ADP−Fluoroaluminate Complexes Are Formed Cooperatively at Two Catalytic Sites of Wild-Type and Mutant α3β3γ Subcomplexes of the F1-ATPase from the Thermophilic Bacillus PS3

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In the crystal structure of the bovine heart mitochondrial The proton-translocating F 0 F 1 -ATP synthases couple proton electrochemical gradients to condensation of ADP with P i in energy-transducing membranes. The F 0 moiety is a membraneembedded protein complex that mediates proton translocation. F 1 is a peripheral membrane protein complex composed of five different subunits with ␣ 3 ␤ 3 ␥␦⑀ stoichiometry. When removed from the membrane, F 1 is an ATPase containing six nucleotide binding sites. Three are catalytic sites that are predominantly on ␤ subunits at ␣/␤ interfaces. The three other sites, called noncatalytic sites, are located predominantly on ␣ subunits at different ␣/␤ interfaces (1-3).The crystal structures of the bovine heart and rat liver mitochondrial F 1 -ATPases as well as that of the ␣ 3 ␤ 3 subcomplex of the TF 1 -ATPase have been determined (4 -6). In the crystals of the bovine heart enzyme (BH-MF 1 ), 1 which form in media containing AMP-PNP, ADP, Mg 2ϩ , and N 3 Ϫ , noncatalytic sites are homogeneously liganded with MgAMP-PNP, whereas catalytic sites are heterogeneously liganded (4). One, designated ␤ T , contains MgAMP-PMP, another, designated ␤ D , contains MgADP and the third catalytic site, designated ␤ E , is empty. Except for small differences in the regions of the terminal phosphates of bound nucleotides, the arrangements of functional amino acid side chains in catalytic sites in ␤ T and ␤ D are essentially identical. In contrast, these residues are arranged differently in ␤ E . In the crystals of the ␣ 3 ␤ 3 subcomplex of TF 1 that form in media free of Mg 2ϩ and nucleotides, the ␣ subunits are in closed conformations corresponding to the liganded ␣ subunits of the mitochondrial enzymes. The ␤ subunits in the ␣ 3 ␤ 3 structure are in open conformations corresponding to ␤ E of BH-MF 1 (5).In the crystals of the rat liver F 1 -ATPase (RL-MF 1 ), which form in media containing ATP, P i , and EDTA, noncatalytic sites are homogeneously occupied with MgATP. In contrast to the crystals of BH-MF 1 , all three catalytic sites of RL-MF 1 contain bound nucleotides in the absence of Mg 2ϩ (6). One catalytic site contains ADP alone, whereas the other two contain ADP and P i . Since Mg 2ϩ is not associated with ADP and P i bound to catalytic sites of RL-MF 1 , side chains in catalytic sites that interact with the Mg 2ϩ ion in the crystal structure of BH-MF 1 are arranged differently in the crystal structure of RL-MF 1 . However, these differences are minor compared with the difference between the closed conformations of ␤ D and ␤ T and the open conformation of ␤ E in BH-MF 1 . In the crystal structure of RL-MF 1 , the conformations of all three ␤ subunits resemble the closed conformation of ␤ D and ␤ T in the crystal structure of BH-MF 1 . Bianchet et al. (6) suggest that the open conformation of ␤ E in the crystal structure of BH-MF 1 is the consequence of low concentrations of nucleotides in the crystallization medium. Extending this argument, they propose that the RL-MF 1 structure with t...

Section: Discussionmentioning

confidence: 93%

Oxidation of the α3(βD311C/R333C)3γ Subcomplex of the Thermophilic Bacillus PS3 F1-ATPase Indicates That Only Two β Subunits Can Exist in the Closed Conformation Simultaneously

Ren¹,

Dou²,

Stelzer

et al. 1999

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“…It has not yet been located in x-ray structures of F 1 . One school of thought envisages that azide might form a tightly bound MgADP⅐N 3 Ϫ complex at catalytic site(s) (46). However, data in Ref.…”

Section: Resultsmentioning

confidence: 99%

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

Ahmad

Senior

2004

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 ...

“…Inhibition of ATPase Activity of F 1 by Azide-Azide is a potent inhibitor of F 1 -ATPases in general, although its mode of inhibition remains controversial (32,33). It has not yet been located in x-ray structures of F 1 .…”

Section: Resultsmentioning

confidence: 99%

Role of βAsn-243 in the Phosphate-binding Subdomain of Catalytic Sites of Escherichia coli F1-ATPase

Ahmad

Senior

2004

In the catalytic mechanism of ATP synthase, phosphate (P i ) binding and release steps are believed to be correlated to ␥-subunit rotation, and P i binding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue ␤Asn-243 appears centrally located in the P ibinding subdomain of catalytic sites. Here we studied the role of ␤Asn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp. Mutation ␤N243A caused 30-fold impairment of F 1 -ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and P i protected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. ␤N243D F 1 -ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADPfluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated ␤N243D F 1 -ATPase, and P i did not affect activation. We conclude that residue ␤Asn-243 is not involved in P i binding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the "attacking water" and of an associated second water.ATP synthase is the enzyme responsible for ATP synthesis by oxidative phosphorylation or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. In its simplest form, exemplified by the Escherichia coli enzyme studied here, it contains eight different subunit types. Three catalytic sites, located at the interfaces of ␣ and ␤ subunits in the membrane-extrinsic ␣ 3 ␤ 3 hexagon, operate in concert to synthesize ATP. Reactions at the catalytic sites are controlled by rotation of the ␥ subunit within the ␣ 3 ␤ 3 hexagon. The ␥ subunit is part of the "rotor," composed of ␥, ⑀, and a ring of c subunits. Passage of protons down the transmembrane proton gradient, through the membrane-buried interface between the c ring and the a subunit, energizes the rotor. A "stator" immobilizes the catalytic sites during rotation. Consisting of the b 2 ␦ subunits, the stator is attached to a subunit in the membrane and to the ␣ 3 ␤ 3 hexagon. Experimentally, the enzyme can be resolved readily into water-soluble F 1 and water-insoluble F 0 sectors, which correspond to the membrane-extrinsic ␣ 3 ␤ 3 ␥␦⑀ and membrane-located ab 2 c ring subcomplexes, respectively. F 1 retains catalytic ATPase activity (a physiological activity in E. coli) and is used extensively for studies of the catalytic sites.