On the antiporter-like subunits of respiratory chain Complex I - implications for the evolution and coupling mechanism of the NADH:quinone oxidoreductase enzyme complex

Detta är en avhandling från Cecilie Mathiesen, Department of Biochemistry, Lund University

Sammanfattning: Complex I or NADH:quinone oxidoreductase is the largest enzyme complex, but the least understood energy coupling site in the respiratory chain of mitochondria and bacteria. No high-resolution structural information exists for this enzyme and the molecular mechanism that couples electron transfer and proton pumping is not understood. In mammals, about 50% of the mitochondrial DNA is encoding protein subunits of Complex I, and thus there is a strong correlation between defect Complex I and various degenerative diseases. Learning more about Complex I is thus important both for basic science and for medicine. Complex I contains three large membrane-spanning subunits, NuoL, NuoM and NuoN, that are homologous to one particular class of Na+/H+ antiporters and therefore are likely to harbor important components of the proton translocation machinery. MrpA and MrpD belong to this class of Na+/H+ antiporters and are found in a gene cluster containing seven genes, mrpA-G. In this work the transmembrane topology of the antiporter-like subunits was determined and their phylogenetic relationship investigated. In the analysis MrpA and MrpD formed distinct branches where NuoL grouped with MrpA and NuoM/N with MrpD. The result suggest that there are functional differences between the MrpA/NuoL and MrpD/NuoM proteins. The remaining proteins encoded by the mrp gene cluster were analyzed by PSI-BLAST, a bioinformatical search tool that more efficiently detect distant homologies. MrpC was found to be a NuoK homologue, and thus we conclude that NuoK, NuoL and NuoM where recruited together to Complex I, from the antiporter module MrpC, MrpA and MrpD. A functional difference between MrpA and MrpD was confirmed using Bacillus subtilis mrpA and mrpD deletion strains that were more salt and pH sensitive than wild type cells, but to a different extent. Recent work by Julia Steubers group at ETH Zürich showed that Complex I from Escherichia coli could translocate Na+, but this has hitherto not been regarded as a general property of proton pumping Complex I. By expressing the Complex I subunits NuoL and NuoM from the alpha-proteobacteria Rhodobacter capsulatus in the B. subtilis deletion strains we have demonstrated that these subunits are capable of Na+ translocation in vivo. Since R. capsulatus Complex I is more closely related to mitochondrial Complex I than to the E. coli enzyme, it is very likely that all Complex I enzymes have the ability to translocate Na+ as well as H+.

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