2.4.4  Mitochondria and organic acid oxidation

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βOrganic acids produced in the cytosol by processes described above are further oxidised in mitochondria via the tricarboxylic acid (TCA) or Kreb’s cycle and subsequent respiratory chain. Energy released by this oxidation is used to synthesise ATP which is then exported to the cytosol for use in biosynthetic events.

(a)  Mitochondrial structure

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Figure 2.22 Transmission electron micrograph of a parenchyma cell in a floral nectary of broad bean (Vicia faba) showing an abundance of mitochondria, generally circular in proiile and varying between about 0.75 and 1.5 µm in diameter. Each mitochondrion is encapsulated by an outer and inner membrane which is in turn infolded to form cristae. Under higher magnification, ‘knobs’ are seen to protrude from this inner membrane and are now recognised as ATP synthase complexes (refer to Figure 1 in Case study 2.1). Cytoplasmic ribosomes are also apparent, many of which have been organised into polyribosomal helices. Scale bar = 0.5 µm. (Original electron micrograph courtesy Brian Gunning)

Plant mitochondria (Figure 2.22) are typically double-membrane organelles where the inner membrane is invaginated to form folds (‘cristae’). The outer membrane contains relatively few proteins and is permeable to most compounds of less than 5 kDa molecular weight, by virtue of a pore-forming protein (‘porin’). This outer membrane also contains an NADH dehydrogenase and a b-type cytochrome whose function is not understood. The inner membrane is the main permeablity barrier of the organelle and controls the movement of molecules by means of a series of carrier proteins. The inner membrane also houses the redox carriers of the respiratory chain and delineates the soluble matrix which contains the enzymes of the TCA cycle, other soluble proteins and the protein synthesising machinery.

Mitochondria are semi-autonomous organelles with their own DNA and protein synthesising machinery. However, the mitochondrial genome codes for only a small portion of the proteins which make up the mitochondrion; the rest are encoded on nuclear genes and synthesised in the cytosol. These proteins are transported into the mitochondrion and assembled with the mitochondrially synthesised proteins to form respiratory complexes. The number of mitochondria per cell varies with tissue type (from a few hundred in mature differentiated tissue to some thousands in specialised cells such as those in the infected zone of N2-fixing nodules; Millar
et al. 1995b). Understandably, more active cells such as those in meristems are generally equipped with larger numbers and consequently show faster respiration rates.

(b)  Mitochondrial substrates

Two substrates are produced from glycolytic PEP for oxidation in mitochondria: malate and pyruvate (Figure 2.21). These compounds are thought to be the most abundant mitochondrial substrates in vivo. However, amino acids may also serve as substrates for mitochondrial respiration in some tissues, particularly in seeds rich in stored protein. This oxidation may be preceded by transamination within the mitochondrion to produce a TCA cycle intermediate, or in some cases may occur directly. For example, most mitochondria contain glutamate dehydrogenase which oxidises glutamate to α-ketoglutarate and produces NADH (as well as ammonia). Some mitochondria also contain proline and glycine dehydrogenases, enzymes that feature in photorespiration and are largely confined to leaf mitochondria (Section 2.3). β-oxidation of fatty acids can occur in plant mitochondria, although this oxidation is slow compared to that in animal mitochondria (most fatty acid oxidation in plants occurs in microbodies).

(c) Carbon metabolism in mitochondria

Malate and pyruvate enter the mitochondrial matrix across the inner membrane via separate carriers. Malate is then oxidised by two enzymes: malate dehydrogenase (a separate isoenzyme from that in the cytosol), which yields OAA and NADH, and NAD-linked malic enzyme, which yields pyruvate and NADH and releases CO2 (Figure 2.23).

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Figure 2.23 Tricarboxylic acid cycle with appendages. Glycolysis in the cytoplasm yields pyruvate of malate as substrates. Malic enzyme catalyses the decarboxylation of malate to pyruvate within the mitochondria. (Original drawing courtesy David Day)

Pyruvate formed either from malate or transported directly from the cytosol is oxidised by the key enzyme pyruvate dehydrogenase to form CO2, acetyl-CoA and NADH. This enzyme, which requires coenzyme A, thiamine pyrophosphate and lipoic acid as cofactors, effectively links the TCA cycle to glycolysis. It consists of a complex of three enzymes: pyruvate dehydrogenase itself, dihydrolipoyl transacetylase and the flavoprotein dihydrolipoyl dehydrogenase. The pyruvate dehydrogenase complex has a molecular weight in the millions and is subject to sophisticated regulatory mechanisms, including phosphorylation/dephosphorylation by a kinase/phosphatase couple. Reversible phosphorylation by the kinase inactivates the enzyme and various factors regulate the kinase (e.g. pyruvate inhibits it whereas ammonia stimulates it). Pyruvate dehydrogenase is also subject to feedback inhibition from acetyl-CoA and NADH.

(d) Tricarboxylic acid (TCA) cycle

The TCA cycle proper begins with a condensation of acetyl-CoA and OAA, to form the six-carbon molecule citrate and release CoA (Figure 2.23) (a reaction catalysed by citrate synthase). Aconitase catalyses the next step, converting citrate to isocitrate. Both of these enzymes occur as isoenzymes in other cellular compartments, citrate synthase in glyoxisomes of oil seeds and aconitase in the cytosol.

NAD-linked isocitrate dehydrogenase then oxidatively decarboxylates isocitrate to form CO2 and a-ketoglutarate, and reduce NAD+. The a-ketoglutarate thus formed is oxidised further to succinyl-CoA in a reaction catalysed by the enzyme α-ketoglutarate dehydrogenase. This enzyme is a complex that has similarities to pyruvate dehydrogenase and the reaction is analogous to the formation of acetyl-CoA from pyruvate. The reaction mechanisms are also very similar but α-ketoglutarate dehydrogenase is not subject to the complicated control of pyruvate dehydrogenase. Succinyl-CoA synthase then catalyses the conversion of succinyl-CoA to succinate, with the concomitant phosphorylation of ADP to ATP, the only substrate-level phosphorylation step in the mitochondrion. This enzyme in plants differs from its mammalian counterpart in that it is specific for ADP rather than GDP.

Succinate dehydrogenase (SDH), which catalyses the oxidation of succinate to fumarate, is the only membrane-bound enzyme of the TCA cycle and is part of the respiratory electron transport chain (complex II, Figure 2.24). SDH is a complex consisting of a flavoprotein and several other subunits; the former has FAD as a covalently bound cofactor and the enzyme also contains two bi-nuclear iron–sulphur clusters. Electrons from FADH2 are passed on to ubiquinone.

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Figure 2.24 Electron transport chain of plant mitochondria. Roman numerals indicate respiratory complexes equivalent to mammalian counterparts. Complex I, NADH-UQ oxido-reductase; complex II, succinate dehydrogenase; complex III, Cyt b/c1 complex; complex IV, Cyt c oxidase; complexV, ATP synthetase. Arabic numerals indicate special plant features: 1, external NADPH dehydrogenase; 2, internal, rotenone-insensitive NADH dehydrogenase; 3, alternative (cyanide-insensitive) oxidase; UQ, ubiquinone. Unbroken arrows indicate pathways of electron flow; broken arrows indicate proton translocation sites. (Original drawing courtesy David Day)

Fumarase catalyses the conversion of fumarate to malate and is unique to the mitochondrion, making it a convenient marker for the mitochondrial matrix. Malate dehydrogenase catalyses the final step of the TCA cycle, oxidising malate to OAA and producing NADH. The reaction is freely reversible, although the equilibrium constant strongly favours the reduction of OAA, necessitating rapid turnover of OAA and NADH to maintain this reaction in a forward direction.

Overall, during one turn of the cycle, three carbons of pyruvate are released as CO2, one molecule of ATP is formed directly, and four NADH and one FADH2 are produced. The latter strong reductants are oxidised in the respiratory chain to reduce O2 and produce ATP. Although most of the TCA cycle enzymes in plant mitochondria are NAD linked, NADP-dependent isoforms of isocitrate and malate dehydrogenases also exist, and these may play a role in a protective reductive cycle in the matrix.

Regulation of carbon flux through the TCA cycle probably occurs via phosphorylation/dephosphorylation of pyruvate dehydrogenase, which will depend in turn on mitochondrial energy status and feedback inhibition of various enzymes by NADH and acetyl-CoA. The rate of cycle turnover thus depends on the rate of electron flow through the respiratory chain (to reoxidise NADH) and utilisation of ATP. TCA cycle turnover will also depend on the rate of substrate provision by reactions in chloroplasts and cytosol, and in pea leaves this may be a major limitation on the rate of respiration in vivo. For example, in an experiment to estimate respiratory chain capacity, Wiskich and Dry (1985) isolated mitochondria from pea leaves and resuspended them in a known volume of reaction medium. A large proportion of organelles is usually ruptured during isolation, and it is important to estimate yield of intact mitochondria. Therefore the volume of the leaf homogenate obtained upon disruption of the leaves was also measured, and a small aliquot set aside. A mitochondrial marker for enzyme activity (e.g. fumarase) was measured for both isolated mitochondria preparation and crude homogenate. The percentage yield of intact mitochondria was inferred from their comparative activities. Respiration rate by isolated mitochondria was then measured in an oxygen electrode with a mixture of substrates. That value was extrapolated to a mitochondrial capacity of a whole leaf by correcting for the yield of intact mitochondria. In a parallel experiment, the in vivo rate of respiration by intact leaf tissue was measured in a second oxygen electrode. Comparative values were as follows:

Rate of respiration by mitochondrial suspension: 220 µmol O2 h–1
Per cent yield of mitochondria: 50
Fresh mass of leaf tissue: 10 g
Respiratory capacity of tissue: 44 µmol O2 h–1 g–1
Respiratory rate of intact leaf tissue: 23 µmol O2 h–1 g–1

Respiratory capacity as inferred from the activity of isolated mitochondria exceeded actual measured rates of intact leaf tissue. Respiration in vivo is therefore constrained, and such restriction might be due to either substrate supply and/or the ATP/ADP ratio.

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