Energy Metabolism in Anaerobically Functioning Mitochondria

Organisms with anaerobic mitochondria can be divided into two different types: those which perform anaerobic respiration and use an alternative electron acceptor present in the environment, such as nitrate or nitrite, and those which perform fermentation reactions using an endogenously produced, organic electron acceptor, such as fumarate (Martin et al. 2001; Tielens et al. 2002). An example of the first type is the nitrate respiration that occurs in several ciliates (Finlay et al. 1983), and fungi (Kobayashi et al. 1996; Takaya et al. 2003), which use nitrate and/or nitrite as the terminal electron acceptor of their mitochondrial electron-transport chain, producing nitrous oxide as the reduced end product. These specific electron-transport chains contain special terminal oxidoreductases that donate electrons to an acceptor present in the environment, in this case nitrite or nitrate instead of oxygen. Little is known of these mitochondria and they will not be discussed further in this chapter.

The other class of anaerobic mitochondria, those which use fermentation reactions, is present in organisms that are highly adapted for either prolonged survival or continuous functioning in the absence of oxygen, such as parasitic helminths, or in organisms that are adapted to alternating periods in the presence and absence of oxygen, such as mussels, oysters and lugworms, which are intermittently dependent on this process when the tides of the sea force them to function anaerobically (De Zwaan 1991; Grieshaber et al. 1994). The energy metabolism in these anaerobic mitochondria differs principally and significantly from that in aerobic mitochondria, as no external final electron acceptors are used. Therefore, this mitochondrial metabolism has to be truly fermentative, in other words the number of NADH-producing reactions has to equal the number of NADH-consuming reactions without the use of oxygen or other external electron acceptors. Many mitochondrial catabolic pathways produce NADH, and anaerobically functioning mitochondria are adapted in that they also comprise catabolic pathways that consume NADH, processes that can be used as an electron sink. So far, in anaerobic mitochondria two distinct processes have been detected that are used as an electron sink to reoxidize the NADH produced by the oxidative catabolic pathways in these mitochondria. The reduction of fumarate to succinate during the fermentative malate dismutation pathway is the most commonly used electron sink in anaerobic mitochondria. Some organisms, however, do not only use this reduction of fumarate, but also use distinct reactions involved in lipid biosynthesis as an electron-sink during anoxic conditions (see later).

Malate dismutation is a fermentation pathway, which involves the use of an especially adapted electron-transport chain and the reduction of endoge-nously produced fumarate as an electron sink (Tielens and Van Hellemond 1998; Fig. 5.2). In organisms that are adapted to anoxic functioning using malate dismutation, carbohydrates are degraded by the usual glycolytic pathway to PEP, which is then converted to malate (as described before). This malate, produced in the cytosol, is transported into the mitochondria for further degradation (Fig. 5.2). In a split pathway, one portion of this malate is oxidized via pyruvate and acetyl-CoA to acetate and another portion is reduced to succinate, which is often further metabolized to propionate (Tielens 1994). Although several variations of malate dismutation with various end products occur, the use of succinate production from fumarate as an electron sink is universal. The reduction of malate to succinate occurs in two reactions that reverse part of the Krebs cycle, and the reduction of fumarate is the essential NADH-consuming reaction used to maintain redox balance. Fumarate reduction is linked to electron transport via electron-transferring

MALATE

PYRUVATE

MALATE

PYRUVATE

AcCoA

Acetate

ASCT

|Acyl-CoA|

ECRJy

PYR-

AcCoA

Acetate

ASCT

OXAC

NADH NAD*

CITR

FUM SUCC

OXAC

NADH NAD*

CITR

H+

Fig. 5.2. Possible metabolic pathways in facultative anaerobic mitochondria. Shaded boxes show components of the electron-transport chain used during hypoxia, open boxes are components used during aerobiosis, and the hatched boxes (complex I and ATP-synthase) are components used under aerobic as well as anaerobic conditions. ASCT acetate:succinate CoA-transferase, C cytochrome c, CI, CIII and CIV complexes I, III and IV of the respiratory chain, CITR citrate, ECR enoyl-CoA reductase (such as present in Ascaris suum), ETF electron-transfer flavoprotein, ETF:RQ OR electron-transfer flavoprotein:rhodoquinone oxidoreductase, FRD fumarate reductase, FUM fumarate, MAL malate, OXAC oxaloacetate, PYR pyruvate, RQ rhodoquinone, SDH succinate dehydrogenase, SUCC succinate, Succ-CoA succinyl-CoA, TER trans-2-enoyl-CoA reductase (such as present in E. gracilis), UQ ubiquinone f FUM

Fig. 5.2. Possible metabolic pathways in facultative anaerobic mitochondria. Shaded boxes show components of the electron-transport chain used during hypoxia, open boxes are components used during aerobiosis, and the hatched boxes (complex I and ATP-synthase) are components used under aerobic as well as anaerobic conditions. ASCT acetate:succinate CoA-transferase, C cytochrome c, CI, CIII and CIV complexes I, III and IV of the respiratory chain, CITR citrate, ECR enoyl-CoA reductase (such as present in Ascaris suum), ETF electron-transfer flavoprotein, ETF:RQ OR electron-transfer flavoprotein:rhodoquinone oxidoreductase, FRD fumarate reductase, FUM fumarate, MAL malate, OXAC oxaloacetate, PYR pyruvate, RQ rhodoquinone, SDH succinate dehydrogenase, SUCC succinate, Succ-CoA succinyl-CoA, TER trans-2-enoyl-CoA reductase (such as present in E. gracilis), UQ ubiquinone enzyme complexes in an anaerobically functioning electron-transport chain (Fig. 5.2). Characteristic components of these anaerobically functioning mitochondria are fumarate reductase (FRD), the enzyme catalysing the reduction of fumarate to succinate, and rhodoquinone (RQ), the quinone shuttling the electrons from NADH dehydrogenase (complex I) towards FRD for the reduction of fumarate.

Next to fumarate reduction, some organisms use specific reactions in lipid biosynthesis as an electron sink to maintain redox balance in anaerobically functioning mitochondria. In anaerobic mitochondria two variants are known: the production of branched-chain fatty acids and the production of wax esters. The parasitic nematode Ascaris suum reduces fumarate in its anaerobic mitochondria, but instead of only producing acetate and succinate or propionate, like most other parasitic helminths, this organism also use the intermediates acetyl-CoA and propionyl-CoA to form branched-chain fatty acids (Komuniecki et al. 1989). This pathway is similar to reversal of P-oxidation and a complex mixture of the end products acetate, propionate, succinate and branched-chain fatty acids is excreted. In this pathway, the condensation of an acetyl-CoA and a propionyl-CoA, or of two propionyl-CoA molecules, ultimately results in the formation of the excreted end products 2-methylbutanoate and 2-methylpentanoate, respectively (Figs. 5.2, 5.3). After the condensation, the resulting intermediates are first reduced by NADH and are then hydrated in reactions comparable to those occurring in mammalian mitochondrial P-oxidation. In the final reaction of the pathway, the NADH-dependent reduction of 2-methyl branched-chain enoyl-CoAs, both membrane-bound and soluble components are involved. Complex I, rhodoquinone and electron-transport flavoprotein reductase comprise the membrane-bound components (Fig. 5.2, 5.3). The soluble components consist of two flavoproteins: electron-transport flavoprotein reductase and

Flavoprotein Oxidation

Fig. 5.3. The major components involved in mitochondrial NADH oxidation in facultative anaerobic mitochondria. In anaerobically functioning mitochondria, NADH is oxidized either by soluble enzymes (left) or by membrane-bound complexes of the electron-transport chain (middle). Under aerobic conditions, a classic respiratory chain is used to oxidize NADH (right). Proton translocation is indicated by H+ with arrows. Ovals represent the electron transporters RQ, UQ and cytochrome c (cyt. c), and electron transport is indicated by dashed arrows. The vertical bar represents a scale for the standard redox potentials in millivolts. Fum fumarate, NADH-DH NADH dehydrogenase, NADH-ECR soluble NADH:enoyl-CoA reductase, RQH2 rhodoquinol, Succ succinate, UQH2 ubiquinol

Fig. 5.3. The major components involved in mitochondrial NADH oxidation in facultative anaerobic mitochondria. In anaerobically functioning mitochondria, NADH is oxidized either by soluble enzymes (left) or by membrane-bound complexes of the electron-transport chain (middle). Under aerobic conditions, a classic respiratory chain is used to oxidize NADH (right). Proton translocation is indicated by H+ with arrows. Ovals represent the electron transporters RQ, UQ and cytochrome c (cyt. c), and electron transport is indicated by dashed arrows. The vertical bar represents a scale for the standard redox potentials in millivolts. Fum fumarate, NADH-DH NADH dehydrogenase, NADH-ECR soluble NADH:enoyl-CoA reductase, RQH2 rhodoquinol, Succ succinate, UQH2 ubiquinol

2-methyl branched-chain enoyl-CoA reductase (Komuniecki and Harris 1995; Fig. 5.2). This branched-chain fatty acid formation provides an additional pathway for the oxidation of NADH, and could provide increased flexibility in regulating intramitochondrial NADH to NAD+ ratios under the reducing conditions in the gut of the host, by serving as an important sink for excess reducing power (Komuniecki and Harris 1995).

The photosynthetic flagellate Euglena gracilis utilizes another variant of mitochondrial lipid biosynthesis as alternative electron sink to survive hypoxia. This adaptation enables the mitochondrion of this organism to produce ATP also in the absence of oxygen. Under aerobic conditions, Euglena performs a more or less typical oxidative phosphorylation in association with a modified Krebs cycle and respiratory chain. Pyruvate from glycolysis enters the mitochondrion and undergoes oxidative decarboxylation; the resulting acetyl-CoA enters a modified Krebs cycle with a succinate-semialdehyde shunt, circumventing the step catalysed by a-ketoglutarate dehydrogenase. Electrons from glucose breakdown are transferred to oxygen as the terminal electron acceptor, and oxidative phosphorylation generates most of the ATP. During hypoxia, mitochondrial fatty acid synthesis serves in Euglena cells as an electron sink when wax esters are formed from its reserve glucose polymer, paramylon (Inui et al. 1984). Waxes are esters of long-chain saturated or unsaturated fatty acids with long-chain alcohols. In the single mitochondrion of Euglena cells, a malonyl-CoA-independent fatty acid synthetic pathway exists, which has the capability to synthesize fatty acids directly from acetyl-CoA as both primer and C2 donor, using NADH as an electron donor. These fatty acids are synthesized by reversal of P-oxidation in that it proceeds via CoA intermediates instead of via acyl carrier protein (ACP). However, a key difference with P-oxidation is that enoyl-CoA reductase functions instead of acyl-CoA dehydrogenase to reduce the double bond. A portion of the fatty acids produced are reduced to alcohols, esterified with another fatty acid and the wax esters formed are deposited in the cytosol. Upon return to aerobio-sis, these wax esters are degraded rapidly and paramylon is resynthesized then (Inui et al. 1982). This mitochondrial system for fatty acid synthesis produces fatty acids with chain lengths of eight to 16 carbons, with a majority of C14. Synthesis of odd-numbered fatty acids also occurs and starts from propionyl-CoA. Formation of the propionate necessary for this pathway functions in itself as an extra electron sink during hypoxia as this propionate is most likely produced via fumarate reduction in the same pathway that occurs also in the anaerobically functioning mitochondria of many parasitic helminths (Fig. 5.2). The acetyl-CoA used in wax ester formation in Euglena cells stems from pyruvate via an unusual oxygen-sensitive enzyme pyruvate:NADP+ oxidoreductase (PNO) (Inui et al. 1987). The core catalytic component of this PNO is PFO, an enzyme also present in amitochondriate protists and in the hydrogenosomes of trichomonads. PNO exists in Euglena in the mitochondrion alongside a classical mitochondrial PDH, with messenger RNA expression patterns converse to that of PNO in response to hypoxia (Hoffmeister et al. 2004).

The two variants of the use of mitochondrial lipid biosynthesis as an electron sink in the absence of oxygen, as described before, have a very comparable crucial reaction in common. In the production of the branched-chain fatty acids in Ascaris mitochondria as well as in the malonyl-CoA-independent fatty acid biosynthesis in Euglena mitochondria, the final reaction is catalysed by a 2-methyl branched-chain enoyl-CoA reductase. However, the Ascaris enzyme is clearly distinct from the Euglena one (Hoffmeister et al. 2005). As described before, the Ascaris enzyme accepts electrons from NADH via complex I, rhodoquinone and an electron-transporting flavoprotein (Komuniecki and Harris 1995; Figs. 5.2, 5.3), whereas it was recently shown that the Euglena enzyme accepts electrons from NADH directly (Hoffmeister et al. 2005). The gene of the Ascaris enzyme shows sequence similarities to mitochondrial acyl-CoA dehydrogenases found in mitochondria of most eukaryotes (Duran et al. 1993), but for the gene of the Euglena enzyme, no homologues were found among other eukaryotes (Hoffmeister et al. 2005).

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