Substrate-induced Conformational Change in Bacterial Complex I

Abstract

The mechanism coupling electron transfer and proton pumping in respiratory complex I (NADH-ubiquinone oxidoreductase) has not been established, but it has been suggested that it involves conformational changes. Here, the influence of substrates on the conformation of purified complex I from Escherichia coli was studied by cross-linking and electron microscopy. When a zerolength cross-linking reagent was used, the presence of NAD(P)H, in contrast to that of NAD ؉ , prevented the formation of cross-links between the hydrophilic subunits of the complex, including NuoB, NuoI, and NuoCD. Comparisons using different cross-linkers suggested that NuoB, which is likely to coordinate the key ironsulfur cluster N2, is the most mobile subunit. The presence of NAD(P)H led also to enhanced proteolysis of subunit NuoG. These data indicate that upon NAD(P)H binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits. Single particle analysis showed the nature of this conformational change. The enzyme retains its L-shape in the presence of NADH, but exhibits a significantly more open or expanded structure both in the peripheral arm and, unexpectedly, in the membrane domain also.

Evolution of Respiratory Complex I

“…3C. The overall L-shape of the molecule is consistent with previous EM reconstructions (34,40,42), allowing us to assign the horizontal, strongly stain-excluding arm as the hydrophobic membrane domain. This domain is about 200 Å long and 60 Å thick, consistent with previous reconstructions and the recent crystal structure of the T. thermophilus enzyme (15).…”

“…3B). This distribution is similar to that observed with the E. coli (40) and bovine enzymes (41) and likely reflects the asymmetrical nature of the molecule, so that it is absorbed to the carbon layer in a preferred orientation. Because all flip classes were very similar, only two classes (flip and flop) were used for the final analysis, and the best class average (flip), representing about 70% of total particles, is shown in Fig.…”

“…Nevertheless, this was sufficient for the reconstruction and provided a clear outline of the molecule. Classification of images into four classes and averaging in IMAGIC (31) revealed three very similar classes in one orientation on the grid (flip) (40) and one class in the opposite orientation (flop) (Fig. 3B).…”

Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

“…This is supported by the homology of the three major subunits of the arm to subunits of cation/proton antiporters (16,17). Mechanisms based on a conformational link between the redox reaction and the proton translocation have been discussed (4,5,18,19). The recently published crystal structures of complex I from Thermus thermophilus (20) and Yarrowia lipolytica (21) imply that the complex contains a coupling site directly linked to electron transfer as well as another site indirectly coupled with electron transfer.…”

The role of proton and sodium ions in energy transduction by respiratory complex I

“…This type of mechanism is supported by the structural data, conformational changes monitored upon NADH binding and/or quinone reduction (35,36), and the observation that the catalytic and transport activities can be decoupled (6).…”