5.6—
Mitochondrial Electron Transport

5.6.1—
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 Fp_s , 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.

5.6.1.1—
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).

5.6.1.2—
Flavoproteins of NADH Dehydrogenase

Storey (1970c, 1971a) distinguished a flavoprotein, F_PM , which was rapidly reduced upon addition of malate. The reduction of this flavoprotein was inhibited by amytal. He tentatively assigned a midpoint potential, E_m7.2 = –70mV for this flavoprotein. F_PM 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.

F_Pha , 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 F_Pha 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.

5.6.1.3—
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 K_m of the enzyme for succinate was 1 mM . Malonate inhibits competitively, with K_i = 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.

5.4.1.4—
Ubiquinone

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 F_Pha , the high potential flavoprotein, between ubiquinone and the b -cytochromes. F_Pha has no counter-part in the animal mitochondrial respiratory chain.

5.6.1.5—
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.

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 b_T , a transducing b -cytochrome, to differentiate it from cytochrome b– 562, or b_K .

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 (b_T ) 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.

5.6.1.6—
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.

5.6.1.7—
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

[*] The 77°K absorption peak.

+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).

5.6.1.8—
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).

5.6.2—
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 (a_max 560 nm), while Chance and Hackett identified an oxidizable b -component with an a_max 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.