Functional Incorporation of Chimeric b Subunits into F 1 F o ATP Synthase

Abstract

ABSTRACT F 1 F o ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F 1 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 in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Δ b ). Although not all of the chimeric subunits were incorporated into F 1 F o ATP synthase complexes, plasmids expressing either chimeric b E39-I86 or b ′ E39-I86 were capable of functionally complementing strain KM2 (Δ 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 subunit, 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 in strain KM2 (Δ b ) yielded F 1 F o complexes containing heterodimeric peripheral stalks composed of both subunits.