Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli.
Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites. In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation.
Kersten
M V
MV
Fachbereich Chemie/Abteilung Biochemie, Universität Kaiserslautern, 67663 Kaiserslautern, Germany.
Dunn
S D
SD
Wise
J G
JG
Vogel
P D
PD
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003497:Cyclic N-Oxides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007093:Imidazoles / Q000737:chemistry; D048289:Imidazolidines; D008274:Magnesium / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013113:Spin Labels
D003497:Cyclic N-Oxides; D007093:Imidazoles; D048289:Imidazolidines; D013113:Spin Labels; C053920:2-hexyl-2,3,5,5-tetramethyl-4-(3-iodo-2-oxopropylidene)imidazolidine-1-oxyl; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D003545:Cysteine