The F1F0 ATP synthase is the enzymatic complex which interconverts the electrochemical energy of the transmembrane proton gradient, generated by oxidative phosphorylation of metabolic substrates, into the energy currency of ATP. In doing so, it reversibly couples transmembrane proton flow to the catalysis of the high energy terminal phosphate bond of ATP. Even the simplest example of the enzyme, the bacterial F1F0, is a complex assembly of eight subunits in non-unit stoichiometry, five making up the catalytic F1 portion (α3β3γδε) and three the F0, the proton channel (a b2c10‒12) We are attempting to understand the role of subunit interactions in the coupling phenomenon and the catalytic mechanism, which exhibits an extraordinarily high degree of allosteric cooperativity.Three-dimensional reconstruction of negatively stained arrays of the F1 has shown it to be a hexagonal barrel-shaped structure; cryoelectron microscopy of unstained, frozen-hydrated specimens (figure 1a) has revealed an interior cavity partially occluded by an internal structure.Immunolabelling with monoclonal Fab fragments to the α subunit, followed by alignment and averaging of cryoelectron images, has demonstrated the alternating αβ arrangement of subunits around the periphery of the F1. The internal structure has also been localized next to one of the (catalytic) β subunits (see figure 2a,b). Addition of Fab fragments to the carboxy-terminal part of the (single-copy) γ subunit preserves the characteristic triangular appearance and uniform orientation of the Fl-Fab complexes (figure lb).Averages of these images place the additional density of the label at the periphery of the structure, superimposed on a β subunit (figure 2c). Surprisingly, similar results are obtained with readily-binding monoclonal antibodies to each of the single-copy subunits.
Crystallographic insight into the catalytic mechanism of subunit A of the A-ATP synthase and the P-loop switch in evolution
