Mutagenesis of Residue βArg-246 in the Phosphate-binding Subdomain of Catalytic Sites of Escherichia coli F1-ATPase

Abstract

Residues responsible for phosphate binding in F 1 F 0 -ATP synthase catalytic sites are of significant interest because phosphate binding is believed linked to proton gradient-driven subunit rotation. From x-ray structures, a phosphate-binding subdomain is evident in catalytic sites, with conserved ␤Arg-246 in a suitable position to bind phosphate. Mutations ␤R246Q, ␤R246K, and ␤R246A in Escherichia coli were found to impair oxidative phosphorylation and to reduce ATPase activity of purified F 1 by 100-fold. In contrast to wild type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate or MgADP-fluoroscandium, showing the Arg side chain is required for wild-type transition state formation. Whereas 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ϳ50%, although reaction still occurred at residue ␤Tyr-297, proximal to ␤Arg-246 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in ␤E (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type but not in mutants. The results show that phosphate can bind in the ␤E catalytic site of E. coli F 1 and that ␤Arg-246 is an important phosphate-binding residue.ATP synthesis from ADP and P i in oxidative and photophosphorylation occurs in the catalytic sites of F 1 F 0 -ATP synthase. This membrane enzyme uses the energy of an ion gradient (usually protons) to drive rotation of a "rotor" composed of subunits ␥⑀c n (where n can vary from 10 to 14 in different organisms), the rotor serving as a transmission device which upon rotation drives the chemical synthesis of ATP sequentially at three catalytic sites. Catalytic sites are located at ␣/␤ interfaces of the ␣ 3 ␤ 3 subunit hexagon, which are immobilized during rotation by the "stator," consisting of subunits ␦b 2 . The proton motor is composed of the single a and a ring of c subunits. Experimental work in this system has been greatly facilitated by the ability to separate the water-soluble F 1 sector (subunits ␣ 3 ␤ 3 ␥␦⑀) from the membrane-embedded F 0 sector (subunits ab 2 c ring ), with retention of ATP hydrolysis function in the former and of proton conduction in the latter. For recent reviews of structure and function of ATP synthase see Refs. 1-4.Based on kinetic measurements, Boyer and co-workers (5, 6) hypothesized that the affinity of catalytic sites for P i is increased by the proton gradient, and currently the concept that P i binding is enhanced by proton gradient-driven subunit rotation appears well supported. Significant P i binding to Escherichia coli F 1 catalytic sites was not measurable by direct binding assay using radioactive P i (7). The K d value for P i binding was Ͼ10 mM as measured by competition with Mg-AMPPNP or ATP in fluorescence titrations using the ␤Y331W mutant enzyme (8 -10). Calculations based on unisite catalysis kinetics showed that K d P i in unisite catalysis is ...

Modulation of Charge in the Phosphate Binding Site of Escherichia coli ATP Synthase

“…From their work, a K d (P i ) of 0.2 mM was calculated. In recent work we confirmed that this assay was applicable, both with membrane-bound enzyme and with purified F 1 from E. coli (11). Concentration dependence of P i protection against NBD-Cl inactivation in E. coli enzyme was similar to that found by Perez et al (24) in mitochondrial enzyme.…”

“…Concentration dependence of P i protection against NBD-Cl inactivation in E. coli enzyme was similar to that found by Perez et al (24) in mitochondrial enzyme. Studies of NBD-Cl inactivation kinetics and of MgADP protection characteristics confirmed that reaction occurred in the ␤E site in E. coli enzyme (11). Subsequently using mutagenesis we found this assay to be successful in assessing the functional roles of various catalytic site residues in P i binding (11,25,26).…”

“…Studies of NBD-Cl inactivation kinetics and of MgADP protection characteristics confirmed that reaction occurred in the ␤E site in E. coli enzyme (11). Subsequently using mutagenesis we found this assay to be successful in assessing the functional roles of various catalytic site residues in P i binding (11,25,26). X-ray crystal structures of catalytic sites containing the P i analogs AlF 3 (27) and SO 4 2Ϫ (28) were valuable in suggesting residues within the P i binding pocket that were suitable targets for mutagenesis.…”

“…The transition state analog MgADPAlF 4 Ϫ trapped in catalytic sites has been visualized by x-ray crystallography (28), and it is clear that the fluoroaluminate group occupies the position of phosphate in the transition state complex. Contribution of different residues to stabilization of the transition state complex can be compared by assay of inhibition of ATPase activity by MgADP-fluoroaluminate (or MgADP-fluoroscandium) in mutant and wild-type enzymes (11,25). By comparing effects on P i binding and transition state stabilization one can further infer roles of each potential P i residue at early and later steps of the catalytic pathway.…”

“…Our earlier work established that natural Arg residues at positions ␣-376, ␤-182, and ␤-246 were important for P i binding in the ␤E catalytic site of ATP synthase, with the latter playing a key role (11,26). Mutagenesis of ␤Arg-246 to Ala, Gln, or Lys abolished P i binding (11). Residue ␤Asn-243, although totally conserved and located very close to bound P i , was found to be not directly involved in interacting with P i .…”