Prof. Zippora Gromet-Elhanan holds the Marte R. Gomez Chair of Photosynthesis

The photosynthetic ATP synthase: assembly of hybrid complexes
from bacterial and plant subunits defines their roles in catalysis

Zippora Gromet-Elhanan
Tel. (+972)-8-9342729, Fax. (+972)-8-9344118, e-mail: Z.Gromet-Elhanan@weizmann.ac.il
Zippora Gromet-Elhanan, Ziyun Du, Ward C. Tucker
ATP, the chemical fuel which powers all energy requiring processes in biological systems, is synthesized in all cells by a highly conserved very complex F 0F1 ATP synthase-ATPase, which consists of a membrane-embedded F 0 portion, and an extrinsic catalytic F 1 portion (Fig. 1). F 1 is composed of five subunits with a stoichiometry of a 3b 3gde, and functions as a soluble ATPase. The crystal structure of mitochondrial MF1 at 2.8 Å resolution shows an alternating arrangement of all a and b subunits in a closed hexamer (Fig. 1A), with the resolved 40% of the g subunit embedded inside its cavity (Fig. 1B). It also shows that the three catalytic sites of F1 residing mainly on its b subunits at their a/b interfaces, are occupied by different nucleotides and have different conformational states. These findings support the binding change mechanism, which suggested that ATP synthesis and hydrolysis involve transitions among different conformational states of the catalytic sites via rotation of the g subunit relative to the a 3b 3 -subcomplex. The intricate dynamic interactions within and between the contact domains on the a, b , and g subunits, that are required for this rotational catalysis, are still unresolved. We have developed genetic/biochemical assay systems of these subunits in the photosynthetic enzyme and assembled various hybrid complexes, which enable us to study the detailed mechanism of rotational catalysis as well as identify the participating structural elements.

[Figure 1] Fig. 1. A model of the photosynthetic F 0F1 ATP synthase, showing in (A) the resolved MF1-a 3b 3-hexamer and (B) Cross- section of (A) with the resolved part of F 1g. The stalk F1g and e form together with the F0 c subunits the enzyme's rotor. The interaction of the stator F 0 a, b, b', and F1 d subunits with F 1a stabilizes the a 3b 3 -hexamer during rotation.


Stepwise assembly of active a1 b1-dimers and a3b 3-hexamers from refolded monomers of Rhodospirillum rubrum F1a and b subunits

The incubation of both RrF1a and b monomers together led to a rapid appearance of MgATPase activity, which was correlated with the formation of a1 b1-dimers. These dimers could associate into hexamers in presence of the transition state analog AlF X. Both assembled dimers and hexamers have identical low MgATPase activities, indicating that the dimers contain only the catalytic site at their a/b interface. Their inability to associate into a3b 3-hexamers in absence of AlFX reflects a much lower stability of the noncatalytic RrF1a /b interfaces.
Cloned RrF1a was expressed in E.coli cells only in inclusion bodies, whereas RrF 1b could also be expressed in a soluble form. Since the RrF 1a urea-solubilized aggregates were not refolded by any of the published methods for protein folding, we have developed a specific method for their folding into a functional monomeric form. Its efficiency was assayed by the capacity of the RrF1 a monomer to restore, in presence of RrF1 b, ATP synthesis and hydrolysis to LiCl- treated R.rubrum chromatophores, which lost all their F 0F1b subunits but only about 1/ 3 of the a subunits. The remaining a subunits were retained by their specific interaction with the stator subunits (Figs. 1 and 2). The RrF1a folding procedure enabled refolding of all the expressed insoluble RrF1b and also spinach chloroplast CF1b, but not CF1a, into monomers, which by themselves had no ATPase activity.

[Figure 2] Fig. 2. Formation of hybrid membrane-bound F 0F 1 complexes.


Reconstitution of hybrid F1 and F0 F1 complexes from mixtures of native or refolded RrF 1 and CF1 subunits

The activities and functional properties of the dimers and hexamers are very different from those of the whole RrF 1-ATPase, so their further assembly into an a 3b3 g complex is most interesting. There is as yet no recombinant RrF 1g, but since a recombinant CF1 g subunit (g C) is available, we have refolded it together with the RrF1 a(aR ) and b (b R) and obtained an assembled hybrid F1- aR3bR 3 gC complex. It had much higher ATPase activities than the RrF1-dimers or hexamers or even the whole RrF1. But all its catalytic properties were very similar to those of RrF1, except that they were regulated by the gC redox state, which is a specific property of CF1g, not present in any other F1g subunit. This hybrid enables us now to test for the first time rotational catalysis in an engineered photosynthetic F1-a3b3g -ATPase.

Two additional membrane-bound hybrid F0F 1 ATP synthases were obtained by reconstituting the LiCl- treated chromatophores with refolded RrF 1 a and CF1 b monomers, or with a native CF 1 (ab), fully dissociated into a and b monomers (Fig. 2). Detailed assays of their activities revealed that: 1) all three copies of the RrF1 a are required for restoration of proton- coupled ATP synthesis; 2) in presence of RrF1 a, CF1 b can restore ATP synthesis, but is unable to confer sensitivity to the CF 1 specific inhibitor tentoxin; 3) at least one copy of CF 1 a is required together with all three copies of CF 1b for conferral of tentoxin sensitivity, but it stops restoration of proton-coupled activities. The identification of F 1a domains that are essential for efficient coupling and/ or tentoxin sensitivity is being followed in hybrids containing chimeric RrF1 a/CF1 a subunits.

References

Nathanson, L. and Gromet-Elhanan, Z. (1998) Mutagenesis of b-Glu-195 of the Rhodospirillum rubrum F1-ATPase and its role in divalent cation-dependent catalysis. J. Biol. Chem. 273, 10933-10938,

Du, Z. and Gromet-Elhanan, Z. (1998) Assembly of catalytic a1b1-dimers and a3b3-hexamers from recombinant a and b subunits of the Rhodospirillum rubrum ATP synthase. In: "Photosynthesis: Mechanisms and Effects" (Garab, G., ed.). Vol. III, pp. 1735-1738, Kluwer Academic Publishers.

Nathanson, L. and Gromet-Elhanan, Z. (1998) b-subunit Thr-159 and Glu-195 of the Rhodospirillum rubrum ATP synthase are essential for divalent cation dependent catalysis. In: "The Phototrophic prokaryotes" (Peschek, G.A., Loffelhardt, W. and Schmetterer, G., eds.) pp. 375-378, Plenum Publishing Corporation.

Du, Z. and Gromet-Elhanan, Z. (1999) Refolding of recombinant a and b subunits of the Rhodospirillum rubrum FoF1 ATP-synthase into functional monomers that reconstitute an active a1b1-dimer. Eur. J. Biochem. 263, 430-437.

Nathanson, L. and Gromet-Elhanan, Z. (2000) Mutations in the b-subunit Thr-159 and Glu-184 of the Rhodospirillum rubrum FoF1 ATP synthase reveal differences in ligands for the coupled Mg- and decoupled Ca-dependent activities. J. Biol. Chem. 275, 901-905.

Tucker, W.C., Du, Z., Hein, R., Richter, M.L. and Cromet-Elhanan, Z. (2000) Hybrid Rhodospirillum rubrum FoF1 ATP synthases containing spinach chloroplast F1b or a and b subunits reveal the essential role of the a subunit in ATP synthesis and tentoxin sensitivity. J. Biol. Chem. 275, 906-912.

Du, Z., Tucker, W.C., Richter, M.L. and Gromet-Elhanan, Z. (2001) Assembled F1-(ab) and hybrid F1a3b3g-ATPases from Rhodospirillum rubrum a, wild type or mutant b, and chloroplast g subunits. Demonstration of Mg2+ versus Ca2+-induced differences in catalytic site structure and function. J. Biol. Chem. 276, 11517-11523.

Tucker, W.C., Du, Z., Gromet-Elhanan, Z. and Richter, M.L. (2001) Formation and properties of hybrid photosynthetic F1-ATPases. Demonstration of different structural requirments for stimilation and inhibition by tentoxin. Eur. J. Biochem. 268, 2179-2186.

Tucker, W.C., Du, Z., Hein, R., Gromet-Elhanan, Z. and Richter, M.L. (2001) Role of the ATP synthase a-subunit in conferring sensitivity to tentoxin. Biochemistry 40, 7542-7548.

Hein, R., Tucker, W.C., Du, Z., Levine, T., Kuczera, K., Haran, G., Gromet-Elhanan, Z. and Richter, M.L. (2001) ATP synthase dynamics and tentoxin binding probed using hybrid bacterial chloroplast enzyme assemblies. In: Proceedings of the 12th International Congress of Photosynthesis, Brisbane, Australia.

Acknowledgments

This work is supported by Grants from the United- States- Israel Binational Science Foundation (BSF), Jerusalem, and from the Avron-Wilstatter Minerva Center for Research in Photosynthesis, Rehovot.