Functional Incorporation of Chimeric b Subunits into F₁F_O ATP Synthase

Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32605, USA; Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1

^* To whom correspondence should be addressed. Email: bcain{at}biochem.med.ufl.edu.

	Abstract

F₁F₀ ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F₁ sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b' peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b' genes into the E. coli uncF(b) gene. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2(b). Although not all of the chimeric subunits were incorporated into F₁F₀ ATP synthase complexes, plasmids expressing either chimeric b_E39-I86 or b'_E39-I86 were capable of functionally complementing strain KM2(b). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b'_E39-D53 or b_L54-I86 subunits, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b' subunit in vitro. Coexpression of the b_E39-I86 and b'_E39-I86 subunits together in strain KM2(b) yielded F₁F₀ complexes containing heterodimeric peripheral stalks composed of both subunits.