Journal
BIOCHEMISTRY
Volume 40, Issue 35, Pages 10664-10670Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0155697
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- NHLBI NIH HHS [HL07284] Funding Source: Medline
- NIGMS NIH HHS [R01 GM050957, GM50957] Funding Source: Medline
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Structure-function relationships of the gamma-epsilon -c subunit interface of FOF1, ATP synthase, a region of subunit interactions important in coupling between catalysis and transport, were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The EPR line widths and collision accessibilities of 18 spin-labeled, unique cysteine F-1 mutants from yLeu198 to gamma Leu215 indicate an alternating pattern in the mobility and accessibility parameters for positions gamma 201-209, which is reminiscent of a fl-strand. Labels at positions gamma 204 and gamma 210 show tertiary contact upon F-1 binding to F-O and gamma D210C has reduced coupling efficiency. gamma E208C could not be spin labeled, but the uncoupling effects of gamma E208K are suppressed by second-site mutations in the polar loop of subunit c [Ketchum, C. J. and Nakamoto, R. K. (1998) J. Biol. Chem. 273, 22292-222971. The restricted mobility and accessibility of spin labels in the odd-numbered positions between gamma 201 and gamma 207 plus the 2-4-fold higher values in k(cat) for ATP hydrolysis of these same mutant F-1 indicate that the interactions of these residues with the subunit mediate its inhibitory activity. Disrupted interactions with c subunit also cause reduced coupling efficiency. We propose a model for the gamma-epsilon -c interface of Escherichia coli FOF1 ATP synthase in which side chains from the odd-numbered residues of the gamma Lys201-gamma Tyr207 beta -strand directly and functionally interact with the epsilon subunit, while the even-numbered, acidic residues gamma Asp204, gamma Glu208, and gamma Asp210 interact with the F-O sector, probably with subunit c. gamma Subunit interactions with both subunits in this region are important for coupling efficiency.
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