Quinones in aerobic and anaerobic mitochondria

Abstract

Ubiquinone (UQ), also known as coenzyme Q, is a ubiquitous quinone and is known to have several functions. One of these functions is electron carrier in the mitochondrial electron transport chain of aerobically functioning bacteria and eukaryotes. In contrast to this aerobically functioning quinone, rhodoquinone (RQ) is a quinone functioning in electron transport in organisms possessing a (facultative) anaerobic energy metabolism, which is called malate dismutation. The study described in this thesis focuses on the biosynthetic route of RQ, and on the regulation of RQ biosynthesis. Knowledge about RQ biosynthesis can contribute to the development of antiparasitic drugs. RQ biosynthesis is an excellent target for antiparasitic drugs, as the parasitic stages live in anaerobic environments and depend on RQ. RQ is produced endogenously by parasites, while it is absent in their hosts. In addition, knowledge about RQ biosynthesis can provide more information about the evolutionary origin of facultative anaerobically functioning mitochondria. An overview is given of the biochemical and evolutionary aspects of anaerobically functioning mitochondria. Based on the recent functional and phylogenetic studies, it is concluded that true anaerobically functioning mitochondria, such as found in for instance parasitic helminths and some lower marine organisms, most likely did not originate directly from the pluripotent ancestral mitochondrion, but arose later in evolution from the aerobic type of mitochondria after these were already adapted to an aerobic way of life by losing their anaerobic capacities. A study was performed on the mitochondrial proteome and on the quinone content in aerobically and anaerobically grown Euglena gracilis. Quinones were determined using a novel Liquid Chromatography tandem Mass Spectroscopy Method. The RQ:UQ ratio in E. gracilis was increased in anaerobically grown E. gracilis, compared to aerobically grown E. gracilis. The increase of RQ is consistent with the synthesis of odd-numbered wax esters under anaerobic circumstances. Analysis of the mitochondrial proteome of E. gracilis showed several differences between the aerobically and anaerobically grown E. gracilis, as four proteins were missing in the anaerobically grown E. gracilis. The missing proteins are all involved in a typical aerobic metabolism. These results indicate that E. gracilis is prepared for anaerobic functioning before it is encountered. [1’-14C]Ubiquinone-2, [1’-14C]5-demethoxy-5-hydroxy-ubiquinone-2 and [1’-14C]5-demethoxy-ubiquinone-2 were synthesized from [1-14C]acetic acid. A common feature of these benzoquinones is the isoprenoid chain and therefore the 14C-label has been incorporated in this isoprenoid. The products were characterized using mass spectroscopy, 1H-NMR and 13C-NMR. The biosynthesis route of rhodoquinone was studied in Caenorhabditis elegans using RNA-interference and gene-knockout mutants. It was demonstrated that the UQ-biosynthetic-enzymes COQ-6, and most likely COQ-5 as well, are part of the biosynthetic pathway of RQ in C. elegans. This is the first biochemical evidence that UQ and RQ share a large part of their biosynthetic pathway. 5-Demethoxy-1,4-ubiquinone is identified as the last common precursor for both UQ and RQ. A novel bio-informatic approach was used to identify genes involved in the biosynthesis of RQ and not in the biosynthesis of UQ. So far, this strategy did not result in the identification of genes specifically involved in RQ biosynthesis, but it can be successful in future when the genome sequence of more parasitic helminths will become available.