Alternative Oxidase in the Branched Mitochondrial Respiratory Network: an Overview on Structure, Function, Regulation, and Role

Brazilian Journal of Medical and Biological Research (1998) 31: 733-747 Mitochondrial alternative oxidase 733 ISSN 0100-879X

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

F.E. Sluse1 and 1Laboratory of Bioenergetics, Center of Oxygen Biochemistry, W. Jarmuszkiewicz2 Institute of Chemistry B6, University of Liege, Liege, Belgium 2Department of Bioenergetics (Prof. Lilla Hryniewiecka), Adam Mickiewicz University, Poznan, Poland

Abstract

Correspondence Plants and some other organisms including protists possess a complex Key words F.E. Sluse branched respiratory network in their mitochondria. Some pathways • Mitochondria Laboratory of Bioenergetics of this network are not energy-conserving and allow sites of energy • Alternative oxidase Center of Oxygen Biochemistry conservation to be bypassed, leading to a decrease of the energy yield • Structure Institute of Chemistry B6 in the cells. It is a challenge to understand the regulation of the • Regulation University of Liege • Electron partitioning Sart-Tilman, B-4000 Liege partitioning of electrons between the various energy-dissipating and Belgium -conserving pathways. This review is focused on the oxidase side of Fax: (324) 366-28-78 the respiratory chain that presents a cyanide-resistant energy-dissipat- E-mail: [email protected] ing alternative oxidase (AOX) besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the Presented at the XXVI Annual alternative oxidase activity is presented in detail because of its com- Meeting of the Brazilian Society of Biochemistry and Molecular plexity. The alternative oxidase activity is dependent on substrate Biology, Caxambu, MG, Brasil, availability: total ubiquinone concentration and its redox state in the May 3-7, 1997. membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression) or short-term Research supported by the (post-translational modification, allosteric activation) regulated. Elec- Belgian “Fonds de la Recherche tron distribution (partitioning) between the alternative and cytochrome Fondamentale Collective” (No. pathways during steady-state respiration is a crucial measurement to 2.4568.97), the Polish-Belgian Joint Research Program. quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method Received January 22, 1998 (thermokinetic approach). Lastly, the role of the alternative oxidase in Accepted February 17, 1998 non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon) and with harmful reactive oxygen species formation.

Braz J Med Biol Res 31(6) 1998 734 F.E. Sluse and W. Jarmuszkiewicz

Short historical introduction fungi (11,12), in trypanosomes (13), and amoebae (14). Thus, the presence of the Cyanide-insensitive respiration was first cyanide-resistant alternative oxidase is not observed in plants in 1929 by Genevois in limited to plant mitochondria and is quite sweet pea seedlings (1). Most investigations widespread among other types of organisms. made in the past were physiological, utiliz- Isolation of cDNA and genes encoding the ing whole plants or intact tissues, and indi- AOX protein has led to the first primary cated the occurrence of cyanide resistance sequences (15), a transmembrane model linked to heat production (2,3). When iso- (16,17) and structural modeling (9,10,18). lated mitochondria were used, studies were In the last few years considerable progress focused on the relative activities of both has been made in the understanding of the cyanide-insensitive and cytochrome path- regulation and role of AOX. ways by measuring rates of respiration in the The aim of this paper is to provide an presence of an inhibitor of each pathway. In overview of the field and to stress the new 1978, a cyanide-resistant quinol oxidase was achievements. For more detailed comprehen- solubilized from Arum maculatum mitochon- sive reviews of the alternative oxidase field, dria (4,5), and the alternative cyanide-resist- the reader is referred to reviews by Moore and ant respiration was attributed to an enzyme Siedow (16), McIntosh (19), Siedow and called the alternative oxidase (AOX). Partial Umbach (10), Day and Wiskich (20), Day et purification of the alternative oxidase was al. (9), Wagner and Krab (21), Krab (22), and achieved (6,7) but its lability and limited Vanlerberghe and McIntosh (23). activity have hampered enzymological approaches like kinetic and structural studies Branched respiratory network in for a long time. In 1986, monoclonal anti- mitochondria bodies to three induced proteins from Sau- romatum guttatum responsible for cyanide- The respiratory chain of plant mitochon- resistant respiration were obtained (8), dria, taken as an example, differs in several strongly stimulating the field of the alterna- ways from the mammalian mitochondrial tive oxidase research. These monoclonal respiratory chain (Figure 1). The dehydro- antibodies were used to identify AOX pro- genase side presents two additional NAD(P)H teins in a wide variety of plants (9,10), in dehydrogenases, which are not proton pumps

Figure 1 - Organization of the Intermembrane space branched respiratory chain of plant mitochondria. Complex I, NADH dehydrogenase; complex II, succinate dehydrogenase; complex III, cytochrome bc1; complex IV, cytochrome oxidase; AOX, alternative oxidase; c, cytochrome c; Q, ubiquinone; QH2, ubiquinol; INT, internal NAD(P)H dehydrogenase; EXT, external NAD(P)H dehydrogenase.

Matrix

Braz J Med Biol Res 31(6) 1998 Mitochondrial alternative oxidase 735

(and therefore are not conserving energy), Characteristics of AOX protein are not inhibited by inhibitors of the proton- pumping energy-conserving complex I (e.g. Primary structure of AOX rotenone) and allow electrons originating from NADH to bypass complex I. Moreover, The amino acid sequence of a protein can on the oxidase side a cyanide-resistant alter- be derived from the cDNA sequences. By native oxidase is present besides the cyto- analysis of the primary sequence of a protein chrome pathway (complexes III and IV). first insights into its structure can be deduced. This oxidase is not linked to proton gradient AOX primary sequences obtained in such a building and thus dissipates the free energy way contain a mitochondrial transit peptide at released during electron flow into heat (16). the N-terminus. The mature AOX protein from Ubiquinone (Q) has a central position in the S. guttatum, as an example, has a calculated respiratory chain network. This coenzyme molecular mass of 32.2 kDa and contains 283 links the different branches of the network, amino acids (15). Hydropathy plot analysis of receiving electrons from the upstream dehy- several AOX amino acid sequences (15,16) drogenases (reducing pathways) and giving has indicated important conserved features of electrons to the downstream oxidases the protein: a) two hydrophobic regions with a (oxidizing pathways). Thus, during the strong α-helical character and highly conserved oxidation of NADH formed by the Krebs amino acids have been proposed to be mem- cycle, one, two or three sites of energy con- brane-spanning helices, b) these two regions servation may be bypassed (i.e., when the are separated by about 40 amino acids includ- rotenone-insensitive dehydrogenase and ing an amphipathic helix probably exposed to the cytochrome pathway are involved, the intermembrane surface, and c) two hydro- when complex I and AOX are involved, or philic regions of about 100 amino acids on when the rotenone-insensitive dehydrogen- both terminal sides with highly conserved short ase and AOX are involved, respectively), regions at the C-terminus. A topologic model leading to a reduction in protonmotive for the AOX protein has been proposed force and finally in the level of ATP synthe- (9,10,16-19), with the amphipathic helix ex- sis. In mitochondria with a branched net- posed on the cytosolic side and the hydrophilic work, the extent to which the dissipative domains extending into the matrix. Both cyto- pathways are used affects the energy yield in solic and matricial parts are linked by the two the cells. It is obviously of crucial interest for membrane-spanning helices. these cells to be able to control the relative contribution of each pathway of the network Active sites of AOX to the total oxygen uptake and it is a challenge for us to understand the mechanisms The ability of AOX to reduce oxygen to that regulate the partitioning of electron water involves a four-electron reduction re- flow. action (16,28) to avoid production of dam- Electron flow through AOX can be spe- aging partially reduced oxygen species. cifically inhibited by various compounds like Therefore, an active site containing transi- hydroxyamic acids (e.g. salicylhydroxamic tion metals has been predicted (16,29). Evi- acid (SHAM) and benzohydroxamic acid dence that makes iron the leading metal can- (BHAM)) (24), n-propyl gallate (25,26) and didate for the metal associated with the AOX disulfiram (27). Then, the level of the cya- active site comes from experiments with nide-resistant oxygen uptake sensitive to one culture of the yeast Hansenula anomala in of these inhibitors is the diagnosis of AOX which the absence of iron leads to an inac- activity. tive 36-kDa AOX protein (30). Its activity is

Braz J Med Biol Res 31(6) 1998 736 F.E. Sluse and W. Jarmuszkiewicz

recovered by the addition of iron to the immunoblotting using chemical cross-link- culture medium. All of the plant AOX amino ers and oxidizing and reducing agents (32). acid sequences (in the C-terminal domain) These experiments have demonstrated that reveal the presence of two copies of the AOX exists as a dimer of 65 kDa in the inner conserved iron-binding motif (E-X-X-H), mitochondrial membrane and that two dis- providing the additional argument that AOX tinct states of the dimer can be identified: an contains iron. By comparison with coupled oxidized state in which the dimer is co- binuclear iron proteins, an iron-binding four- valently cross-linked by a disulfide bridge helical bundle model of the AOX active site (-S-S-) and a reduced state (-SH HS-) which has been proposed analogous to that of meth- is maintained through non-covalent interac- ane monooxygenase (10,18,31). tions. The intermolecular disulfide bridge is The reducing substrate (i.e. ubiquinol formed at the level of a conserved cysteine

(QH2)) binding site has been postulated to be residue (in the plant AOX) situated in the N- situated on the matrix side of the mitochon- terminal domain exposed to the matrix of drial membrane in a hydrophobic pocket each monomer (Figure 2). A second con- formed by the two membrane-spanning heli- served cysteine residue (in the plant AOX) ces (10,18). The three fully conserved resi- implicated in pyruvate binding is situated in dues (T, E, Y) in the pocket are potential the same N-terminal domain close to the ligands for ubiquinone. Then, in the pro- membrane surface of each subunit (33). posed structural model of AOX, the binuclear iron center where oxygen is reduced to water AOX genetics should be close to the postulated binding site Figure 2 - Topologic model of of reducing QH2 which itself is in the vicin- The AOX protein is encoded in the the dimeric plant AOX in the inner mitochondrial membrane. ity of the binding site for the allosteric effec- nucleus. In many organisms the AOX mono- Highly conserved residues T, Y, tor of the enzyme, pyruvate (see below). mer is revealed as multiple bands of approxi- and E, located in the two trans- mately 36 kDa on immunoblots using a single membrane helices, are proposed to be a potential quinone-bind- Dimeric structure of AOX monoclonal antibody. In the case of plant ing site. The four-helix bundle AOX protein, depending on the species and (postulated binuclear iron center) The structural property of AOX has been tissues, one, two or three bands were ob- is located in the C-terminal hydrophilic domain of the protein revealed by SDS-PAGE electrophoresis and served. Therefore, the question was if these on the matrix side of the inner mitochondrial membrane. On the same side of the membrane, Intermembrane the relative positions of the two space conserved cysteine residues are shown. One, located in the N- terminal hydrophilic domain clos- est to the membrane (near the postulated diiron site and the postulated quinone-binding site), may be the binding site for pyruvate (allosteric effector). Second, Matrix the more N-terminal cysteine residue may be the redox-active group involved in conversion between the more active noncova- lently associated reduced state of the enzyme and the less active oxidized state. Based on Siedow and Umbach (10), Moore et al. (18), and Umbach and Siedow (33).

Braz J Med Biol Res 31(6) 1998 Mitochondrial alternative oxidase 737

bands originate from multiple genes (iso- Control of AOX synthesis forms) or from a single gene by differential processing or by post-translational modifi- Various events can influence the amount cation of the protein. The existence of three of AOX protein present in a cell. The devel- AOX genes has been recently demonstrated opmental state plays a role in plants, as in in soybean using a polymerase chain reac- thermogenic Araceae, where the amount of tion approach (34,35). Differential expres- AOX increases during flowering (36). In sion of these genes has been shown in soy- non-thermogenic soybean cotyledons, AOX bean tissues, explaining the presence of mul- activity increases immediately after germi- tiple bands on immunoblots. Presently, it is nation and during senescence (20). During suggested that AOX multigene families may the phase of growth in amoeba A. castellanii be a common feature in plants (34,35). cell culture, a parallel exists between AOX activity and protein expression that decreases AOX activity with the age of the amoeba culture, reaching a stationary phase (14). Under a large variety The activity of AOX is controlled by of stress treatments like chilling in tobacco, several parameters among which we can wounding in potato tubers, osmotic stress, distinguish regulatory events and substrate etc. the expression of the AOX protein is availability (Figure 3). increased (9,19,37). In cell cultures AOX Regulation of AOX activity can occur at can be induced when the cytochrome path- different levels: i) gene expression that af- way activity is decreased, either with specif- fects the amount of the protein in the mem- ic inhibitors or by disruption of mitochon- brane and differential gene expression that drial protein synthesis (9,19). These results modifies the ratio between isoforms; ii) post- indicate that nuclear gene expression is sen- translational modifications of the protein (i.e. sitive to the activity of the cytochrome path- its redox status that affects the nature of the way and that a communication must exist dimer); iii) the action of allosteric effectors between mitochondria and the nucleus. In like pyruvate. yeast, it has been shown that the ability of an The substrate availability is linked to the inhibitor of the cytochrome pathway to in- concentration of total quinone in the inner duce AOX synthesis (38) is linked to its mitochondrial membrane, the redox state of ability to cause superoxide formation during quinone in the membrane, and O2 concentra- mitochondrial respiration. Superoxide anion tion. itself could induce synthesis of AOX protein

Figure 3 - Mechanism of regula- Gene AOX tion of the alternative oxidase activity. expression synthesis Q concentration

Matrix AOX Electron flux Reducing Post-translational Redox reducing activity Qred/Qtot in respiratory power modification status power chain supply

Cell Allosteric Allosteric Oxygen metabolism activator status supply

Braz J Med Biol Res 31(6) 1998 738 F.E. Sluse and W. Jarmuszkiewicz

(39) and be the messenger between mito- like dithiothreitol (DTT) (20). Reduction of chondria and the nucleus. In tobacco cell the AOX protein can also occur during oxi-

cultures, H2O2 increased levels of AOX dation of specific Krebs cycle substrates like mRNA and AOX activity (40). Salicylic acid, isocitrate and malate that are able to reduce a cellular signal required for the induction of NADP+ in plant mitochondria (44). Thus, it disease resistance in plants, can also be re- has been proposed that the AOX reduction sponsible for AOX synthesis (41,42). It has state depends on NADPH generation and been suggested that salicylic acid-inhibiting involves NADPH-reduced glutathione or the

catalase activity increases the level of H2O2 thioredoxin coupling system (10,20,32,44). which in turn would stimulate gene expres- It has recently been shown that the redox sion including AOX genes (21). state of AOX in vivo is different from that Differential expression of three AOX determined in isolated mitochondria due to genes in soybean has been shown to be spontaneous oxidation of the reduced spe- tissue-dependent and physiological state-de- cies of the protein during organelle isolation pendent (during cotyledon greening) (35). (45). Control of the redox status of AOX Variation in relative abundance of AOX tran- could be a powerful mechanism of regula- scripts is generally correlated with the amount tion of its activity in vivo that may link AOX of protein isoforms detected by immuno- activity to the general redox state of the cell blotting in roots and cotyledons. The three (i.e., an increase in the reducing power isoforms (AOX1-3) are found in cotyledons (NADH, NADPH) induces activation of whereas in roots only AOX3 is present. A AOX). difference in the proportion of AOX isoforms leads to variation in the AOX proper- Regulation by allosteric effectors ties between tissues (basal activity, pyruvate 18 stimulation, O2 discrimination; see below). In plant mitochondria, short ketocarboxy- Moreover, the presence of three types of lic acids such as pyruvate, hydroxypyruvate AOX subunits in the same tissue produces and glyoxylate can activate AOX (9,46,47). heterodimers (32,35) and could provide a Activation by these short ketoacids does not very flexible mechanism of regulation of involve their metabolism as is the case for AOX activity in response to cellular meta- succinate and malate that are not effectors of bolic states. AOX isoforms and their hetero- AOX (46,47). Pyruvate is the most effective dimerism may be a general feature in plants. activator of plant AOX with a half maximal stimulation at 0.1 mM with intact mitochon- Regulation by redox states dria and at less than 5 µM when used with inverted submitochondrial particles (47). It It has been demonstrated that two types has been concluded that pyruvate acts from of dimeric structure of AOX exist, an oxi- the matrix side of mitochondria (47,48). Re- dized form and a reduced form (32). The cent results indicate that activation of AOX reduced form can be four- to five-fold more by pyruvate involves formation of a thio- active than the oxidized form (18,32). The hemiacetal with the sulfhydryl group of the ratio of oxidized to reduced protein varies cysteine residue just upstream of the first considerably between species and tissues. In membrane helix of the protein (Figure 2). cotyledon soybean mitochondria AOX is This is a potential allosteric effector binding mainly reduced, while in root soybean mito- site of the AOX protein (33). Pyruvate stim- chondria it is 50% oxidized (43). In tobacco ulation is reversible and is observed in mito- leaf mitochondria, AOX is largely oxidized chondria where AOX is in its reduced form, and its activity is unmasked by reductants as in soybean cotyledons (20). In tobacco

Braz J Med Biol Res 31(6) 1998 Mitochondrial alternative oxidase 739 leaf mitochondria, with a very low AOX of AOX protein from N. crassa (12) and H. activity largely present in the membrane in anomala (61) indicates lack or displacement the oxidized form, no effect of pyruvate is of the two regulatory cysteine residues con- observed until reduction of the protein by served in all plant AOX proteins. DTT occurs. This observation has led to the conclusion that the two systems of AOX Quinone concentration dependence regulation (i.e., redox state of the protein and pyruvate binding) interact with each other As every enzyme, AOX is controlled by modulating AOX activity. The AOX dimer its substrate-product concentrations. Thus, must be in its reduced form to be activatable the actual concentration of Q and QH2 in the by pyruvate, and the reduced form of the inner mitochondrial membrane is also im- enzyme reveals little activity in the absence portant. The Q content in soybean cotyledon of pyruvate. mitochondria is 3.5 times higher than the Q The effect of pyruvate consists in a low- content in soybean root mitochondria (35,62). ering of the range of the ubiquinone redox For instance, even if succinate can reduce state over which the AOX is active in iso- ubiquinone by 90% in both tissues, the con- lated mitochondria. It has been attributed to centration of ubiquinol present in root mito- lowering the so-called “effective” Km of AOX chondria will be only 30% of that found in for its reduced substrate, ubiquinol (49). This cotyledon mitochondria. This difference in should be understood as an increase in the QH2 concentration may be responsible for a reactivity of the enzyme towards QH2 which markedly lower AOX activity in root mito- does not exclude the effect of pyruvate on chondria. This may indicate that ubiquinone the catalytic constant of the enzyme. The concentration can be a limiting factor of link between the two regulatory mechanisms electron transfer between a dehydrogenase (i.e., redox state of the protein and allosteric and AOX. However, stimulation by pyru- effect of pyruvate) is such that the active vate (see above) is more important in root reduced AOX dimer will slowly oxidize mitochondria whereas it is rather limited in ubiquinol until pyruvate is present on its cotyledon mitochondria in the presence of binding site. the AOX reductant, DTT (35,43). There- In microorganisms such as Acanthamoeba fore, it seems that reduced quinone concen- castellanii (50-52), Euglena gracilis (53), tration is virtually sufficient to saturate AOX Moniliella tomentosa (54,55), Paramecium in cotyledon mitochondria while in root mi- tetraurelia (56), Neurospora crassa (57), tochondria, pyruvate, which lowers the and Hansenula anomala (58) the alternative amount of ubiquinol required for AOX ac- oxidase is stimulated by purine nucleotides tivity (see above), acts in such a way that that probably act from outside the inner mi- ubiquinol also saturates AOX. These results tochondrial membrane (56,59). In mitochon- illustrate the interplay between substrate dria of the amoeba A. castellanii, stimulation (QH2) availability and allosteric effector of AOX activity by GMP resembles the pyru- (pyruvate) presence indicating some interac- vate effect on plant mitochondria (affecting tions between their binding sites. AOX activity vs quinone redox state) (60). In amoeba mitochondria, the AOX activity Quinone redox state dependence which is not sensitive to pyruvate also seems not to be regulated by the redox status of the Early studies using inhibitors (63,64) of protein (14). It may be a general feature of both oxidase pathways indicated that the AOX in microorganisms, especially because AOX pathway was active only when the comparison of known amino acid sequences cytochrome pathway activity was low, i.e., in

Braz J Med Biol Res 31(6) 1998 740 F.E. Sluse and W. Jarmuszkiewicz

the absence of ADP (state 4) or in the pres- NADH and succinate in the kinetic relation- ence of cytochrome chain inhibitors. On the ship between AOX activity and Qred/Qtot other hand, inhibition of AOX apparently did disappears in the presence of pyruvate (70). not modify the activity of the cytochrome

pathway. Thus, AOX activity appeared to be O2 concentration dependence controlled by the activity of the cytochrome

pathway, giving rise to the overflow para- Because of the O2 concentration in the digm: AOX is only active when the cyto- incubation medium widely used to study chrome pathway is saturated or close to being plant mitochondria (about 200-250 µM at air saturated (63-66). Measurements of Q reduc- saturation), oxygen reduction by AOX should

tion state during succinate oxidation by soy- be independent of the O2 concentrations. bean cotyledon mitochondria have shown Indeed, the apparent affinity of AOX for O2 that AOX becomes active only when the Q is high (apparent oxygen Km around 1-2 µm) pool is 40-50% reduced, and then its activity (71-73) and it is lower than the cytochrome

increases sharply and non-linearly with higher oxidase affinity (apparent oxygen Km around Q reduction level (67,68). In contrast, the rate 0.1-0.15 µM) (72,74,75). At variance, much

of state 3 respiration is linearly related to the higher values of apparent Km of AOX for O2 degree of Q reduction (reduced Q (Qred) have been reported (76), ranging from 10 to versus amount of total Q in the membrane 20 µM depending on species, tissues and age (Qtot); Qred/Qtot) and reaches its largest of the plants investigated. It has also been

measured values at a low Qred/Qtot ratio at shown that the apparent Km of AOX for O2 which the AOX activity in the presence of a varied with quinone reduction level: the more cytochrome pathway inhibitor is not detect- reduced the quinone pool the lower is the

able (68). In the absence of inhibitors, the rate affinity for O2. A kinetic model fitting these of state 4 respiration versus Qred/Qtot is data implies two sequential two-electron re- linear until AOX becomes active for a Qred/ duction steps of AOX (four-electron-reduced Qtot ratio higher than 50%, and after this enzyme) and an irreversible activation step

point AOX increases its activity dramati- of AOX occurs before the reduction of O2 to cally. The relationship between respiratory water (76). rate and Qred/Qtot in the cytochrome path- In summary, the regulation of electron way (in state 4 respiration, with BHAM) is flow through the mitochondrial alternative linear (69). To our knowledge, no results pathway is very complex: on the one hand, concerning the dependence of the cytochrome the maximum level of AOX activity is a pathway activity in state 3 respiration (in the function of both the amount of AOX protein presence of BHAM) on ubiquinone reduc- present in the membrane and its redox status tion level have been published. This general (post-translational modification), and on the behavior described above is not identical in other hand, the level of engagement of the mitochondria originating from every plant pathway is dependent on the amount of species or tissue. Indeed, different responses ubiquinone and on its redox state together of AOX activity to the Q redox state may with the protein’s allosteric status as deter- occur depending on the redox state of the mined by pyruvate concentration. Moreover, AOX protein and on the presence of allo- protein redox status and allosteric status are steric activators. Hence, the variety of AOX not independent. A precise understanding of activity widely described with different sub- the interplay and quantitative analysis of strates in different mitochondria cannot only these various levels of regulation can be be explained by changes in Q redox state. For reached only by measurements of the actual instance, the difference between external activities of the two branching oxidases dur-

Braz J Med Biol Res 31(6) 1998 Mitochondrial alternative oxidase 741 ing steady-state respiration (i.e. electron par- oxidizing pathways is no longer considered titioning). valid. Consequently, most of the electron partitioning determinations made in the past Electron partitioning using only rate measurements in the presence or absence of inhibitors must be con- The interplay of the various levels of sidered as misestimated. In order to deter- regulation of AOX will influence not only mine electron partitioning between AOX and AOX activity but also the way the electrons the cytochrome pathway it is necessary to are distributed between the alternative and measure the true contributions of both path- cytochrome pathways (i.e. electron parti- ways to total respiration when both are ac- tioning). Indeed, it is obvious that an in- tive. These measurements must be performed crease in AOX activity at a constant quinone- using well-defined experimental conditions reducing pathway rate due to an increase in order to control all the factors that are either in the affinity of AOX for ubiquinol or known to potentially affect both oxidizing in the AOX maximal rate will modify the pathway activities. Only in this way will an flux of electrons through the cytochrome accurate quantitative description of the res- pathway. Thus, regulation of AOX activity piratory network be obtained. Several strate- also means regulation of electron partition- gies have been developed to measure the ing. The only way to describe quantitatively true contributions of branching pathways electron partitioning is to measure the actual not only in vitro but also in vivo. contribution of each branching oxidase. This determination is hampered by the fact that Kinetic approach both oxidative pathways have the same substrates (QH2, O2) and the same products (Q, A kinetic approach has been developed H2O). Previous estimations of partitioning (69) taking into account the interplay be- were based on the use of inhibitors of both tween quinol-oxidizing and quinone-reduc- pathways (63,64). As mentioned before, the ing electron fluxes in the total steady-state usefulness of inhibitor studies was based on respiratory rate and the redox state of quinone. the concept that the alternative pathway be- This method assumes a homogenous redox haves as an electron “sink” (overflow) (65,66) state of the quinone pool in the inner mito- and that it is kinetically unfavored, being chondrial membrane as the common link unable to compete with the cytochrome path- between the reducing and oxidizing branches way for QH2. This overflow paradigm used of the network and allows a prediction of the to be such a strong dogma that it hid the true contribution of each pathway. Practi- obvious fact that inhibition of one pathway cally, the method implies separate inhibitor inevitably affects the Qred/Qtot ratio and titrations of the dehydrogenase, the cyto- then modifies electron flux in the other path- chrome pathway and AOX together with the way. Fortunately, the recent information on determination of the redox state of ubiquinone regulation of AOX has led to a reexamina- in each of these situations (for a graphic tion of the overflow assumptions (69,77,78). representation see Figure 6C in Van den Numerous studies have demonstrated that Bergen et al. (69)). The dehydrogenase ki- several factors enhance AOX activity (see netic curve versus Qred/Qtot ratio is ob- above) and then enable it to compete with an tained by inhibiting overall electron flux unsaturated cytochrome pathway. Thus, with an oxidase inhibitor (e.g. AOX by measurement of O2 consumption rates in the SHAM or BHAM, and the cytochrome path- presence of inhibitors to ascertain the elec- way by KCN or myxothiazol) that increases tron partitioning between branched quinol- the Qred/Qtot ratio and consequently de-

Braz J Med Biol Res 31(6) 1998 742 F.E. Sluse and W. Jarmuszkiewicz

creases the rate of dehydrogenase activity approach has been useful to emphasize the (product inhibition). The cytochrome path- inadequacy of the classic inhibitor ap- way curve is obtained in the presence of proaches (21,69). SHAM by decreasing the overall electron flux with a dehydrogenase inhibitor (e.g. Oxygen isotope differential malonate for the succinate dehydrogenase) discrimination that decreases the Qred/Qtot ratio and consequently the activity of the cytochrome path- Another method based on the fraction- way (decrease in substrate availability). The ation of oxygen isotopes which can occur AOX curve is obtained in the presence of during plant mitochondrial respiration has KCN by decreasing the overall electron flux been developed (79-83). Fractionation takes with a dehydrogenase inhibitor. The sum of place because molecules containing the the cytochrome pathway curve and the AOX lighter isotope react a little more readily than curve gives the total oxidizing pathway curve molecules that contain the heavier isotope. when Qred/Qtot decreases. The total non- Respiratory discrimination is determined by inhibited steady-state respiration is given by observing progressive changes in isotopic the cross-point of the dehydrogenase curve composition of oxygen within a closed sys- and the total oxidizing pathway curve. This tem (i.e. the build-up of 18O16O relative to intersection gives the Qred/Qtot ratio in the 16O16O after consumption of a substantial absence of inhibitor when both pathways are proportion of the available oxygen). A sub- active. The steady-state when AOX is blocked stantial difference found in the oxygen iso- is given by the intersection of the dehydro- tope discrimination of AOX and cytochrome genase curve and the cytochrome pathway oxidases, with AOX showing a higher frac- curve, while the steady-state when the cyto- tionation factor (79), has led to development chrome pathway is blocked is given by the of a mass spectrophotometric technique that intersection of the dehydrogenase curve and is used to estimate directly steady-state par- the AOX curve. The true contribution of the titioning of electrons between the two oxi- cytochrome pathway to non-inhibited steady- dases. This noninvasive method allows the state respiration is the rate obtained on the measurement of the contribution of both cytochrome pathway curve for Qred/Qtot oxidizing pathways to total respiration (in without inhibitors, when both oxidizing path- the absence of inhibitors) in isolated mito- ways are active, while the true contribution chondria (43,79,80,83) as well as in intact of the alternative pathway is the rate ob- tissues (81,82). tained on the AOX curve for the same Qred/ In order to determine the degree of elec- Qtot ratio. Thus, the kinetic approach de- tron partitioning to the AOX and cytochrome scribed above allows a prediction of the real pathways, the discrimination (D) factors for contribution of each quinol-oxidizing path- the cytochrome pathway (Dc) and for AOX way at any given uninhibited total steady- (Da) must be measured separately in the state rate. presence of an inhibitor of the other pathway However, although this method is pow- (Dc measured in the presence of SHAM, Da erful, a new set of curves must be rebuilt for measured in the presence of KCN) (79). every chosen or encountered experimental These factors represent the limits within condition. Apart from this heavy work, the which the overall respiratory discrimination main default of this method is its basic hy- factor (in the absence of inhibitors) (Dn) pothesis of a quinone homogenous pool must be found. Various D factors are calcu- that has never been demonstrated and lated from the isotopic ratios according to never disproved. Nevertheless, the kinetic the equation:

Braz J Med Biol Res 31(6) 1998 Mitochondrial alternative oxidase 743

lnR/Ro ADP/O method D (‰) = ______x 1000 - lnf Recently, a method based on the non- where R is the isotopic ratio of oxygen at the phosphorylating property of AOX has been sampling time, Ro is the initial isotopic ratio, successfully developed and used to deter- and f is the fraction of remaining oxygen in mine contributions of the two quinol-oxidiz- the reaction cell (79). Knowing the values of ing pathways to total respiration in isolated Dn, Da, and Dc, the partitioning coefficient amoeba mitochondria (84,85). This method A can be calculated according to the equa- involves pair measurements of the ADP/O tion: ratio in the presence or in the absence of an (Dn - Dc) AOX inhibitor (e.g. BHAM) and measure- A (%) = ______x 100 (Da - Dc) ments of the overall state 3 respiration in the presence of GMP (an activator of AOX in Measurements in material such as Chlamy- amoebae) and succinate as oxidizable sub- domonas reinhardtii which lacks cytochrome strate (plus rotenone). This method was first oxidase by mutation confirm that the unin- proposed some time ago (63,86) but never hibited discrimination factor (Dn = 25‰) used with success although it avoids the use represents the AOX discrimination factor of the rates of electron transport of both (Da = 24.2‰), indicating that respiratory pathways in the presence of inhibitors and flux is solely through AOX and that the moreover it is not based on the homogenous obtained values are no artifacts caused by quinone pool hypothesis. If inhibitors (82). V = V + V The oxygen isotope fractionation tech- 3 cyt alt nique showed that AOX can be engaged in where V3 is steady-state 3 respiration, Vcyt is respiration under state 3 conditions when the contribution of the cytochrome pathway, Valt cytochrome pathway is not saturated (83), is contribution of AOX and if and it quantified the effect of several regula- ADP tory factors on electron partitioning into ------(ADP/O)overall Ocyt + Oalt Ocyt Vcyt AOX. Moreover, this method allows the il- α = ------= ------= ------= ------(ADP/O)cyt (+BHAM) ADP Ocyt + Oalt Vcyt +V alt lustration of tissue dependence of this parti------O tioning and the comparison of intact tissue cyt and isolated mitochondria partitioning coef- where Ocyt, Oalt = amount of oxygen taken up ficients (43,83). related to the activity of the cytochrome and This very elegant method is, at AOX pathways, respectively; then, present, the only one that can be used V V x α with intact tissues or with whole plants. cyt = 3 V V - V However, the calibration used in this alt = 3 cyt method requires the use of inhibitors for The ADP/O method is valid only if: i) the each respiratory pathway and assumes that ADP/O ratio in the presence of cyanide is change in the total respiration rate does equal to zero, ii) BHAM does not induce a not change the discrimination factor. proton leak (has no uncoupling effect), and Moreover, the narrowness between the thereby does not affect mitochondrial trans- end points (i.e., Dc and Da) limits the sensi- membrane potential, iii) the ADP/O ratio in tivity of the method and the time-consuming the presence of BHAM is independent of the measurements will prevent its use for kinetic state 3 respiratory rates (within the applied studies of electron partitioning in various range), and iv) isolated mitochondria are conditions. well coupled and stable during the experi-

Braz J Med Biol Res 31(6) 1998 744 F.E. Sluse and W. Jarmuszkiewicz

Figure 4 - Contributions of the intact tissues. It has been used with isolated 200 cytochrome pathway (Vcyt) and phosphorylating mitochondria and could be of the alternative pathway (Valt) to state 3 respiration (V3). applied to permeabilized cells. 150 In summary, at present, three different methods are available to study electron par- alt + V Vcyt titioning between branching oxidizing path- 100 V cyt ways. Each of the described methods has its

Contributions own domain of application because of its

50 own limitation or restraining basic hypoth- Valt esis. Nevertheless, together they offer the best possibilities to study in depth the mech- 0 anism of regulation of electron partitioning. 0 50 100 150 200 V3 Role of the alternative oxidase

mental procedure. All these requirements The only obvious physiological function are positively verified for amoeba mitochon- of AOX can be recognized in specialized dria (84,85). The ADP/O method allows the plant thermogenic tissues (spadices of determination of the contributions of both Araceae) as heat generation related to in- pathways to overall state 3 respiration when crease in temperature taking part in the re- it is decreased by increasing the concentra- productive processes (87). In non-thermo- tion of n-butyl malonate, a non-penetrating genic tissues or cells the role of AOX is now inhibitor of succinate uptake by mitochon- beginning to be better understood thanks to dria. When overall state 3 respiration de- new information concerning its mechanisms clines the AOX contribution decreases of regulation. Indeed, in these cells AOX sharply and becomes negligible (when state must play a more fundamental role at the 3 is lower than 100 nmol O min-1 mg pro- level of metabolism, given the biochemical tein-1) while the cytochrome pathway contri- controls to which the activity of this enzyme bution first increases, then passes through a is submitted (described above). Metabolic maximum (at state 3 rate around 160 nmol O conditions that lead to an increase of the min-1 mg protein-1) and sharply decreases for reducing power in the cell will increase the lower state 3 respiration (Figure 4). These Qred/Qtot ratio, the level of mitochondrial results show that AOX is significantly en- NADPH (therefore the level of reduced AOX gaged before saturation of the cytochrome diner) and pyruvate (allosteric activator) and pathway, and consequently can compete for thus will increase electron partitioning to electrons with the unsaturated cytochrome AOX. Situations where the phosphate po- pathway. This represents the first attempt to tential is high (high ATP/ADP ratio) will examine in detail the steady-state kinetics of lead to the decrease of electron flux into the the two quinol-oxidizing pathways when both cytochrome pathway due to proton-electro- are active and to describe electron partition- chemical potential back pressure and conse- ing between them when the steady-state rate quently increase the partitioning to AOX. of the quinone-reducing pathway is varied. Such conditions (high reducing power and The ADP/O method, perhaps the best for ATP/ADP ratio) are consequences of imbal- detailed kinetic and regulation studies of ances between supply of reducing substrates branching oxidases since it is readily appli- and energy-carbon demand for biosynthesis, cable, cannot, however, be applied to state 4 both being coupled by the respiratory chain respiration and could hardly be used with activity. Operation of AOX may counteract

Braz J Med Biol Res 31(6) 1998 Mitochondrial alternative oxidase 745 these imbalances because it is not directly preventing over-reduction that leads to harm- controlled by the energy status of the cell ful reactive oxygen species production. AOX and may prevent fermentation and favor bio- could also play a role in mitochondrial-chlo- synthesis. These imbalances could be coun- roplastic interactions during photophospho- teracted by fast regulation of AOX activities rylation which increases the reducing power (allosteric and redox status of the protein) or (NADPH) and phosphate potential (ATP) in as a result of slow changes in the environ- the cell (90). In conclusion, AOX appears to ment (various stresses), by modification of have a central role in the balance of cell the amount of AOX protein (gene expres- energy metabolism. sion). The activity of AOX has been shown to prevent the formation of reactive oxygen Acknowledgments species by mitochondria at the level of ubiquinol when the cytochrome pathway is We thank FAPESP and the Brazilian So- slowed down (21,88,89). Thus, AOX may ciety of Biochemistry and Molecular Biol- play a protective role in mitochondria by ogy for their support and invitation.

References

1. Genevois ML (1929). Sur la fermentation alternative oxidase in plants and fungi. alternative oxidase of plant mitochondria. et sur la respiration chez les végétaux Australian Journal of Plant Physiology, 22: Biochimica et Biophysica Acta, 1059: 121- chlorophylliens. Revue Génétique Botani- 497-509. 140. que, 41: 252-271. 10. Siedow JN & Umbach AL (1995). Plant 17. Siedow JN, Whelan J, Kearns A, Wiskich 2. Lance C (1972). La respiration de l’Arum mitochondrial electron transfer and mo- JT & Day DA (1992). Topology of the alter- maculatum au cours du développement lecular biology. Plant Cell, 7: 821-831. native oxidase in soybean mitochondria. de l’inflorescence. Annales des Sciences 11. Lambowitz AM, Sabourin JR, Bertrand H, In: Lambers H & van der Plas LHW (Edi- Naturelles Botaniques, 12: 477-495. Nickels R & McIntosh L (1989). Immuno- tors), Molecular, Biochemical and Physi- 3. Meeuse BJD (1975). Thermogenic respi- logical identification of the alternative oxi- ological Aspects of Plant Respiration. SPB ration in Aroids. Annual Review of Plant dase of Neurospora crassa mitochondria. Academic Publishing, The Netherlands, Physiology, 26: 117-126. Molecular Cell Biology, 9: 1362-1364. 19-27. 4. Huq S & Palmer JM (1978). Isolation of a 12. Sakajo S, Minagawa N, Komiyama T & 18. Moore AL, Umbach AL & Siedow JN cyanide-resistant duroquinol oxidase from Yoshimoto A (1991). Molecular cloning of (1995). Structure-function relationships of Arum maculatum mitochondria. FEBS cDNA for antimycin A-inducible mRNA the alternative oxidase of plant mitochon- Letters, 95: 217-220. and its role in cyanide-resistant respira- dria: a model of the active site. Journal of 5. Rich PR (1978). Quinol oxidation in Arum tion in Hansenula anomala. Biochimica et Bioenergetics and Biomembranes, 27: maculatum mitochondria and its applica- Biophysica Acta, 1090: 102-108. 367-377. tion to the assay, solubilisation and partial 13. Clarkson AB, Bienen EJ, Pollakis G & 19. McIntosh L (1994). Molecular biology of purification of the alternative oxidase. Grady RJ (1989). Respiration of blood- the alternative oxidase. Plant Physiology, FEBS Letters, 96: 252-256. stream forms of the parasite Trypanoso- 105: 781-786. 6. Bonner Jr WD, Clarke SD & Rich PR ma brucei brucei is dependent on a plant- 20. Day DA & Wiskich J (1995). Regulation of (1986). Partial purification and character- like alternative oxidase. Journal of Biologi- alternative oxidase activity in higher ization of the quinol oxidase activity of cal Chemistry, 264: 17770-17776. plants. Journal of Bioenergetics and Arum maculatum mitochondria. Plant 14. Jarmuszkiewicz W, Wagner AM, Wagner Biomembranes, 27: 379-385. Physiology, 80: 838-842. JM & Hryniewiecka L (1997). Immuno- 21. Wagner AM & Krab K (1995). The alterna- 7. Elthon TE & McIntosh L (1986). Charac- logical identification of the alternative oxi- tive respiration pathway in plants: role and terization and solubilization of the alterna- dase of Acanthamoeba castellanii mito- regulation. Physiologia Plantarum, 94: tive oxidase of Sauromatum guttatum mi- chondria. FEBS Letters, 411: 110-114. 318-325. tochondria. Plant Physiology, 82: 1-6. 15. Rhoads DM & McIntosh L (1991). Isola- 22. Krab K (1995). Kinetic and regulatory as- 8. Elthon TE, Nickels RL & McIntosh L tion and characterization of a cDNA clone pects of the function of the alternative (1989). Monoclonal antibodies to the al- encoding an alternative oxidase protein of oxidase in plant respiration. Journal of ternative oxidase of higher plant mito- Sauromatum guttatum (Schott). Proceed- Bioenergetics and Biomembranes, 27: chondria. Plant Physiology, 89: 1311- ings of the National Academy of Sciences, 387-396. 1317. USA, 88: 2122-2126. 23. Vanlerberghe GC & McIntosh L (1997). 9. Day DA, Whelan J, Millar AH, Siedow JN 16. Moore AL & Siedow JN (1991). The regu- Alternative oxidase: from gene to func- & Wiskich JT (1995). Regulation of the lation and nature of the cyanide-resistant tion. Annual Review of Plant Physiology

Braz J Med Biol Res 31(6) 1998 746 F.E. Sluse and W. Jarmuszkiewicz

and Molecular Biology, 48: 703-734. chondrial alternative oxidase. Plant Physi- alternative oxidase of plant mitochondria. 24. Schonbaum GR, Bonner WD, Storey BT & ology, 114: 455-466. FEBS Letters, 329: 259-262. Bahr JT (1971). Specific inhibition of the 36. Elthon TE, Nickels RL & McIntosh L 47. Millar AH, Hoefnagel MHN, Day DA & cyanide-insensitive respiratory pathway in (1989). Mitochondrial events during de- Wiskich JT (1996). Specificity of the or- plant mitochondria by hydroxamic acids. velopment of thermogenesis in Sauroma- ganic acid activation of alternative oxidase Plant Physiology, 47: 124-128. tum guttatum (Schott). Planta, 180: 82- in plant mitochondria. Plant Physiology, 25. Parrish DJ & Leopold AC (1978). Con- 89. 111: 613-618. founding of alternate respiration by lip- 37. Laties GG (1982). The cyanide-resistant, 48. Day DA, Millar AH, Wiskich JT & Whelan oxygenase activity. Plant Physiology, 62: alternative pathway in plant mitochondria. J (1994). Regulation of alternative oxidase 470-472. Annual Review of Plant Physiology, 33: activity by pyruvate in soybean mitochon- 26. Siedow JN & Grivin ME (1980). Alterna- 519-555. dria. Plant Physiology, 106: 1421-1427. tive respiratory pathway. Its role in seed 38. Minagawa N, Koga S, Nakano M, Sakajo S 49. Umbach AL, Wiskich JT & Siedow JN respiration and its inhibition by propyl gal- & Yoshimoto A (1992). Generation of su- (1994). Regulation of alternative oxidase late. Plant Physiology, 65: 669-674. peroxide anion detected by chemilumi- kinetics by pyruvate and intermolecular 27. Grover SD & Laties GG (1978). Character- nescence method in the cyanide-insensi- disulfide bond redox status in soybean ization of the binding properties of disul- tive mitochondria and the induction of the seedling mitochondria. FEBS Letters, 348: firam, an inhibitor of cyanide-resistant res- cyanide-resistant respiration in the yeast 181-184. piration. In: Ducet G & Lance C (Editors), Hansenula anomala. In: Yagi H, Kondo M, 50. Hryniewiecka L, Jenek J & Michejda J Plant Mitochondria. Elsevier/North Hol- Niki E & Yoshikawa T (Editors), Oxygen (1978). Cyanide resistance in soil amoeba land Biomedical Press, Amsterdam, 259- Radicals. Elsevier, New York, 203-206. Acanthamoeba castellanii. In: Ducet G & 266. 39. Minagawa N, Koga S, Nakano M, Sakajo S Lance C (Editors), Plant Mitochondria. 28. Huq S & Palmer JM (1978). Superoxide & Yoshimoto A (1992). Possible involve- Elsevier/North-Holland, Amsterdam, New and hydrogen peroxide production in the ment of superoxide anion in the induction York, Oxford, 307-314. cyanide resistant Arum maculatum mito- of cyanide-resistant respiration in Hanse- 51. Hryniewiecka L & Michejda J (1988). In chondria. Plant Science Letters, 11: 351- nula anomala. FEBS Letters, 302: 217- amoeba mitochondria phosphate mono- 358. 219. nucleotides stimulate specially the alter- 29. Siedow JN (1982). The nature of the cya- 40. Vanlerberghe GC & McIntosh L (1996). native respiratory pathway but not the ro- nide-resistant pathway in plant mitochon- Signals regulating the expression of the tenone-sensitive NADH dehydrogenase. dria. Recent Advancements in Phy- nuclear gene encoding alternative oxidase 5th European Bioenergetic Conference tochemistry, 16: 47-83. of plant mitochondria. Plant Physiology, Reports, Aberystwyth, UK, 20-26 July 30. Minagawa N, Sakajo S, Komiyama T & 111: 589-595. 1998, 286 (Abstract). Yoshimoto A (1990). A 36-kDa mitochon- 41. Rhoads DM & McIntosh L (1992). Salicylic 52. Edwards SW & Lloyd D (1978). Properties drial protein is responsible for cyanide- acid regulation of respiration in higher of mitochondria isolated from cyanide- resistant respiration in Hansenula ano- plants: alternative oxidase expression. stimulated and cyanide-sensitive cultures mala. FEBS Letters, 264: 149-152. Plant Cell, 4: 1131-1139. of Acanthamoeba castellanii. Biochemical 31. Siedow JN, Umbach AL & Moore AL 42. Rhoads DM & McIntosh L (1993). Cyto- Journal, 174: 203-211. (1995). The active site of the cyanide-re- chrome and alternative pathway respira- 53. Sharpless TK & Butow RA (1970). An in- sistant oxidase from plant mitochondria tion in tobacco: Effects of salicylic acid. ducible alternative terminal oxidase in contains a binuclear iron center. FEBS Let- Plant Physiology, 103: 877-883. Euglena gracilis mitochondria. Journal of ters, 362: 10-14. 43. Ribas-Carbo M, Lennon AM, Robinson Biological Chemistry, 245: 58-70. 32. Umbach AL & Siedow JN (1993). Cova- SA, Giles L, Berry JA & Siedow JN (1997). 54. Hanssens L & Verachtert H (1976). Aden- lent and non-covalent dimers of the cya- The regulation of electron partitioning be- osine 5’-monophosphate stimulates cyanide-resistant alternative oxidase protein tween the cytochrome and alternative nide-insensitive respiration in mitochon- in higher plant mitochondria and their re- pathways in soybean cotyledon and root dria of Moniliella tomentosa. Journal of lationship to enzyme activity. Plant Physi- mitochondria. Plant Physiology, 113: 903- Bacteriology, 125: 829-836. ology, 103: 845-854. 911. 55. Vanderleyden J, Van den Eynde E & 33. Umbach AL & Siedow JN (1996). The re- 44. Vanlerberghe GC, Day DA, Wiskich JT, Verachtert H (1980). Nature of the effect action of the soybean cotyledon mito- Vanlerberghe AE & McIntosh L (1995). of adenosine 5’monophosphate on the chondrial cyanide-resistant oxidase with Alternative oxidase activity in tobacco leaf cyanide-insensitive respiration in mito- sulfhydryl reagents suggests that α-keto mitochondria: dependence on tricarboxy- chondria of Moniliella tomentosa. Bio- acid activation involves the formation of a lic acid cycle-mediated redox regulation chemical Journal, 186: 309-316. thiohemiacetal. Journal of Biological and pyruvate activation. Plant Physiology, 56. Doussiere J & Vignais PV (1984). AMP- Chemistry, 271: 25019-25026. 109: 353-361. dependence of the cyanide-insensitive 34. Whelan J, Millar AH & Day DA (1996). The 45. Umbach AL & Siedow AL (1997). Changes pathway in the respiratory chain of Para- alternative oxidase is encoded in a multi- in the redox state of the alternative oxi- mecium tetraurelia. Biochemical Journal, gene family in soybean. Planta, 198: 197- dase regulatory sulfhydryl/disulfide sys- 220: 787-794. 201. tem during mitochondrial isolation: impli- 57. Vanderleyden J, Peeters C, Verachtert H 35. Finnengan PM, Whelan J, Millar AH, cations for inferences of activity in vivo. & Bertrandt H (1980). Stimulation of the Zhang Q, Smith K, Wiskich J & Day DA Plant Science, 123: 19-28. alternative oxidase of Neurospora crassa (1997). Differential expression of the mul- 46. Millar AH, Wiskich JT, Whelan J & Day by nucleoside phosphates. Biochemical tigene family encoding the soybean mito- DA (1993). Organic acid activation of the Journal, 188: 141-144.

Braz J Med Biol Res 31(6) 1998 Mitochondrial alternative oxidase 747

58. Sakajo S, Minagawa N & Yoshimoto Y 69. Van den Bergen CWM, Wagner AM, Krab 442-453. (1997). Effects of nucleotides on cyanide- K & Moore AL (1994). The relationship 81. Robinson SA, Yakir D, Ribas-Carbo M, resistant respiratory activity in mitochon- between electron flux and the redox poise Giles L, Osmond CB, Siedow JT & Berry dria isolated from antimycin A-treated of the quinone pool in plant mitochondria. JA (1992). Measurements of the engage- yeast Hansenula anomala. Bioscience of European Journal of Biochemistry, 226: ment of cyanide-resistant respiration in Biotechnology and Biochemistry, 61: 397- 1071-1078. crassulacean acid metabolism plant Ka- 399. 70. Hoefnagel MHN & Wiskich JT (1996). Al- lanchoe daigremontiana with the use of 59. Vanderleyden J, Kurth J & Verachtert H ternative oxidase activity and the ubiqui- on-line oxygen isotope discrimination. (1979). Characterization of cyanide-insen- none redox level in soybean cotyledon Planta, 164: 415-422. sitive respiration in mitochondria and sub- and Arum spadix mitochondria during 82. Robinson SA, Ribas-Carbo M, Yakir D, mitochondrial particles of Moniliella NADH and succinate oxidation. Plant Giles L, Reuveni Y & Berry JA (1995). tamentosa. Biochemical Journal, 182: Physiology, 110: 1329-1335. Beyond SHAM and cyanide: opportuni- 437-443. 71. Ikuma H, Schindler FJ & Bonner WD ties for studing the alternative oxidase in 60. Jarmuszkiewicz W, Wagner AM & (1964). Kinetic analysis of oxidases in plant respiration using oxygen isotope dis- Hryniewiecka L (1996). Regulation of the tightly coupled plant mitochondria. Plant crimination. Australian Journal of Plant activity of the alternative oxidase in Physiology, 39: S-1x. Physiology, 22: 487-496. amoeba A. castellanii mitochondria. Bio- 72. Bendall DS & Bonner WD (1971). Cya- 83. Ribas-Carbo M, Berry JA, Yakir D, Giles L, chimica et Biophysica Acta, 9th European nide-insensitive respiration in plant mito- Robinson SA, Lennon AM & Siedow JN Bioenergetic Conference Reports, chondria. Plant Physiology, 47: 236-245. (1995). Electron partitioning between the Louvain la Neuve, Belgium, 17-22 August 73. Millar AH, Bergersen FJ & Day DA (1994). cytochrome and alternative pathways in 1996, 36 (Abstract C. V-5). Oxygen affinity of terminal oxidases in plant mitochondria. Plant Physiology, 109: 61. Li Q, Ritzel RG, McLean LLT, McIntosh L, soybean mitochondria. Plant Physiology 829-837. Ko T, Bertrand H & Nargang FE (1996). and Biochemistry, 32: 847-852. 84. Jarmuszkiewicz W, Sluse-Goffart CM, Cloning and analysis of the alternative oxi- 74. Barzu O & Satre M (1970). Determination Hryniewiecka L, Michejda J & Sluse FE dase gene of Neurospora crassa. Genet- of oxygen affinity of respiratory systems (1996). Determination of the respective ics, 142: 129-140. using oxyhemoglobin as oxygen donor. contributions of the cytochrome and al- 62. Ribas-Carbo M, Wiskich JT, Berry JA & Analytical Biochemistry, 36: 428-433. ternative oxidase pathways in Acantha- Siedow JN (1995). Ubiquinone redox be- 75. Rawsthorne S & LaRue TA (1986). Me- moeba castellanii mitochondria. In: haviour in plant mitochondria during elec- tabolism under microaerobic conditions Westerhoff HV, Snoep JL, Wijker J, Sluse tron transport. Archives of Biochemistry of mitochondria from cowpea nodules. FE & Kholodenko BN (Editors), and Biophysis, 317: 156-160. Plant Physiology, 81: 1097-1102. BioThermoKinetics of the Living Cell. BTK 63. Bahr JT & Bonner WD (1973). Cyanide- 76. Ribas-Carbo M, Berry JA, Azcon-Bieto J & Publishing, Amsterdam, The Netherlands, insensitive respiration. I. The steady Siedow JN (1994). The reaction of the 31-34. states of skunk cabbage spadix and bean plant mitochondrial cyanide-resistant al- 85. Jarmuszkiewicz W, Sluse-Goffart CM, hypocotyl mitochondria. Journal of Bio- ternative oxidase with oxygen. Biochimi- Hryniewiecka L, Michejda J & Sluse FE logical Chemistry, 248: 3441-3445. ca et Biophysica Acta, 1188: 205-212. (1998). Electron partitioning between the 64. Bahr JT & Bonner WD (1973). Cyanide- 77. Day DA, Krab K, Lambers H, Moore AL, two branching quinol-oxidizing pathways insensitive respiration. II. Control of the Siedow JN, Wagner AM & Wiskich JT in Acanthamoeba castellanii mitochondria alternative pathway. Journal of Biological (1996). The cyanide-resistant oxidase: to during steady-state 3 respiration. Journal Chemistry, 248: 3446-3450. inhibit or not to inhibit, that is the ques- of Biological Chemistry (in press). 65. Lambers H (1980). The physiological sig- tion. Plant Physiology, 110: 1-2. 86. Lambowitz AM, Smith EW & Slayman CW nificance of cyanide-resistant respiration 78. Millar AH, Atkin OK, Lambers H, Wiskich (1972). Oxidative phosphorylation in Neu- in higher plants. Plant Cell and Environ- JT & Day DA (1995). A critique of the use rospora mitochondria. Studies on wild- ment, 3: 293-302. of inhibitors to estimate partitioning of type, poky and chloramphenicol induced 66. Lambers H (1982). Cyanide-resistant res- electrons between mitochondrial respira- wild type. Journal of Biological Chemis- piration: a non-phosphorylating electron tory pathways in plants. Physiologia try, 247: 4859-4865. transport pathway acting as an energy Plantarum, 95: 523-532. 87. Meese BJD (1975). Thermogenic respira- overflow. Physiologia Plantarum, 55: 478- 79. Guy RG, Berry JA, Fogel ML & Hoerinng tion in aroids. Annual Review of Plant 485. TC (1989). Differential fractionation of oxy- Physiology, 26: 117-126. 67. Moore AL, Dry IB & Wiskich JT (1988). gen isotopes by cyanide-resistant and cya- 88. Purvis AC & Shewfelt RL (1993). Does Measurement of the redox state of the nide-insensitive respiration in plants. the alternative pathway ameliorate chill- ubiquinone pool in plant mitochondria. Planta, 177: 483-491. ing injury in sensitive plant tissues? Physi- FEBS Letters, 235: 76-80. 80. Guy RG, Berry JA, Fogel ML, Turpin DH & ologia Plantarum, 88: 712-718. 68. Dry JB, Moore AL, Day DA & Wiskich JT Weger HG (1992). Fractionation of the 89. Purvis AC (1997). Role of the alternative (1989). Regulation of alternative pathway stable isotopes of oxygen during respira- oxidase in limiting superoxide production activity in plant mitochondria: nonlinear tion by plants - the basis for a new tech- by plant mitochondria. Physiologia Planta- relationship between electron flux and the nique. In: Lambers H & van der Plas LHW rum, 100: 165-170. redox poise of the quinone pool. Archives (Editors), Molecular, Biochemical and 90. Kromer S (1995). Respiration during pho- of Biochemistry and Biophysics, 273: 148- Physiological Aspects of Plant Respira- tosynthesis. Annual Review of Plant Phys- 157. tion. SPB Academic Publishing, Hague, iology and Molecular Biology, 46: 45-70.

Braz J Med Biol Res 31(6) 1998