Abstract
H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.
Highlights
H؉-translocating pyrophosphatase (H؉-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells
Recombinant H؉-PPase was observed in inner membrane vesicles, where it catalyzed both PPi hydrolysis coupled with H؉ transport into the vesicles and PPi synthesis
In the photosynthetic bacterium Rhodospirillum rubrum, Hϩ-PPase is capable of sustaining both PPi hydrolysis coupled with uphill proton translocation and PPi synthesis in conjunction with downhill proton translocation at significant rates under physiological conditions [7,8,9,10,11]
Summary
Hϩ-PPase, proton translocating inorganic pyrophosphatase; AMDP, aminomethylenediphosphonate; IMV, inner membrane vesicles; R-PPase, R. rubrum Hϩ-PPase; RPP-IMV, inner membrane vesicles containing R-PPase; pET-IMV, inner membrane vesicles lacking R-PPase; contig, group of overlapping clones. The R. rubrum Hϩ-PPase (R-PPase) is often referred to as PPi synthase by analogy with ATP synthase [12] Both PPase and proton translocation activities are associated with a single polypeptide of 66 –90 kDa [13,14,15], which possibly forms a dimer [16, 17]. The first Hϩ-PPase discovered was of bacterial origin [7, 8], the proteins from plant and protozoa have been characterized in greater detail by genetic engineering techniques [2, 3] Four of these protein orthologs have been heterologously expressed in yeast Saccharomyces cerevisiae in forms capable of both PPi hydrolysis and Hϩ translocation [15, 19, 20, 31]. Phylogenetic analyses performed in this study reveal a unique relationship between the mersalyl-reactive Cys residues and Kϩ dependence in Hϩ-PPases
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