Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis

Lubov Nathanson,Zippora Gromet-Elhanan

doi:10.1074/jbc.273.18.10933

Lubov Nathanson, Zippora Gromet-Elhanan

Open Access

https://doi.org/10.1074/jbc.273.18.10933

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Abstract

We introduced mutations at the fully conserved residue Glu-195 in subunit beta of Rhodospirillum rubrum F1-ATPase. The activities of the expressed wild type (WT) and mutant beta subunits were assayed by following their capacity to assemble into the earlier prepared beta-depleted, membrane-bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8742-8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, beta-E195K, beta-E195Q, and beta-E195G, were found to bind as the WTbeta into the beta-depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only beta-E195G could restore some of these activities when assayed in the presence of 100 mM sulfite and high MgCl2 or MnCl2 concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrum F1beta-E195 is equivalent to the mitochondrial F1beta-E199, which points into the tunnel leading to the F1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). Our findings indicate that this residue, although not an integral part of the F1 catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F1 catalytic sites between different conformational states.

Highlights

Two recently published x-ray crystallographic structures of rat liver mitochondrial MF1 at 3.6 Å [7] and bovine heart MF1 at 2.8 Å [8] have confirmed the alternate arrangement of the six large ␣ and ␤ subunits in a closed hexamer
Since the equivalent MF1␤-E199 is described in the crystal structure as pointing into the conical tunnel leading to the catalytic nucleotide binding site [8], it was interesting to check whether the E195G mutation affects activity
Expression and Purification of wild type (WT) and Mutant RrF1␤ Subunits—Cloned WT and mutant ␤ genes were ligated into the expression vector pBTacI, and the recombinant plasmid was transformed into E. coli LM3115 [28]

Summary

The abbreviations used are

RrF1␤, RrF1␣, CF1␤, CF1␣, EcF1␤, MF1␤, and TF1␤, ␤ and ␣ subunits of the F1-ATPase of R. rubrum, chloroplasts, E. coli, mitochondria, and thermophilic Bacillus PS3, respectively; Bchl, bacteriochlorophyll; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; DCCD, dicyclohexylcarbodiimide. A large number of in vitro assays of activity have been developed for the RrF1␤ subunit that was isolated in functional form from the chromatophore membrane-bound RrF0F1. A detailed comparison of the activities of these mutants with those of the expressed WT ␤ subunit revealed that RrF1␤-E195 plays an important role in divalent cation-dependent ATP synthesis and hydrolysis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION