The Membrane-Spanning Domain of Complex I Investigated with Fusion Protein Techniques

Abstract: NADH:quinone oxidoreductase, or complex I, is a large, complex and poorly understood bioenergetic enzyme in the respiratory chain of living organisms. The enzyme has a conserved core structure, comprising fourteen protein subunits: Seven subunits protrude from the membrane and contain a flavin prosthetic group and eight iron-sulfur clusters that guide electrons from the oxidation of NADH towards the quinone binding site(s). The remaining seven subunits make up the membrane domain of the enzyme complex. These proteins are lacking prosthetic groups and thus have no color or other spectral features, and their hydrophobic nature imposes a plethora of other technical obstacles. Nevertheless, it is important to learn more about this part of complex I, since it must harbor important parts of the energy coupling machinery. In this work a novel type of fusion protein was created, to facilitate studies of the membrane-spanning domain of complex I. The added fusion domain comprises a cytochrome c where an N-terminal membrane anchor helix has been removed to be replaced by a transmembrane segment in the protein to be tagged. The fusion proteins could be quantified with high accuracy using the extinction coefficient for the cytochrome c, but the red color could also be followed with the naked eye. The heme prosthetic group in cytochrome c is covalently bound to the polypeptide, and thus the fusion proteins could also be monitored under denaturing conditions such as after SDS-PAGE. In case of the three large antiporter-like protein subunits NuoL, NuoM and NuoN, unprecedented amounts of the individual proteins could be produced in Escherichia coli, suggesting that the cytochrome domain also protected the proteins from proteolysis. To further improve purification efficiency, a c-terminal his-tag was added, allowing a one step purification process for the individually expressed proteins. Since complex I is notoriously unstable, it is extremely useful to be able to readily monitor the whereabouts of both the soluble domain and the membrane-spanning domain during purification and handling. Therefore, the gene encoding the fused cytochrome c domain was introduced into the nuo-operon in the E. coli chromosome, enabling the production of cytochrome-tagged whole complex I enzyme. The only limit to the cytochrome c-tagging method is that the c-terminal end of the fusion protein must be periplasmic for heme insertion to occur. A subsequent spin-off from the previous studies was that the transmembrane topology of the small NuoA subunit had to be revised. This revision was also supported by conventional alkaline phosphatase fusion protein techniques.