Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation.
A spin-labeled photoaffinity ATP analog, 2-N3-2',3'-SL-ATP (2-N3-SL-ATP) was specifically loaded at catalytic (exchangeable) or noncatalytic (nonexchangeable) nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase. Photolysis of the enzyme complexes resulted in the specific modification of beta-Tyr-345 when the catalytic sites were occupied and beta-Tyr-368 when noncatalytic sites were filled. These are the same amino acid assignments that were made previously using 2-N3ATP. The results demonstrate that the attachment of a spin label moiety to the ribose ring does not prevent proper binding of the analog at both types of nucleotide sites on F1-ATPase and suggest that the probe can be used for investigations of the nucleotide-binding sites using ESR spectroscopy. Enzyme that is in complex with the 2-N3-SL-ATP exhibits an ESR spectrum that is typical for highly immobilized nitroxyl radicals both in the dark or after photolysis. Additional peaks in the high- and low-field regions arise due to dipolar spin interactions most likely involving a pair of catalytic and noncatalytic sites. The two sites are calculated to be approximately 15 A apart. This distance, obtained through ESR spectroscopy, combined with the finding that the 2 labeled amino acids are only 23 residues apart from each other, further supports an adenylate kinase-like arrangement of nucleotide binding sites on F1-ATPase where catalytic and noncatalytic sites are in close proximity (Vogel, P. D., and Cross, R. L. (1991) J. Biol. Chem. 266, 6101-6105).
Vogel
P D
PD
Fachbereich Chemie der Universität, Kaiserslautern, Federal Republic of Germany.
Nett
J H
JH
Sauer
H E
HE
Schmadel
K
K
Cross
R L
RL
Trommer
W E
WE
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D008027:Light; D008929:Mitochondria, Heart / Q000201:enzymology*; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013113:Spin Labels
D000227:Adenine Nucleotides; D000345:Affinity Labels; D001386:Azides; D013113:Spin Labels; C075134:2-azido-2'-3'-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester)ATP; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases