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Chapter 5— Plant Mitochondria
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Chapter 5—
Plant Mitochondria


Mitochondria are the sites of non-photosynthetic energy transduction in eukaryotic cells which, carry out aerobic metabolism. Energy transduction includes those processes by which the chemical potential energy of organic substrates is transformed into a readily mobilized form, adenosine-5'-triphosphate (ATP). Organic substrates are oxidized and the free energy of oxidation is conserved by processes which are common to all mitochondria, regardless of source. Thus, with regard to the oxidative and phosphorylative processes, information obtained from studies in animal mitochondria is applicable to plant mitochondria.

Notable differences between plant mitochondria and animal mitochondria do occur, although these differences do not contradict the basic similarities in the mechanism of energy transduction. For example, plant mitochondria possess external reduced nicotinamide adenine dinucleotide (NADH) dehydrogenases which oxidize exogenous NADH; mitochondria from animal sources lack this capability. Mitochondria from many plant sources are relatively insensitive to cyanide inhibition, a feature not found in animal mitochondria. On the other hand, the b -oxidation pathway of fatty acids is located in animal mitochondria, whereas in plants, the enzymes of fatty acid oxidation occur in the glyoxysomes.

In this chapter, the morphology and function of plant mitochondria are discussed. In almost all cases, information is drawn from studies with mitochondria from higher plants. Emphasis is placed on the components of the plant mitochondria respiratory chain and their interactions with each other. Current ideas on oxidative phosphorylation are discussed with reference to knowledge gained from studies with animal and yeast mitochondria. Reversed electron flow and ion transport activities are considered with reference to studies in plant mitochondria. Structure and function relationships are sought, but in many instances, sufficient evidence is not available or available only from studies with mammalian or avian systems; it seems unwarranted, however, to draw exact parallels between animal and plant systems.


Morphology in Situ

Mitochondria in living cells are highly pleomorphic, as shown by phase contrast microcinematography by Hongladarom et al., (1965). Pleomorphism is reflected


also in thin section electron microscopy, in which mitochondria appear as roughly circular profiles as well as highly elongated or irregular cross sections (Fig. 5.1a). The circular sections may represent transverse or oblique sections through an otherwise elongated organelle. The diameter of the elongated mitochondrion appears to be about 0.4 to 0.5 µm, while the length may be several micrometers long. Although rods or apparent spheres are the most common profiles seen, sections derived from branched or cup shaped organelles have also been discerned (Bagshaw et al., 1964). The recent analysis of serial sections of yeast cells by Hoffman and Avers (1973), which showed that yeast contains a single, giant, branched mitochondrion, suggests that the irregular cross sections of mitochondria of other cells might also be sections of a single branched organelle.

The mitochondrion consists of a double membrane system with an inner convoluted membrane enclosing the matrix, and surrounded by a smooth outer membrane (Fig. 5.1a, 5.1b). High resolution electron micrographs of material

Figure 5.1a
Mitochondria in phloem parenchyma cells of a maize leaf. Magnification bar = 1 m m.
(Micrograph courtesy of O. E. Bradfute and Diane C. Robertson.)

fixed with glutaraldehyde and post-fixed with osmium tetroxide show the tripartite nature of both the inner and outer membranes. Each membrane has a thickness of approximately 9 nm (Baker et al., 1968).


Figure 5.1b
Isolated mitochondria from mung bean hypocotyls. Mitochdria have been
suspended in 0.3 M  mannitol prior to fixation. Magnification × 26,000.
(Micrograph courtesy of W. D. Bonner, Jr.)

Morphology of Isolated Mitochondria

Electron micrographs of isolated mitochomdria show circular cross sections, presumably reflecting a spherical shape when released from their cellular environment. The electron micrographs of the intact isolated mitochondrion show clearly the two membrane systems, as well as the tripartite organization of each membrane. The fine structure of isolated mitochondria is highly dependent upon the osmolarity of the suspending medium (Baker et al., 1968). When mitochondria are suspended in 0.3 to 0.4 M sucrose or mannitol, the matrix appears contracted and electron dense (Fig. 5.1b), but when suspended in 0.2 M sucrose, the matrix appears more expnaded and less electron dense, and resembles that of mitochondria seen in sity . The dense matrix of mitochondria suspended in 0.3 to 0.4 M sucrose or mannitol is due to the hypertonicity of the suspending medium. Since the inner membrane is generally regarded as the osmotic barrier, the dense nature of the matrix reflects a water loss, which is reversible when the organelles are suspended in 0.2 M sucrose.

Negatively-stained water-lysed mitochondria show that the inner membranes have the characteristic stalked particles similar to those reported for mammalian mitochondrial membranes (Fernandez-Moran, 1962; Parsons et al., 1965). The particles have a headpiece with a diameter of 10 nm, attached to a stalk 3.5 to 4.5 nm wide and 4.5 nm long (Fig. 5.2). These resemble the particles identified with ATPase function in heart mitochondria (Racker et al., 1969).


Figure 5.2
Portion of a surface spread and negatively stained summer squash
mitochondrion. The large areas of membrane (IM) are presumed to be
part of the inner membrane forming the shell of the mitochondrion.
The cristae (C) appear as smaller pieces of membrane of rounded shape
connected together by narrower (possibly tubular) pieces. The membranes
are coated with projecting knob-like subunits which are best seen lying in
the plane of the object at the edge of the pieces of membrane (arrow). The
dimension of the head of the subunit is 10 nm and the stem is 3.5–4.0 nm wide
and 4.5 nm long. (Parsons  et al.,  1965). (Reproduced by permission of the
National Research Council of Canada from the Canadian
Journal of Botany, Volume 43, 1965. pp. 647–55.)


Isolation and Purification

Techniques of Isolation and Purification

Mitochondria have been obtained from a large number of plant sources including roots, storage tissue, stems and photosynthetic tissues. The usual problems of isolation, regardless of the source, are (a) the rupture of a rather rigid cell wall and (b) the prevention of damage to organelles through the release of intracellular, particularly vacuolar, contents. Ikuma (1970) listed a number of conditions for successful isolation of tightly-coupled mitochondria. These include (a) gentle tissue disruption, (b) rigorous exclusion of contaminating particles and (c) the use of a buffered grinding medium isotonic with mitochondria and containing a variety of protective reagents. Most investigators employ some device to reduce quickly the tissue to a coarse slurry, which is passed through a cloth filter to remove large debris. The fraction which sediments between 1,000 g and 10,000 g is collected as the mitochondrial fraction. This fraction will oxidize all the intermediates of the Krebs tricarboxylic acid cycle, exhibit respiratory control and yield ADP to O ratios approaching the theoretical value for the substrate used. The mitochondrial fraction can be further purified by density gradient centrifugation. This may be done in discontinuous sucrose gradients (Baker et al., 1968; Douce et al., 1972a) or Dextran-40 gradients (Solomos et al., 1973). Mitochondria form a band at the interface between 1.2 and 1.5 M sucrose (Douce et al., 1972a). This is recovered and diluted slowly to 0.3 M sucrose. This procedure yields mitochondria with intact outer and inner membranes as shown by electron microscopy. The integrity of the outer membrane is also shown enzymatically by the inability to reduce exogenous cytochrome c with NADH or succinate as substrates, unless the mitochondria have been subjected to mild osmotic shock which renders the outer membrane permeable to high molecular weight solutes.

During the disruption of cells and throughout the isolation procedures, a number of protective reagents must be present. Inclusion of sodium ethylenediamine-tetracetate (EDTA) in the isolation medium has been shown to give mitochondria with high respiration rates (Lieberman & Biale, 1955). EDTA probably removes cationic inhibitors although the specific cation complexed is unknown.

Many plant cells release phenolic compounds when ruptured. These phenolic compounds are oxidized in the presence of air and form polymers which are inhibitory to mitochondrial respiration and coupled phosphorylation. To prevent the damaging effects of phenolic compounds, a variety of reagents have been used successfully. Polyvinyl pyrrolidone is a competitive inhibitor of purified phenolase (Walker & Hulme, 1965), and has been used extensively as a protecting agent for mitochondrial isolation (Jones et al., 1965; Hulme et al., 1964; Wiskich, 1966). Other reagents include morpholinopropane sulphonate, cysteine, and sodium metabisulphite (Stokes et al., 1968). Morpholinopropane


sulphonate is thought to form complexes with phenolic compounds, while sulphydryl reagents inhibit phenoloxidases.

Free fatty acids are known to uncouple oxidative phosphorylation from electron transport (Borst et al., 1962; Baddeley & Hanson, 1967). The uncoupling activity of fatty acids is reversed by the addition of bovine serum albumin. Bovine serum albumin also reverses the uncoupling activity of many other uncoupling agents, such as nitro- and halo-substituted phenols, dicumarol, and carbonyl-cyanide m -chloro-phenylhydrazone in rat liver mitochondria (Weinbach & Garbus, 1966). Dalgarno and Birt (1963) showed that free fatty acids were present in mitochondrial preparations from carrot root tissue. These included oleic, stearic, palmitic and some short chain fatty acids, as well as polyunsaturated C18 acids. Mitochondria isolated from such tissues were uncoupled as shown by a P/O ratio less than 0.1. When bovine serum albumin was included in the isolation medium, mitochondria became well coupled, with a P/O ratio greater than 1.6 with succinate as substrate. As a matter of routine, most isolation procedures include 0.1% (w/v) of bovine serum albumin (Cohn Fraction V, low in free fatty acids). Bovine serum albumin binds fatty acids, and other lipophilic uncoupling agents, but the nature of the binding is not clear.

Isolation from Green Tissues

Mitochondria isolated from photosynthetic tissues are rarely free from chloroplasts or chloroplast fragments. Rocha and Ting (1970) subjected spinach leaf material to linear sucrose gradients (40 to 80% w/v) and obtained fractions after equilibrium. They found, nonetheless, that the mitochondrial fraction was contaminated with 13% intact chloroplasts and 6% broken chloroplasts. The degree of contamination was estimated from the activities of characteristic marker enzymes. Malate dehydrogenase and cytochrome c oxidase served as mitochondrial markers, while chlorophyll content and triose-phosphate dehydrogenase were chloroplast markers.

Mitochondrial Membranes

Structure of Membranes

Electron micrographs of plant mitochondria show clearly the tripartite nature of both the outer and inner membranes. This may be interpreted as a lipid bilayer with the hydrophobic fatty acid chains oriented toward the interior of the bilayer (see chapter 2). The lipids of mitochondrial membranes are largely phospholipids. It is the current view that phospholipid bilayers are highly dynamic, with a high degree of fluidity in the fatty acid region, as well as high lateral mobility of the phospholipids in the plane of the membrane.


The membrane proteins may form loose interactions with the lipid bilayer, or may be very tightly associated with the membrane. The loosely associated proteins most likely have exposed hydrophilic side chains and are easily extracted from membranes. The proteins tightly associated with membranes presumably have exposed hydrophobic side chains and are pictured as partially or wholly embedded in the lipid bilayer. These proteins are extracted from membranes with difficulty. Indeed, they may require a lipid environment for optimal activity.

Membrane Lipids

A survey of the lipid composition of mitochondrial membranes reveals great differences depending upon the source of mitochondria. McCarty et al., (1973) investigated the phospholipid composition of the inner and outer membranes of mung bean (Phaseolus aureus ) and potato tuber (Solanum tuberosum ) mitochondria which were prepared in such a way as to exclude contaminating particles. Phospholipids comprised 90% or more of the mitochondrial membrane lipids. The main phospholipids were phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl glycerol, except in the outer membrane which did not contain significant amounts of phosphatidyl glycerol. The phospholipid composition is shown in Table 5.1 together with that of beef heart mitochondria


Table 5.1. Phospholipid composition of mitochondrial membranes (McCarty et al., 1973.)


% total lipids


mung bean inner membrane

potato inner membrane

potato outer membrane

beef heart mitochondria

Phosphatidyl choline





Phosphatidyl ethanolamine





Phosphatidyl glycerol



none detected


for comparison. Minor amounts of lysophosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl glycerol were also found. The fatty acids of the three principal phospholipids of mung bean mitochondria were palmitic, linoleic and linolenic acids. Stearic acid occurred in conjunction with phosphatidyl glycerol. Schwertner and Biale (1973), by contrast, found that phospholipids comprised only about 50% of the total lipids of mitochondria from avocado (Persea sp.), cauliflower (Brassica oleracea ) and potato tubers, and found more phosphatidyl inositol than phosphatidyl glycerol. The principal fatty acids of the phospholipids of avocado, cauliflower and potato mitochondrial membranes were palmitic, linoleic and linolenic. The fatty acids of the neutral lipids of cauliflower


mitochondria were C16 and shorter chain acids, while those of potato mitochondria included palmitic, oleic, linoleic and linolenic. The fatty acid composition of the membrane lipids can be highly variable and may reflect the growth conditions of the tissue. Mitochondria from cold grown mung beans (15°C) have a higher amount of unsaturated fatty acids than mitochondria isolated from mung beans grown at 25°C (see also chapter 2).

Sterols comprise about 2.6% of the total lipids of maize shoot mitochondria (Kemp & Mercer, 1968). These may be esterified, of which cholesterol and b -sitosterol are the principal compounds. Of the unesterified sterols, stigmasterol and b -sitosterol are the principal ones. The fatty acids esterified to the sterols include lauric, myristic, palmitic, and linolenic as the most abundant.


Enzymes of the Tricarboxylic Acid Cycle

Mitochondria contain all the enzymes of the tricarboxylic acid cycle. Isolated mitochondria oxidize all of the acids of the cycle, and chromatographic analysis shows that the products are those expected for the reactions of the tricarboxylic acid cycle (Lieberman & Biale, 1956; Avron & Biale, 1957; Bogin & Erickson, 1965).—
Citrate Synthetase

Citrate synthetase (Citrate oxalacetate-lyase (CoA-acetylating)) activity is associated with a particulate fraction of leaf and root tissue from tobacco, bean, and soybean, which sediments at 10,000 g. This fraction consisted largely of mitochondria (Hiatt, 1962). Citrate was formed in the presence of acetyl CoA and oxalacetate. Citrate synthetase has been isolated from wheat scutellum mitochondria (Barbareschi et al., 1974) with minimal contamination by glyoxysomes. The mitochondrial citrate synthetase was released by sonic disruption of the mitochondria and recovered in the supernatent fluid. The purified enzyme had a molecular weight of 96,000 daltons as determined by its elution volume in Sephadex G-100 gel filtration. The Km for acetyl CoA and for oxalacetate were 4 µM and 34 µM respectively. The enzyme was inhibited competitively with respect to acetyl CoA by ATP. In these respects, the mitochondrial citrate synthetase from wheat scutellum mitochondria is similar to that from mammalian sources.—
Pyruvate Oxidase

Pyruvate is oxidized by mitochondria with a requirement for catalytic amounts of one of the TCA cycle acids (Millerd, 1953; Walker & Beevers, 1956). TCA


cycle acids were added to castor bean (Ricinus communis ) mitochondria at a concentration of 0.001 M . Oxygen consumption ceased after 60 minutes. When pyruvate at a concentration of 0.01 M was then added, oxygen consumption continued at rapid rates and linearly up to three hours. Other cofactors required were NAD, coenzyme A, ATP, and thiamin pyrophosphate.—
Isocitrate Dehydrogenase

A NAD-specific isocitrate dehydrogenase [L-iso-citrate:NAD oxidoreductase (decarboxylating)] has been purified from pea shoot (Pisum sativum var Alaska) mitochondria (Cox & Davies, 1967). The enzyme was released from pea shoot mitochondria ruptured by extrusion through a French pressure cell at 3,000 lbs in–2 . The enzyme, whose Km for NAD was 0.22 µM , was activated by Mn2+ and Mg2+ and to a lesser extent by Zn2+ , and inhibited by NADH, with Ki = 0.19 mM . The mitochondrial isocitrate dehydrogenase was specific for NAD which differentiates it from the cytosolic isocitrate dehydrogenase, which requires NADP as the electron acceptor.—
Malate Dehydrogenase

Malate dehydrogenase (L-malate:NAD oxidoreductase) exists in both mitochondrial and cytosolic forms. Moreover, the mitochondrial malate dehydrogenase may occur as several isozymes. Ting et al., (1966) separated two isozymes from young maize (Zea mays ) mitochondria after sonic disruption. Starch gel electrophoresis revealed a faint fast moving (toward anode) band and a major slow moving band. Grimwood and McDaniel (1970) also found a major slow moving band in polyacrylamide gel electrophoresis with several lighter fast moving bands. Boulter and Laycock (1966) attributed the minor bands to complexes of the mitochondrial malate dehydrogenase with other proteins, since re-electrophoresis of the eluted bands always gave a band in the main mitochondrial fraction as well as the original minor band. They determined the molecular weight of the main malate dehydrogenase to be 74,000 daltons.

Plant mitochondria oxidize malate readily, but glutamate must be included in the reaction mixture to remove the accumulated oxalacetate, due to the unfavourable equilibrium of the reaction. The oxidation of malate with endogenous NAD+ is inhibited by rotenone and antimycin (Day & Wiskich, 1974), but is insensitive to these inhibitors when exogenous NAD+ is supplied.—
Malic Enzyme

Malate may be oxidized by a NAD+ -dependent malic enzyme [L-malate:NAD oxidoreductase (decarboxylating)] with the formation of pyruvate. Macrae and Moorhouse (1970) showed that pyruvate accumulated during malate oxidation


by cauliflower bud mitochondria, unless thiamin pyrophosphate was included in the reaction medium. Under the latter conditions, malate was probably oxidized by both malic enzyme and malate dehydrogenase so that in the presence of cofactors for pyruvate oxidation and citrate formation, the latter accumulates. Malic enzyme is the main pathway for malate oxidation by wheat shoot mitochondria, since oxalacetate did not inhibit malate oxidation, although it did inhibit transiently the oxidation of citrate and pyruvate (Brunton & Palmer, 1973). The activities of malic enzyme and malate dehydrogenase differ in mitochondria from various sources (Macrae, 1971b). While the relative activity of malate dehydrogenase has in all cases been greater than the activity of malic enzyme, the accumulation of pyruvate vs oxalacetate may vary considerably. Pyruvate was accumulated in preference to oxalacetate by a ratio of 27.0 in cauliflower bud mitochondria, while the pyruvate/oxalacetate ratio for wheat shoot mitochondria was 0.13. When malic enzyme activity is high, mitochondrial NADH levels are raised, and thereby reduce oxalacetate accumulation by product inhibition of malate dehydrogenase. A strong pH dependence of the activities of the two enzymes was also observed (Macrae, 1971a). At pH 6.0 to 7.0, pyruvate accumulates, while at pH values between 7.0 and 8.0 pyruvate accumulation drops and oxalacetate accumulation rises. The pathway may reflect the pH profiles of malic enzyme and malate dehydrogenase. Below pH 7.0, the activity of malic enzyme would maintain a high internal concentration of NADH which would favour the conversion of oxalacetate to malate; above pH 7.0, the decreased activity of malic enzyme and the consequent drop in the NADH levels would favour the oxidation of malate by malate dehydrogenase.

Enzymes of Fatty Acid Oxidation

Fatty acids were oxidized by a particulate preparation from peanut cotyledons (Stumpf & Barber, 1956). This fraction was identified as the mitochondrial fraction. Cooper and Beevers (1969a,b) have separated the particulate fraction from castor bean and have shown that the enzymes of the b -oxidation pathway as well as the enzymes of the glyoxylate pathway are associated instead with a heavy particle, the glyoxysome. Mitochondria from castor beans contained less than 5% of the glyoxylate cycle enzymes and virtually none of the b -oxidation enzymes.

Enzymes of Fatty Acid Biosynthesis

Isolated mitochondria from avocado mesocarp, flowerlets of cauliflower, and from white potato tubers were capable of incorporating radioactive acetate, acetyl-CoA, malonate, or malonyl-CoA into long-chain fatty acids (Yang & Stumpf, 1965; Mazliak et al., 1972). Cofactor requirements included coenzyme A, NADPH, ATP, and Mg2+ or Mn2+ . The principal acids formed were palmitic


and stearic acids by avocado mesocarp mitochondria, while mitochondria from cauliflower and white potato tubers synthesized some mono-unsaturated fatty acids as well, i.e., palmitoleic (9-hexadecanoic acid) and, with longer incubation times, oleic (9-octadecanoic) and cis-vaccenic (11-octadecanoic) acids. With the appearance of the C18 mono-unsaturated acids, stearic acid is found only in trace amounts, indicating that the unsaturated C18 acids were formed from stearic acid.

Enzymes of Phospholipid Biosynthesis

The synthesis of phospholipids proceeds via the following reaction:


Mitochondria isolated from flowerlets of cauliflower contain all of the enzymes necessary for the formation of CDP-diglyceride from glycerol-3-phosphate, when coenzyme A, ATP, CTP and fatty acids are provided (Douce, 1971). Radioactivity from 32 P-ATP was found in phosphatidic acid in peanut cotyledon mitochondria (Bradbeer & Stumpf, 1960). The incorporation of 32p into phosphatidic acid was stimulated by the presence of small amounts of a ,b -diglyceride, indicating the presence of a mitochondrial diglyceride phosphokinase. 14 C-CTP was incorporated into CDP-diglyceride by cauliflower mitochondria (Sumida & Mudd, 1968). The radioactivity of CDP-diglyceride declines in the presence of a -glycerol phosphate or inositol, with the expected formation of phosphatidyl glycerol phosphate or phosphatidyl inositol. Using preparations carefully purified in sucrose density gradients from mung bean hypocotyl mitochondria, Douce et al. (1972b) showed that the CTP:phosphatidic acid cytidyl transferase activity was associated with the inner membrane fraction. Since the activity was not released upon sonication, it was assumed that the transferase was a membrane bound enzyme.

Mitochondrial Electron Transport

Components of the Respiratory Chain

Reducing equivalents derived from the oxidation of the TCA cycle acids are oxidized in a stepwise manner in the mitochondrial electron transport chain. The electron transport chain, or respiratory chain, is a series of functionally


linked election carriers which undergo alternate reduction and oxidation, with molecular oxygen as the terminal election acceptor. Elections are donated by carriers of low redox potential to carriers of high redox potential. It is via these oxidation-reduction reactions that the main oxidative cellular energy transduction occurs, either through the formation of intermediate states which can be coupled to cellular work, (e.g., ion transport) or through the phosphorylation of ADP to form ATP, which can then mediate cellular endergonic reactions. The main components of the respiratory chain have been identified, both by characteristic reaction toward inhibitors and by spectral analysis. In its basic form, the respiratory chain in plant mitochondria is very similar to that of mitochondria from fungal or animal sources. The chain, (shown in Fig. 5.3) can be functionally separated into (a) NADH: coenzyme Q oxidoreductase; (b) succinate: coenzyme Q oxidoreductase; (c) reduced coenzyme Q: cytochrome c oxidoreductase; and (d) cytochrome oxidase. With refinements in detection systems, additional components have been identified. The concentration and molar ratios of the principal components have been determinded by Lance and

Figure 5.3
The generlized mitochondrial electron transport chain. The components are NAD, nicotinamide
adenine dinucleotide; Fp and Fps , the flavoproteins associated with NADH dehydrogenase
and with succinate dehydrogenase respectively; Q, Coenzyme Q or the quinone containing
carrier; cyt b , the complex of b -cytochromes; cyt c 1 , the tightly bound c-cytochrome (cyt
c-549 in plant mitochondria); cyt c, the salt extractable c-cytochrome; and cyt  a+a3 , the
a -cytochromes of cytochrome oxidase. Not shown are non-heme iron proteins which
have been tentatively identified in plant mitochondria, and bound copper of cytochrome
oxidase, which has been identified in plant mitochondria, and bound copper of cytochrome
oxidase, which has been identified in cytochrome oxidase from animal sources only.


Bonner (1968) for mitochondria isolated from a number of sources and are given in Table 5.2. The concentrations of the cytochromes are quite similar to those of animal mitochondria.


Table 5.2. Concentration and stoichiometry of respiratory chain components in plant mitochondria (Lance & Bonner, 1968.)


Conc. of carriers


cyt aa3

cyt b

cyt c





nM per mg protein


Helianthus tuberosus






Phaseolus aureus






Solanum tuberosus






Brassica oleracea






Symplocarpus foetidus












Stoichiometry based on cyt c as unity

Helianthus tuberosum






Phaseolus aureus






Solanum tuberosum






Brassica oleracea






Symplocarpus foetidus




Nicotinamide Adenine Dinucleotide

Malate dehydrogenase and isocitrate dehydrogenase are NAD+ linked enzymes. The oxidation of substrates is accompanied by the reduction of endogenous NAD. This is shown by the strong fluorescence of reduced NAD. Carefully isolated mitochondria contain sufficient endogenous NAD to oxidize malate or isocitrate. The oxidation of malate or isocitrate by endogenous NAD is inhibited by rotenone or amytal. Malate oxidation is stimulated by the addition of NAD, but the oxidation then becomes insensitive to rotenone inhibition (Wiskich et al., 1960; Day & Wiskich, 1974), just as the oxidation of exogenous NADH is insensitive to rotenone or amytal (Wilson & Hanson, 1969; Day & Wiskich, 1974) suggesting more than one pathway of NADH oxidation. Douce et al., (1973) and Day and Wiskich (1974) delineated three mitochondrial NADH dehydrogenases, one located on the outer membrane, a second located on the outer surface of the inner membrane, and a third on the inner surface of the inner membrane. Each dehydrogenase has a characteristic response to inhibitors. The outer membrane dehydrogenase is characterized by an antimycin insensitive NADH: cytochrome c reductase with added cytochrome c. In intact mitochondria, the NADH dehydrogenase of the outer surface of the inner membrane is coupled to cytochrome oxidase and goes through the antimycin sensitive site,


but by-passes the rotenone sensitive site. This dehydrogenase shows NADH: cytochrome c reductase activity (antimycin sensitive) at low osmolarity only, due to the impermeability of the intact outer membrane to added cytochrome c. These first two dehydrogenases oxidize exogenous NADH. The NADH dehydrogenase of the inner surface of the inner membrane oxidizes the NADH linked to malate and isocitrate dehydrogenases. Electrons must go through the rotenone and the antimycin sensitive sites to cytochrome oxidase. In the intact mitochondrion, these various pathways interact, as demonstrated by the relief of antimycin or rotenone inhibition of malate: cytochrome c reductase activity by added NAD+ . Further evidence for the delineation of the NADH dehydrogenases is obtained through the P/O or ADP/O ratios of tightly-coupled mitochondria. Oxidation of malate or isocitrate gives ratios approaching 3.0, while the oxidation of NADH gives ratios approaching 2.0. The by-pass of the rotenone sensitive portion of the respiratory chain results in by-passing one of the phosphorylation sites as well (Wilson & Hanson, 1969).—
Flavoproteins of NADH Dehydrogenase

Storey (1970c, 1971a) distinguished a flavoprotein, FPM , which was rapidly reduced upon addition of malate. The reduction of this flavoprotein was inhibited by amytal. He tentatively assigned a midpoint potential, Em7.2 = –70mV for this flavoprotein. FPM is the flavoprotein involved in the first energy conservation site and hence is most likely the flavoprotein involved in the NADH dehydrogenase located in the inner surface of the inner membrane.

FPha , a high potential non-fluorescent flavoprotein is rapidly reduced upon the addition of exogenous NADH in mung bean mitochondria (Storey, 1970d). This reduction is insensitive to amytal (Storey, 1970c). Its midpoint potential is approximately +110mV (Storey, 1971a). Flavoprotein FPha could be the flavoprotein associated with the dehydrogenases which oxidize exogenous NADH, but it is not possible to determine if it is the flavoprotein of the outer membrane dehydrogenase or the inner membrane dehydrogenase from the information available.—
Flavoprotein of Succinate Dehydrogenase

Isolated mitochondria from a number of plant sources oxidize succinate readily, with oxygen consumption rates of about 450 nM O2 min–l mg–l protein (Douce et al., 1972a). Activation by ATP is required to obtain maximal rates of respiration with succinate as substrate, as well as for rapid response to addition of ADP (Drury et al., 1968). The activation by ATP is often attributed to the removal of inhibitory amounts of bound oxalacetate (Wiskich & Bonner, 1963) but the mechanism of the activation is not clear. The ATP effect is not due to phosphorylation mechanisms since neither oligomycin nor dinitrophenol affect the ATP activation (Singer et al., 1973).


Hiatt (1961) reported the partial purification of succinate dehydrogenase from mitochondria from bean roots and tobacco (Nicotiana tabacum ) leaves. The Km of the enzyme for succinate was 1 mM . Malonate inhibits competitively, with Ki = 0.24 mM . The apparent Michaelis constant of isolated mitochondria for succinate, in the presence of ADP and phosphate was 0.4 mM (Ikuma & Bonner, 1967). Singer et al., (1973) studied the succinate dehydrogenase of submitochondrial particles prepared by sonication of isolated mung bean and cauliflower mitochondria. The enzyme in submitochondrial particles was activated by a number of agents including substrate or Br . With Br activation, oxalacetate was removed, although it cannot be assumed that the oxalacetate was uniquely associated with the succinate dehydrogenase of the particles. Other activators included CoQ10 , NADH, NAD-linked substrate (i.e., malate plus pyruvate), and ADP (Oestreicher et al., 1973). Succinate reducible flavoprotein was not detected spectrally (Storey, 1970a), but a flavoprotein associated with succinate dehydrogenase was determined chemically. Singer et al., (1973) found that succinate dehydrogenase contained covalently bound flavin as a histidyl-a -FAD. The flavin content was approximately 0.2 nM per mg protein. The molar ratio of flavin to enzyme was not determined.—

Studies on the role of ubiquinone in the plant mitochondrial electron transport chain have not been extensive. Beyer et al., (1968) extracted ubiquinone from mung bean submitochondrial particles. A single ubiquinone was found which co-chromatographed with ubiquinone-10. The spectrum of the extracted ubiquinone-10 has an absorption peak at 275 nm in the oxidized form and at 290 in the reduced form. The ubiquinone was reduced by succinate and NADH; at an aerobic steady state, 38% of the ubiquinone was reduced by succinate, while 56% was reduced by NADH. At anaerobiosis induced by succinate or NADH, 88% and 84% respectively were reduced. Sodium hydrosulphite (dithionite) gave additional reduction (i.e., about 93% reduction). The quinones were virtually 100% oxidized in aerobic suspension in the absence of substrates. Ubiquinone is generally acknowledged as part of the mitochondrial respiratory chain and is placed at the juncture of succinate dehydrogenase and NADH dehydrogenase. This placement is based on the considerable work with animal mitochondria. Storey and Bahr (1972), basing their conclusions upon measurements of the half times of reduced to oxidized transitions, and the times for 50% reduced to the fully reduced state, suggested that ubiquinone is in the main respiratory chain of mung bean mitochondria. Ubiquinone is the link between the dehydrogenases and the cytochromes, as usually regarded in animal mitochondria, but Storey and Bahr (1972) in addition placed FPha , the high potential flavoprotein, between ubiquinone and the b -cytochromes. FPha has no counter-part in the animal mitochondrial respiratory chain.

Cytochrome b

The cytochromes are hemo-proteins. Three classes of cytochromes, distinguished by their spectral properties, as well as by the nature of their prosthetic groups are found in mitochondria (see Fig. 5.4). The a -cytochrome contains as its prosthetic group, heme a , while the b- and c -cytochromes contain a heme closely related to protoporphyrin IX. In the c -cytochromes, the heme is covalently linked to the protein via sulphur atoms in a thio-ether linkage. In the reduced state, the cytochromes exhibit strong absorption bands in the visible region of the spectrum which have been useful in their identification and in the analysis of their function. In addition, both the oxidized and reduced forms absorb strongly in the region around 400 nm, which is a characteristic of all heme compounds.

The b -cytochromes are best resolved when their spectra are determined at low temperatures, e.g., 77°K. Three b -cytochromes have been identified and two others are suggested. Their spectral properties are summarized in Table 5.3.


Table 5.3. The b- cytochromes of plant mitochondria.


a -peak, 25°C nm

a -peak, 77°K nm



b– 556


553 554

+75 to + 100




+ 75


b– 560



+ 40 to + 80






b– 558

557 558

553 555

70 to 105


b– 566


561 563







S2 O4 2– reducible

557 561

554, 560



References: (a) Lambowitz & Bonner, 1974; (b) Dutton & Storey, 1971.

Considerable variability exists in the nomenclature of the b -cytochromes. In conformity to the International Union of Biochemistry, the b -cytochromes are designated according to the a -peak of their reduced spectrum at room temperature (25°C). It should be noted that there is a blue shift of about 3 nm in the spectrum at 77°K relative to the spectrum at room temperature. The use of the a -absorption peak is further complicated by the fact that some authors use the absorption maximum at 77°K to designate the various b -cytochromes. In older nomenclature, mammalian cytochrome b –562, as orginally described by Keilin, was designated cytochrome b. As other b -cytochromes were discovered with an a -absorption peak significantly different from 562 nm, these were designated with subscripts. More recently, cytochrome b– 566 was thought to be directly involved in energy transduction and was designated cytochrome bT , a transducing b -cytochrome, to differentiate it from cytochrome b– 562, or bK .


Figure 5.4
Prosthetic groups of the cytochromes.


The multiplicity of b -cytochromes is due to different b -cytochromes in mitochondria rather than a splitting of the absorption bands at low temperature, since the peak heights do not change in synchrony in the presence of reducing agents, inhibitors or uncouplers (Lance & Bonner, 1968). The b -cytochromes are placed in the respiratory chain according to the following sequence (Storey, 1973):


Cytochrome b– 560 (557) was placed on the oxygen side of cytochrome b –556 (553) as a result of the determination of the rates of oxidation of the 556 and 560 components by an oxygen pulse of an anaerobic suspension of mitochondria. The 560 component was oxidized with a half-time of oxidation of 6 to 8 msec while the 556 component was oxidized with a half-time of 150 to 200 msec. The reduction by succinate of these two components in anaerobiosis showed, however, that b– 560 was reduced more slowly than b– 556 which was contrary to the expected rates in view of the rates of oxidation (Storey & Bahr, 1972; Storey, 1973). The slow reduction was ascribed to the more negative redox potential of b –560. The midpoint potentials of b –560 and b –556 would predict that b– 560 would be on the substrate side of b –556. Further resolution of the sequence of the b -cytochromes is necessary.

Cytochrome b– 566 was thought to be analogous to the b– 566 (bT ) of mammalian mitochondria. Cytochrome b– 566 from animal mitochondria was found to undergo a midpoint potential shift as well as an enhanced reduction in anaerobic suspension when the respiratory chain was energized (Chance et al., 1970). This was interpreted as the formation of a high energy intermediate of phosphorylation directly involving cytochrome b– 566. In plant mitochondria, the midpoint potential shift of b– 566 was not observed (Dutton & Storey, 1971; Lambowitz et al., 1974). Although enhanced reduction of b– 566 by ATP or by energization of the respiratory chain could be demonstrated in plant mitochondria, it could be explicable by reverse electron flow through the b -cytochromes (Lambowitz et al., 1974; Lambowitz & Bonner, 1974). Thus the status of a transducing b -cytochrome in plant mitochondria is in question. In fact, cytochrome b –566 was excluded from the main sequence of the respiratory chain, since it remains oxidized in anaerobic suspensions (succinate reduced) while other b -components, pyridine nucleotides and fluorescent flavoproteins are reduced (Storey, 1969, 1974). There was a lack of equilibration between the low redox potential carriers with cytochrome b– 566. The function of cytochrome b –566 is left uncertain.—
Cytochrome c

Two c -type cytochromes have been detected in plant mitochondria. They have the same relationship as cytochrome c and c1 in animal mitochondria (Lance & Bonner, 1968). The room temperature spectrum shows a large peak at 550 mn which shifts to 547 nm at liquid nitrogen temperature (77°K). As with cytochrome


c in animal mitochondria, the cytochrome c (cyt c –547[*] ) is easily extracted by salt solutions. A second component with a low temperature absorption peak at 549 nm remains after extensive washing with phosphate buffer. The 547 nm absorbing component is recovered in the phosphate buffer extract while the 549 nm absorbing component remains in the pellet. Both are reducible by ascorbate, which differentiates them from the b -cytochromes. The spectral properties of the 549 absorbing c component and its strong binding to mitochondrial membranes relate this c component to cytochrome c1 of animal and yeast mitochondria. The midpoint potentials of the two c-cytochromes of mung bean mitochondria have been determined to be +235 mV in both cases (Dutton & Storey, 1971). The half-time of oxidation of cytochrome c– 547 and c –549 are 3.0 and 3.1 msec respectively when KCN treated anaerobic mitochondria were pulsed with 14 µM O2 . The electron transfer sequence of the c -cytochromes was given as cyt c– 549 to cyt c– 547 (Storey & Bahr, 1972).

Cytochrome c (cyt c– 547) is essentially identical to cytochrorne c from all eukaryotic sources. Cytochrome c from one source will react with the reductase and oxidase from quite distantly related sources. The amino acid sequence of cytochrome c from a large number of sources is shown to have a high degree of homology (Nolan & Margoliash, 1968; Dickerson et al., 1971). This homology is all the more striking when the tertiary structure is considered. For example, the amino acids about the heme show a high degree of conservatism among the cytochrome c proteins examined. Those amino acid residues important to the structure and function of the protein have suffered few substitutions in the course of evolutionary history.—
Cytochrome Oxidase

The cytochrome oxidase of plant mitochondria contains cytochromes a and a3 , as does that from animal mitochondria. These are two spectroscopically differentiated components, although two separate chemically different entities have not been isolated. The optical properties are well differentiated in the presence of cyanide or azide, which binds to cytochrome a 3 . The a -band of cytochrome oxidase at room temperature is located at 602 nm; at 77°K, there is a blue shift to 598 nm. The reduced spectrum of cytochrome a is revealed in the difference spectrum of an azide treated aerobic suspension minus an aerobic suspension. All components are oxidized except for cytochrome a, which shows a symmetrical reduced a -band at 598 nm. The a3 spectrum is shown in a difference spectrum of an anaerobic (succinate reduced) plus azide suspension, minus an aerobic plus azide suspension. The reduced a -peak of cytochrome a cancels and the reduced a -peak of cytochrome a3 is shown with its maximulm at 603 nm (Lance & Bonner, 1968). The midpoint potentials of cytochromes a and a3 are


+190 and +380 mV respectively (Dutton & Storey, 1971). Half-times of oxidation in oxygen pulsed anaerobic suspension are 2.0 msec and 0.8 msec respectively for cytochromes a and a3 (Storey, 1970b).—
Non-Heme Iron Proteins

Few investigations have been carried out on the occurrence and nature of non-heme iron proteins in higher plant mitochondria. Electron paramagnetic resonance (epr) signals characteristic of iron sulphur centres (non-heme iron proteins) were observed by Cammack and Palmer (1973) in mitochondria from Helianthus tuberosus and Arum maculatum with components at g = 2.02 and 1.93, 2.05 and 1.92, and at 2.10 and 1.87. Schonbaum et al., (1971) obtained an epr signal in NADH-reduced skunk cabbage (Symplocarpus foetidus ) mitochondria for a component at g = 1.94 and some complex components near g = 2.00. These iron sulphur centres no doubt participate in electron transport, since they undergo oxidation and reduction. At present, their precise functions are not known. They may be analogous to the iron sulphur centres identified in the NADH dehydrogenase segment of yeast and pigeon heart submitochondrial particles, which are believed to be closely involved in energy coupling (Ohnishi, 1973).

Cyanide Resistant Respiration

An unusual characteristic of respiration in plant mitochondria is a partial insensitivity to cyanide inhibition. Partial insensitivity is exhibited to inhibition by azide, antimycin and 2-heptyl hydroxy-quinolin-N-oxide, all of which are potent inhibitors of oxygen uptake in animal mitochondria. This cyanide insensitivity may be almost 100% as in the spadix mitochondria from some aroid species, notably of Arum maculatum and Symplocarpus foetidus, partial as in mung bean mitochondria, or completely lacking as in the mitochondria from fresh, dormant white potato tubers (Bahr & Bonner, 1973a). In the latter, a cyanide insensitivity, and indeed a cyanide stimulation of respiration, may be induced upon vigorously aerating slices of potato tuber tissue in water for 24 hours. Mitochondria isolated from such aged potato tuber slices are much less inhibited by antimycin or cyanide (Hackett et al., 1960).

Because of the insensitivity to cyanide at concentrations which completely inhibit the respiration of animal mitochondria, it is unlikely that the cyanide insensitive respiration is due to an incomplete inhibition of cytochrome oxidase. This 'excess' oxidase hypothesis has been considered by various workers and received strong support from the finding that cytochromes a and c were incompletely reduced in the presence of cyanide (Chance & Hackett, 1959). The insensitivity to antimycin, which inhibits electron transport between the cytochrome b to cytochrome c region favours the argument that the cytochrome system is by-passed entirely and that there exists an alternate oxidase in mito-


chondria showing cyanide insensitivity, although mitochondria of A. maculatum and S. foetidus have the conventional cytochrome complement (Bendall & Hill, 1956; Chance & Hackett, 1959). By and large, cytochrome c and cytochrome oxidase are reduced in the presence of cyanide (Bendall & Bonner, 1971). The incomplete reduction of cytochromes found by Chance and Hackett could be attributed to (a) the fact that the cytochromes are not always reduced by substrates relative to the reduction by dithionite; (b) possible spectral interference in the measurement of cytochrome c due to an oxidized b -cytochrome; or (c) in coupled mitochondria, significant reverse electron transport may cause the carriers on the oxygen side of the respiratory chain coupling site to become partially oxidized (Bonner & Bendall, 1968; Bendall & Bonner, 1971). Chance and Hackett (1959) and Bendall and Hill (1956) reported, however, that a b –type cytochrome becomes oxidized in the presence of cyanide and oxygen. Bendall and Hill called their b -component from A. maculatum cytochrome b7 (amax 560 nm), while Chance and Hackett identified an oxidizable b -component with an amax at 558.5 nm. It was hypothesized that a b -cytochrome functioned as the shunt to the alternate oxidase. Such a role for a b -cytochrome is favoured, since Bahr and Bonner (1973b) reported that the known flavoproteins and ubiquinone had equal access to oxygen by either pathway, based on observations of their oxidation in the presence or absence of cyanide, and Storey and Bahr (1969a) found no identifiable carriers among flavoprotein, ubiquinone, the known b -cytochromes or c -cytochromes which could mediate electron transfer to the alternate oxidase. Cytochrome b7 was identified with cytochrome b –557 (77°K) (Chance et al., 1968), but since the oxidation rate of cytochrome b– 557 was unaffected by m-chlorobenzhydroxamic acid, an inhibitor of the alternate oxidase (Schonbaum et al., 1971) it was felt that cytochrome b– 557 does not play a part in the alternate pathway, and should not be equated with Bendall and Hill's cytochrome b 7 (Erecinska & Storey, 1970). Bendall and Bonner (1971) showed that thiocyanate and other metal binding agents also inhibit the alternate pathway, suggesting a role for non-heme iron proteins or other metalloproteins. Efforts to identify non-heme iron proteins which may be the alternate oxidase have not been successful (Cammack & Palmer, 1973). Strong epr signals characteristic of iron-sulphur proteins in mitochondria of aged Jerusalem artichoke (H. tuberosus ) and in A. maculatum mitochondria were found. However, these are most likely the iron sulphur proteins of NADH: ubiquinone reductase since they were not reducible by succinate, and were, moreover, unaffected by hydroxamic acids. Schonbaum et al., (1971) found that the epr signals in NADH reduced skunk cabbage submitochondrial particles were enhanced by treatment with m-iodobenzhydroxamic acid. In addition to a signal at g = 1.94, which is characteristic of NADH dehydrogenase, a set of complex signals near g = 2.0 was detected at 77ºK. It was thought that the g = 2.0 signal may originate from the alternate pathway.

The possibility that the alternate oxidase may involve a flavoprotein has been explored. Flavoprotein oxidases which reduce oxygen with the formation of


hydrogen peroxide are known, but such oxidases have not been identified for cyanide resistant mitochondria (Bendall & Bonner, 1971).

The function of cyanide resistant respiration is unclear. In the spadix tissue of maturing flowers of Arum and Symplocarpus, it may serve the function of thermogenesis. A rise in temperature of the spadix tissue ten degrees above the ambient has been recorded. This thermogenesis may aid these plants in pollination, since flowering occurs in early spring.

Energy Linked Reactions of Mitochondria

Oxidative Phosphorylation—
Coupling Sites

Oxidative phosphorylation, the process whereby the reaction


is coupled to the oxidation of reduced electron carriers of the respiratory chain occurs in plant mitochondria in the same manner as does the reaction in animal mitochondria. The sites of phosphorylation are identical (see Baltscheffsky & Baltscheffsky, 1974). These are the sites I in the NADH: ubiquinone reductase segment, II in the cytochrome b: cytochrome c reductase segment and III in the cytochrome oxidase segment of the respiratory chain. It has not been possible to identify precisely those components of the respiratory chain which are the energy transducers in each of the three sites, although these have been approximated from the change in redox potentials (D E0 ') between adjacent carriers (Lehninger, 1965), by application of the crossover theorem (Chance & Williams, 1955), and from shifts in the midpoint potential of certain electron carriers upon the addition of ATP to uncoupled anaerobic suspensions of mitochondria (Wilson & Dutton, 1970a,b; Lindsay & Wilson, 1972; Chance et al., 1970; Ohnishi, 1973; Devault, 1971). The D E0 ' indicates the thermodynamic feasibility of coupling between two adjacent components. Thus, coupling sites were thought to be located between endogenous NAD and the flavoprotein of NADH: ubiquinone reductase; between cytochrome b and cytochrome c of the cytochrome b :cytochrome c reductase; and between cytochrome a and oxygen of cytochrome oxidase. The application of the crossover theorem by and large confirms the general location based on thermodynamic grounds. According to the crossover theorem, electron transport through the coupling site is the rate limiting step in tightly coupled mitochondria. The carrier on the substrate side of the coupling site should become reduced, while the carrier on the oxygen side would become oxidized. Upon the addition of the phosphate acceptor, ADP,


there would be observed a rapid transient oxidation of the carrier on the sub-strate side, and a reduction of the carrier on the oxygen side of the coupling site, concomitant with the release of controlled respiration in the state 4-state 3 transition (see Table 5.4).


Table 5.4. Definition and values of steady states in mitochondria (Chance & Williams, 1955.)


(O2 )

ADP levels


Respiratory rates

Rate-limiting substance


> 0






> 0






> 0




respiratory chain


> 0











Wilson and Dutton (1970a) reported that the midpoint potential (Em ) of cytochrome a3 becomes more negative upon energization of an anaerobic suspension of rat liver mitochondria by ATP. Similarly they report that the Em of one of the b cytochromes (amax 564 nm) increases upon energization by ATP (Wilson & Dutton, 1970b; Chance et al., 1970). These shifts of the midpoint potentials were attributed to changes in ligand interaction energy of the heme iron upon energization, and that the iron atoms of these cytochromes were directly involved in energy coupling.

Such a change in the midpoint potential was postulated by Wang (1970). In his model, the phosphoimidazole group becomes a much weaker coordinating ligand for the Fe(II) than imidazole, and hence should lower the midpoint reduction potential of the corresponding electron carrier. Similar experiments in the iron-sulphur centres of the site I region of the respiratory chain showed that of the five iron sulphur centres identified, ATP affected the reduction potential of only one of these centres, designated as centre I (Ohnishi, 1973). Addition of ATP caused a partial oxidation of centre I when the reduction potential was poised at a value where the iron was almost completely reduced. These observations suggested that the reduction potential of centre I was dependent upon the phosphate potential, and that the addition of ATP caused a lowering of the midpoint potential of the centre I iron sulphur protein. The significance of the changes in the midpoint potential was questioned, at least for the site II region, since none of the b -cytochromes showed an ATP-induced potential shift when investigated in plant mitochondria (Dutton & Storey, 1971; Lambowitz et al., 1974). One must conclude that there is a fundamental difference in the function of the b -cytochromes in plant and animal mitochondria or that the changes in the midpoint potential do not reflect the formation of an energy transducing species. Lambowitz et al., took the latter view and attributed


the midpoint potential changes to a reverse electron flow with cytochrome b –566 and cytochrome a3 equilibrating with the redox mediators by way of cytochrome c, while the iron sulphur centre of site I equilibrated through the nicotinamide adenine nucleotide pool.—
ADP:O Ratios

The general location of the three coupling sites means that for tightly coupled mitochondria, predictable ratios of the moles of ADP phosphorylated to the gram-atoms of oxygen consumed (ADP/O or P/O) may be obtained for each substrate. Thus succinate should give a ratio of 2.0, NAD-linked substrates a ratio of 3.0 and a -ketoglutarate a ratio of 4.0 (one phosphorylation via succinyl-CoA, three phosphorylations via the respiratory chain NADH). These ratios are largely confirmed in plant mitochondria. Early attempts gave results much below the predicted ratios, but with refinements in the procedure for the isolation of plant mitochondria, the inclusion of required cofactors, and the exclusion of competing reactions, predicted ratios have been approached, as shown in Table 5.5. Exogenous NADH does not interact with respiratory chain NAD,


Table 5.5. Oxidative phosphorylation by isolated mitochondria (Douce et at., 1972a).




Respiratory control index

Phaseolus aureus












Solanum tuberosum












but with flavoproteins of the external dehydrogenase. These ultimately enter the respiratory chain at the level of cytochrome b and hence electrons go through two coupling sites only (Douce et al., 1973).—
Energy Coupling in Cyanide Resistant Respiration

Mitochondria of Symplocarpus are fully capable of energy conservation in the uninhibited state. Hackett and Haas (1958) found P/O ratios greater than 3 for a -ketoglutarate oxidation by skunk cabbage mitochondria. In the presence of cyanide, the energy coupling sites associated with the cytochromes are by-passed, and energy coupling involves only the NADH:ubiquinone reductase portion of the respiratory chain. Storey and Bahr (1969b) obtained ADP/O ratios of 1.3 with succinate as substrate in skunk cabbage mitochondria in the absence


of cynide; the ratio was zero in the presence of cyanide. With malate as the substrate, the ADP/O ratios were 1.9 and 0.7 in the absence and presence of 0.3 mM KCN respectively, showing that coupling sites II and III were effectively by-passed while coupling site I, which could be activated by malate but not by succinate, was still functioning in the presence of cyanide.

Wilson (1970a), however, has proposed that energy coupling occurs in the alternate pathway as well. He found that cyanide treated mitochondria of A. maculatum and Acer pseudoplatanus produced ATP, with an ATP/O ratio of 0.5 at high O2 concentration (greater than 100 µM ) with succinate as the substrate. Submitochondrial particles of A. maculatum which lacked malate dehydrogenase activity oxidized succinate with P/O ratios of 0.2 to 0.6 (Wilson 1970b). Since malate dehydrogenase-depleted particles were still capable of phosphorylation, the possibility that the phosphorylation was due to the oxidation of a product of succinate oxidation, i.e., malate, could be eliminated, therefore suggesting that a phosphorylation site exists in the alternate pathway which functions only at high C2 concentration. Cyanide inhibited mitochondria from spadices of A. maculatum and from mung bean hypocotyls still retain an energy related function through the cytochromes. Bonner and Bebdall (1968) showed that a substrate linked reverse electron flow is active in spadix mitochondria. Cytochrome c and cytochrome oxidase of cyanide treated mitochondria were reduced by ascorbate plus, N,N,N',N' -tetramethyl-p -phenylenediamine (TMPD), an electron donor to cytochrome c. Subsequent addition of succinate caused a reoxidation of cytochrome c and cytochrome a with a partial reduction of cytochrome b. The reoxidation of cytochromes c and a was prevented by uncoupling concentrations of carbonylcyanide-phenyl-hydrazone or 2,4-dinitrophenol. Wilson and Moore (1973) also showed that cyanide inhibited mung bean mitochondria could carry out oxidation of ascorbate plus TMPD. The oxidation was thought to involve a reverse electron flow through coupling site II and energized by ATP or the high energy intermediate.—
Mechanism of Coupling

The mechanism of energy coupling remains an intractable problem in spite of the impressive array of workers in this area. Three outstanding hypotheses are under active consideration: (a) the chemical intermediate hypothesis; (b) the chemiosmotic hypothesis; and (c) the conformation coupling hypothesis.

The operation of the chemical intermediate hypothesis may be summarized as follows:


where A and B are adjacent electron carries at the coupling site, X and I are unknown couplers common to all coupling sites (see Greville, 1969). The scheme postulates the existence of both non-phosphorylated and phosphorylated intermediates. A non-phosphorylated intermediate accounts for the action of uncouplers, which is independent of the presence of phosphate. The nonphosphorylated intermediate can also be coupled to work functions such as ion transport, which is sensitive to uncouplers, but not to the phosphorylation inhibitor, oligomycin. The phosphorylated intermediate transfers a phosphoryl group to ADP. Hill and Boyer (1967) showed that the bridge oxygen between the b and g phosphorus of ATP is furnished by ADP. Hence, the mechanism of phosphorylation involves an activation of phosphate.

The chemiosmotic hypothesis in its simplest form as first proposed by Mitchell (1961) involves a vectorial metabolism in which the elements of water are transported to opposite sides of the mitochondrial membrane (Fig. 5.5).

Figure 5.5
Representation of the Mitchell chemiosmotic
scheme for oxidative phosphorylation.
(Redrawn from Mitchell, 1966.)

Thus oxido-reduction reactions create a pH gradient as well as a potential difference (outside positive) which tends to drive H+ back across the membrane into the inner compartment. This force is the proton-motive force, and it is this flow of protons through the coupling site (a reversible ATPase) which drives ATP synthesis. The phosphorylation reaction is represented as follows:


It can then be shown that in the absence of a transmembrane electrical potential, a pH differential of 3.5 units is required to poise the ratio of ATP/ADP = 1 while a potential difference of 210 mV would be required in the absence of pH gradients (Mitchell, 1966). The translocation of protons and equivalent OH is effected by ionizable groups which are designated XH and IOH, corresponding to components of an ATPase. The proposed intermediate X-I of the ATPase must have a sufficiently low hydrolysis constant at the high electrochemical


potential of H+ in the outer phase to come to reverse equilibrium with water according to the reaction


On the other hand, the hydrolysis constant of the intermediate X ~ I must be in the order of 105M , and the intermediate X ~ I must be in equilibrium with the ATP/(ADP + Pi ) couple in the inner phase, so that


The system vibrates between states in which the intermediate X – I is alternately accessible to the outer and inner phases, being X – I when in contact with the outer phase and X – I when in contact with the inner phase. The transition of X – I to X ~ I is due not to the pumping of energy into X–I, but to a lowering of the ground state energy for X – I hydrolysis by some 10,000 calories per mole on translocation through an anisotropic membrane.

While experimental verification of the chemiosmotic mechanism has been realized only in chloroplasts by acid-base trasitions (Jagendorf & Uribe, 1966; see also chapter 4), similar experiments have not been successful in mitochondria. Cockrell et al., (1967) have shown ATP synthesis after establishing a K+ gradient by valinomycin-treated mitochondria. However, Glynn (1967) argued that ATP synthesis via a K+ gradient could be explained equally well on the basis of a membrane potential rather than cation transport down a chemical gradient through an ATPase.

In their general aspects, the chemical intermediate hypothesis and the chemiosmotic hypothesis differ primarily in the nature of the initial driving force for coupled phosphorylation. The two hypotheses converge at the level of the ATPase in that both call for an unknown intermediate, X ~ I (Greville, 1969).

The conformational coupling hypothesis of Boyer (1967) differs in that the ATPase has a high affinity for Pi and ADP. Tightly bound ATP is formed via a nucleophilic attack by ADP on orthophosphate in a SN2 reaction with the displacement of water (Korman & McLick, 1970). According to the model, there exists an energy requirement for the release of bound ATP from the complex, by changing the complex from one having a high affinity for ATP to one having low affinity (Boyer et al., 1973). The advantages of this hypothesis are that it explains most of the exchange reactions observed in coupled phosphorylation, and the coupling of ATP to energy yielding reactions of mitochondria.

Reverse Electron Flow

Electron flow and energy transduction through the coupling sites of the respiratory chain are reversible processes. These are shown by the reduction of endogenous NAD and cytochrome b when substrates of higher reduction


potentials are oxidized as well as the ATP induced oxidation of reduced cytochrome a + a3 when the terminal oxidase is inhibited by sulphide. The properties of energy linked NAD reduction by plant mitochondria are similar to those reported in animal mitochondria (Chance, 1961; Chance & Hollunger, 1963). These include the requirement for succinate oxidation and for ATP (Storey, 1971b). Although ATP is required, the reduction of NAD is not sensitive to oligomycin. Hence NAD is reduced by reverse electron flow through the coupling site but without the participation of the ATPase as such. Uncouplers either inhibit the reduction when added before succinate, or reverse the reduction when added after a steady state reduction is attained. This can be interpreted as an effect upon the coupling site in preventing reverse electron flow, as well as a general release of controlled respiration so that the endogenous NADH is now oxidized rapidly through the coupling site. It is not possible to distinguish between the two alternatives.

Reverse electron flow through coupling sites II and III has been demonstrated by Storey (1972) and Lambowitz et al., (1974). When ATP is added to sulphideinhibited or to anaerobic suspensions of mung bean mitochondria, the b -cytochromes become reduced while cytochrome c and cytochrome a + a3 become oxidized. The effect is reversed by uncouplers or by phenazine methosulphate (PMS) which mediates electron flow between cytochromes b and c. The reverse electron flow through coupling site II involves an ATPase (Lambowitz et al., 1974). Hydrolysis of ATP is observed in an anaerobic suspension of mung bean mitochondria supplemented with ascorbate plus TMPD and ATP. Addition of PMS caused an increase in the rate of ATP hydrolysis which is to be expected if PMS formed a shunt of electrons from reduced cytochrome b to cytochrome c.

Ion Transport

Mitochondria contain two compartments, one of which is readily accessible to low molecular weight solutes such as sucrose or mannitol, and a second in-accessible to sucrose or mannitol. The former is identified with the inter-membrane space, and the latter, the mitochondrial matrix. The volume enclosed by the inner membrane behaves as an osmometer (Lorimer & Miller, 1969; Overman et al., 1970). Selective permeability to solutes and active transport are properties of the inner mitochondrial membrane. The movement of solutes across the inner membrane may be determined by assay for net increases in intramitochondrial contents. If the movement results in the net increase or decrease in the osmolarity of the sucrose-inaccessible volume, such solute movements will be reflected in volume changes of the inner compartment which can be monitored by changes in the light scattering properties of a mitochondrial suspension. Thus mitochondrial swelling is accompanied by a decrease in light scattering, while a contraction is reflected by an increase in light scattering.

Monovalent Cations

Permeability of the inner membrane of plant mitochondria to potassium and chloride ions is restricted. Slow, passive permeability to K+ and C1 may be observed on transfer of mitochondria to isosmotic KCl, but when mitochondria are energized by the addition of substrate (NADH), rapid loss of both K+ and Cl occurs, accompanied by mitochondrial contraction (Wilson et al., 1969; Kirk & Hanson, 1973). In contrast to the energized extrusion of K+ and C1 , mitochondria undergo an NADH induced swelling' in potassium acetate solution, and K+ and acetate ions are taken up. This has been interpreted as an active transport of acetate, while K+ penetrates along an electrochemical potential and chloride is not transported. The NADH-induced swelling in potassium acetate is inhibited by uncouplers of oxidative phosphorylation, by ADP plus Pi , or by respiratory chain inhibitors (Wilson et al., 1969; Lee & Wilson, 1972), but is restored when oligomycin is present with ADP plus Pi . An intermediate of oxidative phosphorylation possibly mediates active transport of acetate, but not of chloride. This is strengthened by the observation that mitochondria respire with expected respiratory control and ADP/O ratio when suspended in isotonic KCl, but lose respiratory control in potassium acetate.

Ionophorous antibiotics, valinomycin and gramicidin D, increase the permeability of the mitochondrial membrane to potassium ion as shown by the increased rate of passive swelling in potassium chloride solutions (Kirk & Hanson, 1973; Miller et al., 1970a). Valinomycin also increases the rate of NADH-induced swelling in potassium acetate, as well as in chloride, phosphate, sulphate and nitrate salts of potassium (Kirk & Hanson, 1973; Wilson et al., 1972) although the swelling due to acetate and phosphate reverses on exhaustion of NADH. This has been interpreted to mean that the swelling is due to the enhanced permeability of the inner membrane to potassium, as well as an active transport of acetate or phosphate. In the case of the latter anions, swelling is thought to be due to an enhanced permeability toward K+ while the anions diffuse along an electric potential. On cessation of NADH-supported respiration, acetate and phosphate leak out according to their chemical potential, followed by potassium ions, while no leakage of chloride, nitrate or sulphate occurrs as they are distributed along their chemical potential (Wilson et al., 1972). The picture which emerges is that the movement of potassium ion is controlled by the movement of anions, but K+ flux can be modified by ionophores such as valinomycin or gramicidin D. Valinomycin may stimulate K+ uptake via a H+ exchange as found by Rossi et al., (1967) in liver mitochondria, but such K+ uptake due to H+ exchange is not accompanied by swelling of the mitochondria.—
Divalent Cations

Ca2+ does not cause marked stimulation of respiration in plant mitochondria as it does in animal mitochondria, except with exogenous NADH as substrate


(Miller et al., 1970b; Miller & Koeppe, 1971). A slight release from controlled respiration is detected with malate plus pyruvate or with succinate as substrates. This is associated with the accumulation of calcium and inorganic phosphate by mitochondria (Miller et al., 1970b). Extensive Ca2+ uptake by plant mitochondria occurs only in the presence of inorganic phosphate (Hodges & Hanson, 1965; Elzam & Hodges, 1968; Earnshaw et al., 1973; Chen & Lehninger, 1973). The phosphate dependent Ca2+ transport is energy dependent and may be supported by substrate oxidation or by ATP (Elzam & Hodges, 1968). The energy dependence seems to be at the level of an intermediate of oxidative phosphorylation. Substrate-supported Ca2+ transport is sensitive to uncouplers and respiratory inhibitors (Chen & Lehninger, 1973) as well as to ADP (Elzam & Hodges, 1968). The ATP supported Ca2+ transport is inhibited by oligomycin. Similar observations have been reported for Sr2+ transport by mitochondria (Johnson & Wilson, 1972). Other anions, e.g. acetate, arsenate, sulphate, chloride or nitrate promote neither Ca2+ nor Sr2+ uptake (Hodges & Hanson, 1965; Johnson & Wilson, 1972) although all will produce a metabolically independent swelling, while arsenate and acetate produce an active swelling as well, indicating that mitochondrial membranes are permeable to these anions, and will actively transport arsenate or acetate, as well as phosphate (Hanson & Miller, 1967; Johnson & Wilson, 1972; Lee & Wilson, 1972). Uptake of Ca2+ and inorganic phosphate results in the deposition of electron dense material in mitochondria which is dependent upon the concentrations of both Ca2+ and phosphate, and the time of incubation (Peverly et al., 1974). The composition of the deposits has not been ascertained, but is believed to be a form of calcium phosphate. The deposition of the phosphate salt of divalent alkaline earth metal ions may account for the contraction of mitochondria induced by Ca2+ . Since the volume changes of mitochondria are measured by light scattering changes, the formation of crystals within the mitochondria may increase the ight scattering properties of the suspension, and be interpreted as a contraction.—
Anion Transport

The importance of anion transporters in the movement of solutes across the mitochondrial inner membrane has been studied extensively in mammalian mitochondria (Chappell & Haarhoff, 1967; Harris, 1969). In these studies, the role of the phosphate transporter and the malate transporter is emphasized. Similar investigations were carried out by Phillips and Williams (1973b) and by Wiskich (1974). The presence of anion transporters was demonstrated by the spontaneous swelling of mitochondria in solutions of the ammonium salts of phosphate or malate. Ammonium salts were used because the mitochondrial membrane is readily permeable to ammonium ion. Mitochondrial swelling under these conditions is indicative of an osmotic adjustment due to the net influx of solutes into the mitochondrial matrix (Overman et al., 1970; Wilson et al., 1973). The swelling in ammonium phosphate was inhibited by


N -ethylmaleimide which inhibits the phosphate-hydroxyl antiporter while the swelling in ammonium malate as well as the malate supported respiration was inhibited by 2-butylmalonate, 2-phenylsuccinate, benzylmalonate or p -iodo-benzylmalonate, all inhibitors of the dicarboxylate carrier (Phillips & Williams, 1973a,b). The anion transport system is interpreted as follows: (a) a phosphatehydroxyl antiporter which transports phosphate in exchange for hydroxyl ion; (b) a malate-phosphate antiporter which transports malate in exchange for phosphate; (c) a tricarboxylate-malate antiporter which transports tricarboxylate anions in exchange for malate; (d) other dicarboxylate anions enter by exchange with malate. It is the prevailing view that anions are actively transported, and that cations follow the anion transport along an electric gradient (Hanson & Miller, 1967). The essential role of anion transport in determining cation movement, however, is modified by the presence of cation ionophores.

Further Reading

Bonner W.D., Jr. (1965) Mitochondria and electron transport. In Plant Biochemistry (eds. J.F. Bonner & J.E. Varner). Academic Press.

Bonner W.D., Jr. (1973) Mitochondria and plant respiration. In Phytochemistry, vol. 3 (ed. L.P. Miller). Van Nostrand Reinhold.

Dawson A.P. & Selwyn M.J. (1974) Mitochondrial oxidative phosphorylation. In Companion to Biochemistry (eds. A.T. Bull, J.R. Lagnado, J.O. Thomas & K.F. Tipton). Longman.

Goddard D.R. & Bonner W.D., Jr. (1960) Cellular respiration. In Plant Physiology, vol. 1A (ed. F.C. Steward). Academic Press.

Greenberg D.M. (ed.) (1967) Metabolic Pathways, vol. 1. Energetics, Tricarboxylic Acid Cycle and Carbohydrates. Academic Press.

Hanson J.B. & Hodges T.K. Energy linked reactions of plant mitochondria. In Current Topics in Bioenergetics, vol. 2 (ed. D. Rao Sanadi). Academic Press.

Lehninger A.L. (1964) The Mitochondrion: Molecular Basis of Structure and Function. W.A. Benjamin, Inc.

Munn E.A. (1974) The Structure of Mitochondria. Academic Press.

Öpik H. (1974) Mitochondria. In Dynamic Aspects of Plant Ultrastructure (ed. A.W. Robards). McGraw-Hill.

Sato S. (ed.) (1972) Mitochondria. In Selected Papers in Biochemistry, vol. 10. University Park Press.

Slater E.C., Zaniuga Z. & Wojtczak L. (eds.) (1967) Biochemistry of Mitochondria. Academic Press.

Wainio W.W. (1970) The Mammalian Mitochondrial Respiratory Chain. Academic Press.


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