Tài liệu Báo cáo khoa học: A point mutation in the ATP synthase of Rhodobacter capsulatus results in

A point mutation in the ATP synthase of Rhodobacter capsulatus

results in differential contributions of DpH and Du in driving

the ATP synthesis reaction

Paola Turina and B. Andrea Melandri

Department of Biology, Laboratory of Biochemistry and Biophysics, University of Bologna, Italy

The interface between the c-subunit oligomer and the

a subunit in the F0 sector of the ATP synthase is believed to

form the core of the rotating motor powered by the protonic

flow. Besides the essential cAsp61 and aArg210 residues

(Escherichia coli numbering), a few other residues at this

interface, although nonessential, show a high degree of

conservation, among these aGlu219. The homologous resi￾due aGlu210 in the ATP synthase of the photosynthetic

bacterium Rhodobacter capsulatus has been substituted by a

lysine. Inner membranes prepared from the mutant strain

showed approximately half of the ATP synthesis activity

when driven both by light and by acid-base transitions. As

estimated with the ACMA assay, proton pumping rates in

the inner membranes were also reduced to a similar extent in

the mutant. The most striking impairment of ATP synthesis

in the mutant, a decrease as low as 12 times as compared to

the wild-type, was observed in the absence of a transmem￾brane electrical membrane potential (Du) at low trans￾membrane pH difference (DpH). Therefore, the mutation

seems to affect both the mechanism responsible for coupling

F1 with proton translocation by F0, and the mechanism

determining the relative contribution of DpH and Du in

driving ATP synthesis.

Keywords: ATP synthase; mutagenesis; Rhodobacter cap￾sulatus; DpH; Du.

Membrane-bound F0F1-ATPases (ATP synthases) catalyze

ATP synthesis in bacteria, chloroplasts and mitochondria at

the expenses of an electrochemical potential gradient of

protons (or Na+ ions in some species). The membrane￾embedded hydrophobic F0 sector is involved in proton

translocation across the membrane, and the hydrophilic F1

sector contains the catalytic sites (reviewed in [1–3]). A

wealth of high resolution structural information for the

soluble part has appeared since the first crystal structure of

the mitochondrial F1 was reported in 1994 [4], paralleled by

an increasing amount of experimental evidence supporting a

rotational mechanism of catalysis (reviewed in [5]).

In the most investigated Escherichia coli enzyme, F1

consists of five types of subunits in stoichiometry a3b3cde

and F0 consists of three types of subunits in stoichiometry

ab2c9)12. The c subunit monomers span the membrane as a

hairpin of two a helices [6] and are arranged in a oligomer in

the form of a ring (see, for example, the crystallographic

evidence in [7]). Subunit a most likely consists of five

transmembrane helices [8–10], the fourth of which has been

shown by extensive cross-linking analysis to pack against

the second transmembrane segment of subunit c [11]. The

fourth and fifth transmembrane helices, residues 206–271,

house the most conserved regions of the subunit.

In view of the ATP-driven rotation of the c- and

e-subunit shaft within the a3b3 subunit barrel in F1, it is

proposed that the c subunit ring in F0, which is connected to

the ce shaft [12–14], rotates against the a subunit, which is

connected to the a3b3 barrel through the b and d subunits

[15,16]. Experimental evidence consistent with this idea has

been presented [17–19].

A few mechanistic models for torque generation in F0

have been proposed, which emphasize the role of electro￾static interactions [20–22] or the role of conformational

changes within the c subunit [23]. All models include a

central role for the essential carboxyl group of the c subunit

and for the essential Arg residue in the a subunit (cAsp61

and aArg210, respectively, in E. coli).

Besides the cAsp61/aArg210 couple in the middle of the

membrane, the remaining a/c interface regions are believed

to form the access pathways for protons. Probably lining the

acidic access pathway is residue aGlu219, based on cross￾linking data [11]. Several lines of evidence support a close

spatial and functional interaction between aGlu219 and

aHis245, including the fact that in the ATP synthases of

mitochondria and of photosynthetic bacteria the position of

these two amino acids in the primary sequence are inverted

[24], the fact that the E. coli double mutant aGlu219 fi

His/aHis245 fi Glu has an ATP synthase activity signifi￾cantly higher than that of either of the single mutation

strains [25], and their close position in the proposed

topological models [8–10]. Although these residues were

shown to be nonessential by extensive mutagenic analysis

Correspondence to B. A. Melandri, Laboratory of Biochemistry and

Biophysics, Department of Biology, University of Bologna, Via

Irnerio, 42, I-40126 Bologna, Italy. Fax: + 39 051 242576,

Tel.: + 39 051 2091293, E-mail: [email protected]

Abbreviations: GTA, gene transfer agent; Bchl, bacteriochlorophyll;

ACMA, 9-amino-6-chloro-2-methoxyacridine; RC, photosynthetic

reaction center;
l~Hþ , transmembrane difference of electrochemical

potential of protons; Du, bulk-to-bulk transmembrane electrical

potential difference; Dw, surface electrical potential difference.

Enzyme: ATP synthase (EC 3.6.3.14).

Note: a website is available at http://www.biologia.unibo.it/

(Received 12 November 2001, revised 21 February 2002, accepted 21

February 2002)

Eur. J. Biochem. 269, 1984–1992 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02843.x