factor one have been studied using paramagnetic Mn(II), an effective ATPase cofactor which can be monitored by magnetic resonance techniques (1,2). The environment of bound Mn(II) can be characterized by the magnetic field dependence of the paramagnetic contribution to the nuclear magnetic resonance relaxation rate of solvent nuclei. Values for the hydration number of the bound ion, the distance between the water protons and the ion, and the correlation times for the water-ion interaction have been deduced for several metal-enzyme complexes (3). We have examined the magnetic field dependence of proton spin-lattice relaxation induced by Mn(II) bound to latent CF1 in order to characterize the Mn(II) binding sites on CF1 in the absence and presence of added ADP.

Photosystem II reaction centers evolve O 2 in the dark when H 2 O 2 is added as a substrate. Although some of this activity can be attributed to catalase, as much as 75% of the activity was not affected by the addition of 1 mM KCN. Several lines of evidence demonstrate that this KCN-insensitive O 2 evolution from H 2 O 2 in the dark is catalyzed by the cycling of S states in the oxygen-evolving complex including: (1) inactivation of H 2 O 2 -mediated O 2 evolution by Ca/EDTA washing; (2) susceptibility of the activity to inhibition by amines like ammonia and Tris; (3) inhibition by CCCP which is known to accelerate the rate of deactivation of the S 2 state and; (4) a direct dependence of the rate of O 2 evolution on the presence of calcium and (5) chloride.

The extrinsic 33-kDa protein (P33) was cross-linked covalently to the binding site on P33-depleted PSII preparations which is responsible for reconstitution of photosynthetic water oxidation after PSII preparations have been washed with 1 M CaCl2. Conditions were found in which more than half of the cross-linked protein complexes formed in the PSII preparations retained the ability to catalyze the oxidation of water. The complex is composed of the P33 cross-linked to the D1 and D2 proteins and a 34-kDa protein, which is present in lower abundance than the other three proteins. After solubilization of the membranes with SDS and purification by preparative SDS-PAGE, the complex retains bound manganese and can catalyze the conversion of H2O2 to O2. Calcium and chloride increased the catalase activity of the purified cross-linked complex while lanthanum or hydroxylamine abolished the activity. By use of the specific activity of the H2O2-dependent reaction to follow the extent of purification of the cross-linked complex, the most highly purified complex was determined to contain 0.34 microgram of manganese/180 micrograms of protein. The mole ratio of Mn/protein was calculated to range from 3.6 to 4.5 depending on the assumed stoichiometry of the protein subunits. The results presented here provide direct evidence that one or more of the three proteins that have cross-linked to the P33 are responsible for binding the manganese of the oxygen-evolving complex.

A significant portion of the catalytic site of CF0-CF1 is located on the β subunit (for a review, see (1)). This enzyme contains three copies of the β subunit which suggests that there are three active sites. Experiments that involve isotope exchange (2), substrate trapping (3) and binding of Mn (4) suggest that at least two of the active sites are coupled. Soluble CF1 is a latent ATPase that can be activated by a variety of procedures (5), some of which are believed to require the exchange of disulfide bonds of the γ subunit (6).

The identification of the amino acids on coupling factor one (CF1) which participate in binding substrate during the synthesis of ATP has been limited thus far to studies employing the use of reagents that covalently modify specific amino acids on the enzyme (Vallejos, 1981). In cases where this technique has been successful, the reagent will cause an irreversible loss of enzymatic activity which is prevented by the presence of high concentrations of substrates during the modification. These experiments suggest that a carboxyl group, a lysine, an arginine and a tyrosine are in the proximity of the active site of CF1.

The application of spin-echo methods (1) in NMR provides a means of selecting resonances on the basis of spin-spin relaxation, which in turn is related to the mobility of residues or molecules involved. Spin-echo spectroscopy has been applied previously in this laboratory to the selective observation of mobile amino acid sidechains on CF1, the proton translocating ATPase of the chloroplast thylakoid membrane (2, 3). These experiments have demonstrated the existence of a small subset of amino acid sidechains, approximately 25 in number, which extend into the aqueous phase and exhibit mobility much higher that that of the protein as a whole. Two of the sidechains, one aspartate and one glutamate, are immobilized in a highly selective manner by nucleotide binding to the active site. The immobilized groups lie at a distance of approximately 15Â from the metal cofactor bound at the active site.