Abstract
F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.
Highlights
IntroductionF1Fo-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP
From the Department of Wiochemistry, the §Moiecular Genetics Program, and the Wepartment of Botany, University of Kansas, Lawrence, Kansas 66045-2106
To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the f3 subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase
Summary
F1Fo-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the f3 subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. The cysteine residue at position 63 in chloroplast f3 is known to be located at the interface between a and f3 subunits and to be conformationally coupled, in vitro, to the nucleotide binding site >40 A away. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may involve an interaction, via conformational changes, between the amino-terminal domains of the a and f3 subunits. The FIFo-type ATP synthases utilize the energy of a transmembrane proton gradient to drive the conversion of ADP plus Pi to ATP.
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