The evolution of respiratory chain complex I from smaller functional modules

Abstract: NADH:quinone oxidoreductase, or complex I, is a large membrane bound enzyme in the respiratory chain of living organisms, that has evolved from the combination of smaller functional building blocks. The enzyme has a conserved core structure, comprising 14 protein subunits: Seven subunits protrude from the membrane and contain a flavin and eight iron-sulfur clusters. The remaining seven subunits in the membrane domain are lacking prosthetic groups and thus have no color or other spectral features. 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 cytochrome c fusion protein was created, to facilitate studies of these subunits. The fusion proteins remained functional, and could be quantified spectroscopically or detected using anti-cyt c antibodies. Using whole genome sequences, the distribution of different types of complex I in the tree of life was surveyed. A compact, 11-subunit complex I was found both in the archeal and the eubacterial kingdoms, whereas the full size complex I was only found in some eubacterial phyla. Bacillus subtilis strains genetically deleted for their antiporter subunits MrpA or MrpD were used to study the function of the homologous ion transporter proteins in complex I and its evolutionary progenitors. By comparing the complementation capacity of the Na+ and pH sensitive growth phenotypes, the function of the individual subunits could be deduced. It was concluded that MrpA and MrpD are single ion transporters that exhibit antiporter activity when working together. NuoN could only rescue the ΔMrpD strain whereas NuoL primarily improved the growth of the ΔMrpA strain. Thus, complex I most likely contain one Na+ transporter and two H+ transporters. The corresponding subunits from an 11-subunit complex I showed essentially the same ion specificity as the full size complex I, whereas the hydrogenase transporter subunit corroborated the theoretical prediction that these proteins are degenerate, less specific forms.