Multiple - subunit Interactions Modulate Rotational Catalysts in the Escherichia Coli FoF1 ATP Synthase

Negrey, Natalie Carol, Department of Molecular Physiology and Biological Physics, University of Virginia
Nakamoto, Robert, Department of Molecular Physiology and Biological Physics, University of Virginia
Bushweller, John, Department of Molecular Physiology and Biological Physics, University of Virginia

The subunit of the F 1 sector of the Escherichia coli ATP synthase is part of the rotor complex (--c 10 ). The subunit has been implicated in coupling, as its presence allows for the maintenance of the proper rate limiting transition state structure thought to be necessary for coupling of proton transport to ATP hydrolysis or synthesis (Peskova, Y.B. and Nakamoto, R.K., Biochemistry 39: 11830-11836, 2000). The subunit has also been shown to modulate ATP hydrolysis activity. The two structural domains of the subunit have been thought to play different roles: the N-terminal -sandwich domain plays a structural role in attaching the F 1 complex to the membrane-bound F O and maintaining coupling between the two domains, while the C-terminal -helical domain plays a role in the inhibition of hydrolysis activity. By characterizing steady state ATPase activity and isokinetic analysis of a series of mutant enzymes of F 1 and F O F 1 , including point mutants, site-directed disulfide cross-linked mutants, and truncation mutants that disrupt or stabilize the interactions of the subunit domains with the  subunit, we show that two functions of the subunit are largely separable by domain. The proper interaction of the N-terminal domain with  is necessary not only for structural attachment but also for the optimal transition state structure, while the C- terminal domain interactions with  and the -hexamer modulate hydrolysis activity. Disrupting the interactions of one domain and maintaining those of the other results in disruption of that domain's role without disrupting the role of the other domain. Furthermore, we show that the conformation of the subunit C-terminal domain in the active state of the enzyme changes upon binding of F 1 to F O , resulting in the observed  increase in hydrolysis activity. The mechanism of action of lauryldimethylamine oxide (LDAO) is also explored, and it is shown that LDAO has two major effects on ATP hydrolysis function, one that is -dependent, and that is capable of disrupting interactions of the N and C-termini with , and one that is -independent, which increases the activity of ATP hydrolysis without affecting the transition state structure.

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PHD (Doctor of Philosophy)
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