An EPR analysis of cyanide-resistant mitochondria isolated from the mutant poky strain of Neurospora crassa.

An analysis of the paramagnetic components present in mitochondria isolated from the poky mutant of Neurospora crassa is described. The study was undertaken with a view to shedding light on the nature of the cyanide- and antimycin A-resistant alternative terminal oxidase which is present in these preparations. Of the ferredoxin-type iron-sulfure centers, only Centers S-1 and S-2 of succinate dehydrogenase could be detected in significant quantities. Paramagnetic centers attributable to Site I were virtually absent. In the oxidized state, at least two 'high potential iron sulfur' centers could be distinguished and these were attributed to Center S-3 of succinate dehydrogenase and a second component analogous to that found in mammalian systems. Much of the Center S-3 signal was in a highly distorted state which was apparently dependent upon the presence of an accompanying free radical species. At lower field positions, a succinate-reducible signal peaking around g = 3.15 was found. This signal is caused by a low spin heme species, presumably the cytochrome c which is the only major cytochrome in these mitochondria. At even lower field positions, signals attributable to iron in a field of low symmetry at g = 4.3 and multiple high spin heme species around g = 6, could be distinguished. The effects of salicylhydroxamic acid, an inhibitor of the alternative oxidase, were tested on these components. Effects could be seen on at least one high spin heme component and also partially upon the distorted Center S-3 signal converting part of it to a signal indistinguishable from center S-3. Some increase in the g = 4.3 iron signal was also noted. No effects of the inhibitor on the ferredoxin-type centers were detected.     

An EPR analysis of cyanide-resistant mitochondria isolated from the mutant poky strain of Neurospora crassa

An analysis of the paramagnetic components present in mitochondria isolated from the poky mutant of Neurospora crassa is described. The study was undertaken with a view to shedding light on the nature of the cyanide- and antimycin A-resistant alternative terminal oxidase which is present in these preparations.

Of the ferredoxin-type iron-sulfur centers, only Centers S-1 and S-2 of succinate dehydrogenase could be detected in significant quantities. Paramagnetic centers attributable to Site I were virtually absent. In the oxidized state, at least two ‘high potential iron sulfur’ centers could be distinguished and these were attributed to Center S-3 of succinate dehydrogenase and a second component analogous to that found in mammalian systems. Much of the Center S-3 signal was in a highly distorted state which was apparently dependent upon the presence of an accompanying free radical species. At lower field positions, a succinate-reducible signal peaking around g = 3.15 was found. This signal is caused by a low spin heme species, presumably the cytochrome c which is the only major cytochrome in these mitochondria. At even lower field positions, signals attributable to iron in a field of low symmetry at g = 4.3 and multiple high spin heme species around g = 6, could be distinguished.

The effects of salicylhydroxamic acid, an inhibitor of the alternative oxidase, were tested on these components. Effects could be seen on at least one high spin heme component and also partially upon the distorted Center S-3 signal converting part of it to a signal indistinguishable from Center S-3. Some increase in the g = 4.3 iron signal was also noted. No effects of the inhibitor on the ferredoxin-type centers were detected.

These results are interpreted with respect to the nature and location of the alternative oxidase and with respect to possible models for the nature of the alternative oxygen-consuming component.     

Thermodynamic and EPR characteristics of two ferredoxin-type iron-sulfur centers in the succinate-ubiquinone reductase segment of the respiratory chain.

Two distinct ferredosin-type iron-sulfur centers (designated as Centers S-1 and S-2) are present in the soulble succinate dehydrogenase in approximately equivalent concentrations to that of bound flavin. Both Centers S-1 and S-2 exhibit electron paramagnetic resonance absorbance in the reduced state at the same magnetic field (gz = 2.03, gy = 1.93, and gx = 1.91) with similar line shape. Center S-2 is reducible only chemically with dithionite and remains oxidized under physiological conditions. Thus, its functional role is unknown; however, thermodynamic and EPR characterization of this iron-sulfur center has revealed important molecular events related to this dehydrogenase. The midpoint potentials of Centers S-1 and S-2 determined in the soluble succinate dehydrogenase preparations are -5 +/- 15 mV and -400 +/- 15 mV, respectively, while corresponding midpoint potentials determined in particulate preparations, such as succinate-cytochrome c reductase or succinate-ubiquinone reductase, are 0 +/- 15 mV and -260 +/- 15 mV. Reconstitution of soluble succinate dehydrogenase with the cytochrome b-c1 complex is accompanied by a reversion of the Center S-I midpoint from -400 +/- 15 mV to -250 +/- 15 mV with a concomitant restoration of antimycin A-sensitive succinate-cytochrome c reductase activity. There observations indicate that, during the reconstitution process, Center S-I is restored to its original molecular environment. In the reconstitutively active succinate dehydrogenase, the relaxation time of Center S-2 is much shorter than that of S-1, thus Center S-2 spectra are well discernible only below 20 K (at 1 milliwatt of power), while the resonance absorbance of Center S-1 is detectable at higher temperatures and readily saturates below 15 K. Over a wide temperature range the power saturation of Center S-1 resonance absorbance is relieved by Center S-2 in the paramagnetic state, and the Center S-2 central resonance absorbance is broadened by Center S-1 spins, due to a spin-spin interaction between these centers. These observations indicate an adjacent location of these centers in the enzyme molecule. In reconstitutively inactive enzymes, subtle modification of the enzyme structure appears to shift the temperature dependence of Center S-2 relaxation to the higher temperature. Thus the EPR signals of Center S-2 are also detectable at higher temperature. In this system a splitting of the central peak of the Center S-2 spectrum due to spin-spin interaction was observed at extremely low temperatures, while this was not observed in reconstitutively active enzymes or in paritculate preparations. This spin-spin interaction phenomena of inactive enzymes disappeared upon chemical reactivation with concomitant appearance of the reconstitutive activity. These observations provide a close correlation between the molecular integrity of the enzyme and its physiological function.     

Regulation of Alternative Pathway Activity in Plant Mitochondria : Deviations from Q-Pool Behavior during Oxidation of NADH and Quinols

External NADH and succinate were oxidized at similar rates by soybean (Glycine max) cotyledon and leaf mitochondria when the cytochrome chain was operating, but the rate of NADH oxidation via the alternative oxidase was only half that of succinate. However, measurements of the redox poise of the endogenous quinone pool and reduction of added quinones revealed that external NADH reduced them to the same, or greater, extent than did succinate. A kinetic analysis of the relationship between alternative oxidase activity and the redox state of ubiquinone indicated that the degree of ubiquinone reduction during external NADH oxidation was sufficient to fully engage the alternative oxidase. Measurements of NADH oxidation in the presence of succinate showed that the two substrates competed for cytochrome chain activity but not for alternative oxidase activity. Both reduced Q-1 and duroquinone were readily oxidized by the cytochrome oxidase pathway but only slowly by the alternative oxidase pathway in soybean mitochondria. In mitochondria isolated from the thermogenic spadix of Philodendron selloum, on the other hand, quinol oxidation via the alternative oxidase was relatively rapid; in these mitochondria, external NADH was also oxidized readily by the alternative oxidase. Antibodies raised against alternative oxidase proteins from Sauromatum guttatum cross-reacted with proteins of similar molecular size from soybean mitochondria, indicating similarities between the two alternative oxidases. However, it appears that the organization of the respiratory chain in soybean is different, and we suggest that some segregation of electron transport chain components may exist in mitochondria from nonthermogenic plant tissues.     

EPR studies of higher plant mitochondria. I Ubisemiquinone and its relation to alternative respiratory oxidations.

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (gmax = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria. The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the Ks of this effect was an order of magnitude higher than the Ki of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria. The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.     

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.     

The spatial relationships and structure of the binuclear iron-sulfur clusters in succinate dehydrogenase.

Two binuclear iron-sulfur clusters (designated S-1 and S-2) are present in succinate dehydrogenase in approximately equal concentration to that of flavin. The large difference in their midpoint potentials (0 and -400 mV, respectively, in the soluble enzyme) permits the acquisition of individual electron paramagnetic resonance spectra characterized by nearly identical rhombic g tensors (gz = 2.025, gy = 1.93, gx = 1.905). Spin-coupling between the two centers is manifested by broadening and splitting of spectra of reconstitutively active and inactive succinate dehydrogenase, respectively, as the temperature is lowered; relief of power saturation of Center S-1 spectra on reduction of Center S 2; and observation of half-field ("delta ms = 2") signals in the dithionite-reduced enzyme. Saturation behavior of fully reduced dehydrogenase is consistent with the presence of S-1 and S-2 at equivalent concentrations/molecule. Simulation of the spin-coupled spectra, assuming dipolar interaction, provides information on molecular structure. Electron paramagnetic resonance spectra of the enzyme in 80% dimethylsulfoxide are nearly identical to the characteristic binuclear spectra obtained with adrenodoxin. These data provide additional evidence for binuclear structure of both Center S-1 and S-2. The extremely fast relaxation of Center S-2 at low temperatures would imply either an anomalously small value of J or an alternative relaxation mechanism, possibly due to the coupling between S-1 and S-2.     

EPR studies of higher plant mitochondria I. Ubisemiquinone and its relation to alternative respiratory oxidations

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (g_max = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria.

The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the K_s of this effect was an order of magnitude higher than the K_i of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria.

The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.     

Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain.

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidezed state analogous to the bacterial high potential iron-sulfur protein, has bwen detected in the reconstitutively active soluble succinate dehydrogenase preparation. Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 +/- 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1:1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating electrons from the flavoprotein subunit to most probably ubiquinone and then to the cytochrome chain.     

Intramitochondrial positions of ubiquinone and iron-sulphur centres determined by dipolar interactions with paramagnetic ions.

E.p.r. (electron-paramagnetic-resonance) spectra of ubisemiquinone (QH) organic radicals and all of the known iron-sulphur centres were studied in normal and 'nickle-plated' pigeon heart mitochondria, submitochondrial particles and submitochondrial particles from which succinate dehydrogenase had been removed. Incubation of pigeon heart mitochondria, submitochondrial particles or succinate dehydrogenase-depleted submitochondrial particles with substrate in the presence of pure O2 results in the accumulation of Q-H. In mitochondria, the e.p.r. spectrum of Q-H is characterized by in-homogeneous line broadening. A heterogeneous population of semiquinones appears to be partly responsible for these effects in mitochondria. Additon of Ni(II) to mitochondria renders saturation of the Q-H resonance more difficult. On the other hand, the resonance in either submitochondrial particles or succinate dehydrogenase-depleted particles is narrower than the same spectrum in mitochondria, and saturates like a homogeneous line. The presence of Ni(II) in either of these preparations, further, has no effect on either the A-H spectrum or the saturation curve. Therefore QH appears to be situated on the exterior surface of the mitochondrion. Likewise, the e.p.r. spectra and saturation curves of iron-sulphur centre N-2 exhibit characteristics of inhomogeneous line broadening, not only in intact mitochondria but also in both submitochondrial particles and succinate dehydrogenase-depleted particles. Because of the small pool size of centre N-2, this effect is likely to arise from a spin interaction with some other component in the membrane. Ni(II) has no effect on the saturation in centre N-2 in mitochondria or submitochondrial particles, and only a marginal effect in the succinate dehydrogenase-depleted preparation. These results are indeterminate with respect to the position of centre N-2 in the membrane; but suggest that its distance from the succinate dehydrogenase binding site is on the order of 1 nm. All of the other ferredoxin-type iron-sulphur centres in both preparations were not affected by paramagnetic ions. Homogeneous e.p.r. spectra and saturation curves are observed for both of the HiPIP-type (high-potential iron-sulphur protein-type) iron-sulphur centres in mitochondrial centres S-3 and bc-3. Addition of No(II) to intact mitochondria results in a dipolar interaction with centre bc-3. No effect was observed on centre S-3 in either preparation. A comprehensive model is presented for the structure of the respiratory electron-transport system in mitochondria, based on e.p.r. relaxation studies in the present and the preceding paper. There is no direct evidence for transmembrane electron flow through any of the known energy-coupling sites in mitochondria, so that direct hydrogen atom transfer across the membrane (as a combination of H+ translocation coupled to electron flow) does not occur...     

Iron-sulphur centres in mitochondria from Arum maculatum spadix with very high rates of cyanide-resistant respiration.

X-band electron-paramagnetic-resonance spectroscopy at 4.2--77K combined with measurements of oxidation-reduction potential was used to identify iron--sulphur centres in Arum maculatum (cuckoo-pint) mitochondria. In the oxidized state a signal with a derivative maximum at g = 2.02 was assigned to succinate dehydrogenase centre S-3. Unreduced particles showed additional signals at g = 2.04 and 1.98 (at 9.2 GHz), which may be due to a spin-spin interaction. In the reduced state a prominent signal at g = 1.93 and 2.02 was resolved into at least three components that could be assigned to centres S-1 and S-2 of succinate dehydrogenase (midpoint potentials -7 and -240 mV respectively at pH 7.2) and a small amount of centre N-1b (e'o= -240 mV) of NADH-ubiquinone reductase. In addition, changes in line shape around -10 mV indicated the presence of a fourth component in this signal. The latter was more readily reduced by NADH than by succinate, suggesting that it might be associated with the external NADH dehydrogenase. The iron-sulphur centres of NADH-ubiquinone reductase were present in an unusually low concentration, indicating that the alternative, non-phosphorylating, NADH dehydrogenase containing a low number of iron-sulphur centres may be responsible for most of the high rate of oxidation of NADH.     

Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.     

Structure-function relationships of the alternative oxidase of plant mitochondria: A model of the active site

A major characteristic of plant mitochondria is the presence of a cyanide-insensitive alternative oxidase which catalyzes the reduction of oxygen to water. Current information on the properties of the oxidase is reviewed. Conserved amino acid motifs have been identified which suggest the presence of a hydroxo-bridged di-iron center in the active site of the alternative oxidase. On the basis of sequence comparison with other di-iron center proteins, a structural model for the active site of the alternative oxidase has been developed that has strong similarity to that of methane monoxygenase. Evidence is presented to suggest that the alternative oxidase of plant mitochondria is the newest member of the class II group of di-iron center proteins.     

Alternative Oxidase Activity and the Ubiquinone Redox Level in Soybean Cotyledon and Arum Spadix Mitochondria during NADH and Succinate Oxidation

In Arum and soybean (Glycine max L.) mitochondria, the dependence of the alternative oxidase activity on the redox level of ubiquinone, with NADH and succinate as substrates, was studied, using a voltametric procedure to measure the ubiquinone redox poise in the mitochondrial membrane. The results showed that when the enzyme was activated by pyruvate the relationship between the alternative oxidase rate and the redox state of the ubiquinone pool was the same for both NADH and succinate oxidations. In the absence of pyruvate the alternative oxidase had an apparent lower affinity for ubiquinol. This was more marked with NADH than with succinate and was possibly due to pyruvate production during succinate oxidation or to an activation of the alternative oxidase by succinate itself. In Arum spadix (unlike soybean cotyledon) mitochondria, succinate oxidation via the alternative oxidase maintained the ubiquinone pool in a partially reduced state (60%), whereas NADH oxidation kept it almost completely reduced. Previous data comparing mitochondria from thermogenic and nonthermogenic tissues have not examined the full range of ubiquinone redox levels in both tissues, leading to the suggestion that the activity of alternative oxidase for Arum was different from nonthermogenic tissues. When the complete range of redox states of ubiquinone is used and the oxidase is fully activated, the alternative oxidase from thermogenic tissue (Arum) behaves similarly to that of nonthermogenic tissue (soybean).     

Stimulation of the Alternative Pathway by Succinate and Malate

Stimulation of the cyanide-resistant oxidation of exogenous NADH in potato (Solanum tuberosum L. cv Bintje) tuber callus mitochondria was obtained with succinate, malate, and pyruvate. Half-maximal stimulation was observed at a succinate or malate concentration of 3 to 4 mM, which is considerably higher than that found for pyruvate (0.128 mM). No effect of succinate or malate addition was found when duroquinone was the electron acceptor. Duroquinol oxidation via the alternative pathway was poor and not stimulated by organic acids. Under stimulating conditions, no swelling or contraction of the mitochondria could be observed. Conversely, variation of the osmolarity did not affect the extent of stimulation. However, the assay temperature had a significant effect: no stimulation occurred at temperatures below 16 to 20[deg]C. Membrane fluidity measurements showed a phase transition at about 17[deg]C. Ubiquinone reduction levels were not significantly higher in the presence of succinate and malate, but the kinetics of the alternative oxidase were changed in a way comparable to that found for stimulation by pyruvate. At low temperatures the alternative oxidase displayed "activated" kinetics, and a role for membrane fluidity in the stimulation of the alternative pathway by carboxylic acids is suggested.     

Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.     

Induction and activation of the alternative oxidase of potato tuber mitochondria

Potato tubers ( Solanum tubersum L. cv. Grata) were stored for atleast 1 week at room temperature and then incubated with an equal amount of apples ( Malus domestica L.) for 2 days. After this treatment, intact tuber mitochondria isolated by Percoll gradient centrifugation showed a high degree of induction of the alternative oxidase, measured as cyanide-resistant, salicylhydroxamic acid-sensitive respiration. With succinate as substrate an activity of more than 130 nmol O₂(mg protein) ¹ min ^t was obtained. An assay of the alternative oxidase using duroquinol as an electron donor was developed. To become reliable the assay required the presence of defatted bovine serum albumin (BSA) and catalase (EC 1. 11. 1. 6). Furthermore, a lowering of the assay temperature to 15°C improved the stability of the duroquinol-based activity. One remarkable finding was that with duroquinol (or external NADH) as substrate the alternative oxidase was synergistically activated by succinate (as well as by malate) even in the presence of the succinate dehydrogenase inhibitor malonate. Our interpretation is that succinate and malate (indirectly) activate the alternative oxidase and that this activation is part of a physiological mechanism for regulation of the alternative oxidase.     

Nature of the effect of adenosine 5''-monophosphate on the cyanide-insensitive respiration in mitochondria of Moniliella tomentosa.

The alternative oxidase of Moniliella tomentosa mitochondria is stimulated by 5'-AMP. This effect may be masked, depending on the isolation procedure of the mitochondria. The preparation of submitochondrial particles results in the expression of the 5'-AMP effect. Two more methods are now described to reveal the 5'-AMP effect whenever it would be masked: (1) switching on the myokinase activity of the mitochondria to deplete them of endogenous 5'-AMP; (2) using detergents (sodium dodecyl sulphate, sodium deoxycholate) in a controlled detergent:protein ratio, or chloroform. The alternative oxidase of detergent-solubilized mitochondria was somewhat less selective towards nucleotides than were intact mitochondria. The effect of nucleotides on quinol oxidation by mitochondrial preparations and on quinol autoxidation was also studied. Mitochondrial oxidation of succinate by the alternative oxidase and autoxidation of quinols behaved similarly in the presence of certain nucleotides. Both reactions were stimulated. Both reactions were also inhibited by salicylhydroxamic acid. These effects on quinol oxidation disappeared when bovine serum albumin or mitochondrial proteins were present. From the results obtained it is not possible to exclude quinol autoxidation as a final step of the alternative oxidase.     

EPR studies on a Hipip type iron-sulfur center in the succinate dehydrogenase segment of the respiratory chain

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a Hipip-type iron-sulfur center (Center S-3) has been detected in the reconstitutively active soluble succinate dehydrogenases. E_m7,4 determined in a particulate, antimycin A sensitive succinate-cytochrome $\text{c}$ reductase is +60 ± 15 mV. This center is extremely labile towards oxygen in a manner similar to the reconstitutive activity of the dehydrogenase. Even freshly prepared reconstitutively active enzyme shows a considerably diminished content of Center S-3 relative to flavin and displays a partly modified spectra. All reconstitutively inactive dehydrogenases give rise to a highly modified or no Center S-3 spectra at all. These observations indicate that Center S-3 is a constituent of succinate dehydrogenase and plays a role in the physiological function of the enzyme, $\text{i.e.}$ transferring electrons most probably to ubiquinone.