Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX.

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°' ∼-450 mV) using NADH (E°' -320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°' -80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

Electron microscopy and image analysis of the complexes I and V of the mitochondrial respiratory chain.

The results of Section IV can be summarized in a simple ATP synthase model. This model implies that either the alpha or the beta subunits must be closer to the membrane. The work of Gao and Bauerlein (1987) indicates that the alpha subunits are closer to the membrane. Although the overall structure is more or less clear, important questions need to be clarified. First, the number and the arrangement of the subunits in the F0 part must be known. Second, the exact shape of F1, and particularly the shape of the large subunits needs to be elucidated. On the basis of fluorescence resonance energy transfer measurements by McCarty and Hammes (1987), a model was presented showing large oblong subunits. Such 'banana-shaped' subunits, which are also presented in the many phantasy models (e.g. Walker et al., 1982), are very unlikely in view of the electron microscopical results, although the large subunits do not need to be exactly spherical. The third and most interesting central question is on the changes in the structure that take place during the different steps in the synthesis of ATP. It can now be taken as proven that the energy transmitted to the ATP synthase is used to induce a conformational change in the latter enzyme, in such a way as to bring about the energy-requiring dissociation of already synthesized ATP (Penefsky, 1985 and reviewed in Slater, 1987). But the way in which the three parts of the ATP synthase are involved is completely unknown. It is rather puzzling that such a long distance exists between the catalytic sites, which are on the interface of the alpha and beta subunits and the F0 part where the proton movements occur, which, according to Mitchell's theory (1961), is the driving force for the synthesis of ATP. Perhaps alternative mechanisms such as the collision hypothesis formulated by Herweijer et al. (1985) are more realistic in describing the mechanism of ATP synthesis. It would bring the complexes I and V close together, not only in the artificial way treated in this paper, but in a useful way for energy conversion.