Introduction of a carboxyl group in the loop of the F0 c-subunit affects the H+/ATP coupling ratio of the ATP synthase from Synechocystis 6803.
The proton translocation stoichiometry (H+/ATP ratio) was investigated in membrane vesicles from a Synechocystis 6803 mutant in which the serine at position 37 in the hydrophilic loop of the c-subunit from the wild type was replaced by a negatively charged glutamic acid residue (strain plc37). At this position the c-subunit of chloroplasts and the cyanobacterium Synechococcus 6716 already contains glutamic acid. H+/ATP ratios were determined with active ATP synthase in thermodynamic equilibrium between phosphate potential (deltaGp) and the proton gradient (deltamuH+) induced by acid-base transition. The mutant displayed a significantly higher H+/ATP ratio than the control strain (wild type with kanamycin resistance) at pH 8 (4.3 vs. 3.3); the higher ratio also being observed in chloroplasts and Synechococcus 6716. Furthermore, the pH dependence of the H+/ATP of strain plc37 resembles that of Synechococcus 6716. When the pH was increased from 7.6 to 8.4, the H+/ATP of the mutant increased from 4.2 to 4.6 whereas in the control strain the ratio decreased from 3.8 to 2.8. Differences in H+/ATP between the mutant and the control strain were confirmed by measuring the light-induced phosphorylation efficiency (P/2e), which changed as expected, i.e., the P/2e ratio in the mutant was significantly less than that in the wild type. The need for more H+ ions used per ATP in the mutant was also reflected by the significantly lower growth rate of the mutant strain. The results are discussed against the background of the present structural and functional models of proton translocation coupled to catalytic activity of the ATP synthase.
Van Walraven
Hendrika S
HS
Department of Structural Biology, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. rieky@bio.vu.nl
Scholts
Marijke J C
MJ
Lill
Holger
H
Matthijs
Hans C P
HC
Dilley
Richard A
RA
Kraayenhof
Ruud
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D025222:Chloroplast Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011522:Protons; D013816:Thermodynamics
D021122:Protein Subunits; D011522:Protons; D000255:Adenosine Triphosphate; D025222:Chloroplast Proton-Translocating ATPases