Hydrogen Peroxide Produced by Mitochondrial Monoamine Oxidase Catalysis: Biological Implications

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Send Orders for Reprints to [email protected] Current Pharmaceutical Design, 2014, 20, 155-160 155 Hydrogen Peroxide Produced by Mitochondrial Monoamine Oxidase Catalysis: Biological Implications

Dale E. Edmondson*

Department of Biochemistry, Emory University School of Medicine, Rollins Research Building, 1510 Clifton Road, Atlanta, Georgia 30322 USA

Abstract: The biological roles of mitochondrial-produced reactive oxygen species continue to receive intensive investigation since one of the products (H2O2) has important cellular signaling roles as well as contributing to apoptotic responses. In general, the source of mitochondrial reactive oxygen species is thought to be the superoxide anion produced from Complex I and Complex III components of the electron transport chain. Superoxide anion readily dismutates to H2O2 with subsequent transformation to the hydroxyl radical by Fenton chemistry. An overlooked source of H2O2 in the mitochondrion is its production as a catalytic reaction product from the outer membrane enzymes: monoamine oxidases A and B. The literature is reviewed to document identified degenerative reactions attributed to H2O2 produced by MAO A and by MAO B catalysis. Available information on the topologies of these enzymes in the mitochondrial outer membrane is also discussed with relevance to H2O2 production and involvement in cell signaling functions as well as degenerative effects. Keywords: Monoamine oxidases A and B, amine oxidation, mitochondrial outer membrane, mitochondrial intermembrane space, membrane topology, reactive oxygen species, hydrogen peroxide, hydroxyl radical.

INTRODUCTION This review examines the properties of both isoenzymes and Although the mitochondrion in eukaryotic cells is usually con- summarizes the studies relating the biological consequences of their sidered to be the organelle of energy production for powering cell production of H2O2. In this context, only the production of H2O2 as metabolism, there is currently renewed emphasis on its role of pro- an ROS species will be considered with the expectation that ducing reactive oxygen species (ROS) that have biological signifi- this molecule can undergo Fenton-type chemistry to form the hy- cance in degenerative processes, cell apoptosis, and in cell signaling droxyl radical: + - [1]. A recent review has thoroughly described the myriad of proc- H + H2O2 + e HO + H2O esses thought to be involved in signaling using one member of In this regard, the author will adhere as closely as possible with ROS; hydrogen peroxide (H2O2) including immunity, autophagy, the recommendations outlined in a recently published perspective differentiation, and adaption to hypoxia [2]. Mitochondria-derived [5]. H2O2 is thought to be a resulting product from the dismutation of .- superoxide anion (O2 ) which occurs either enzymaticaly (mito- CATALYTIC REACTIONS OF MONOAMINE OXIDASES chondrial superoxide dismutase) or non-enzymaticaly following the MAO A and MAO B belong to the class of flavoenzyme oxi- well-known reaction: dases in that the oxidative reactions in catalysis utilize O2 as elec- .- 2 O2 H2O2 + O2 tron acceptor to form H2O2 (Fig.1). Neurotransmitters such as sero- The mitochondrial electron transport chain is thought to be the tonin, dopamine and norepinephrine are among the primary amines .- source of O2 with identified sites being Complex I (either the FMN that serve as electron donors which, on oxidation to their respective of NADH dehydrogenase or CoQ) and Complex III (the cyto- imines, are hydrolyzed to form their respective aldehydes and + chrome b-c1 complex) [1]. Estimates of the electron flux in respira- NH4 . Biogenic amines that are present in food are also substrates .- tion that result in the formation of O2 have been found to range which include tyramine and phenethylamine. MAO A and MAO B from 0.12-2% in vitro and are expected to be far lower in vivo [1]. exhibit differing specificities for these amine substrates with MAO A missing component in the above-cited reviews on the mitochon- A favoring serotonin and norepinephrine while MAO B exhibiting a drial production of H2O2 is the role of the mitochondrial outer higher activity with phenethylamine. Tyramine and dopamine are membrane bound enzymes monoamine oxidases (MAOs) which oxidized at comparable rates by either enzyme under physiological utilize O2 as an electron acceptor forming H2O2 as product. These conditions. Therefore, the primary function of these enzymes is in enzymes are expressed in differing levels among various tissues and the regulation of neurotransmitter levels and in the metabolism of their expression levels are known to be regulated with development ingested amines which could function as false neurotransmitters. [3]. Two forms of the enzyme (MAO A and MAO B) are found in The oxidative reaction in catalysis involves reoxidation of the mammals with ~70 % sequence identities and differing substrate reduced flavin cofactors of MAO A and MAO B by molecular oxy- and inhibitor specificities although there is some overlap in func- gen to form the respective oxidized enzymes and H2O2. MAO A tional properties. Using whole cell systems, Pizzinant et al. [4] have differs from MAO B in that it exhibits a KmO2 of ~10 M [6] while shown that both isoenzymes are capable of producing H2O2 that can MAO B exhibits a relatively high KmO2 of ~240 M [7]. The reac- 5 -1 be detected in the cell medium on addition of amine substrates tion of O2 with the free reduced forms of the enzymes is ~ 10 M -1 which supports the view that these enzymes should be considered min which is increased ~10-fold on reaction of O2 with the re- as sources of mitochondrial ROS. duced flavin-protonated imine complex. No evidence is found for .- the production of any O2 from either MAO A or MAO B catalytic turnover. The O2 reaction with the reduced flavin of either enzyme *Address correspondence to this author at the Department of Biochemistry, specifically results in the stoichiometric formation of H O as reac- Emory University School of Medicine, Rollins Research Building, 1510 2 2 tion product. Clifton Road, Atlanta, Georgia 30322 USA; Tel: 001-404-727-5972; Fax: 001-404-727-2738; E-mail: [email protected]

was also determined [13, 14] thereby providing structural insights O into both isoenzymes. Human MAO B crystallizes as a dimer and exhibits hydrodynamic properties in solution as a dimer of two R-CH2-CH identical subunits [10, 12]. In contrast, human MAO A crystallizes + as a monomer [13, 14] while rat MAO A crystallizes as a dimer O2 H2O2 + [15] (Fig. 2B & C). Probes of the oligomeric states of human MAO NH4 A and MAO B in their membrane bound forms using pulsed EPR R-CH2-CH2-NH2 spectroscopy [16] showed both enzymes as dimeric in the mito- MAO A or MAO B chondrial outer membrane as is rat MAO A. The apparent dissocia- Amine Substrates tion of human MAO A dimers to monomers apparently occurs on Serotonin solubilization and purification in aqueous detergent solutions and Norepinephrine the monomeric form is more readily crystallized while with rat Dopamine MAO A, the dimeric form crystallizes more readily. Tyramine The structural data show that MAO A and MAO B belong to Phenethylamine the class of “tail-anchored” membrane bound proteins [17] in which Fig. (1). The reaction catalyzed by monoamine oxidases A and B. their respective C-terminal domains form -helical trans-membrane structures that anchor the proteins to the mitochondrial outer mem- MOLECULAR PROPERTIES OF HUMAN MONOAMINE brane. The entrances to the catalytic sites for substrate entry appear OXIDASES A AND B to be at the surface of the outer membrane and the substrate and The gene sequences of MAO A and MAO B and their genetic flavin binding protein domains of either protein are in aqueous locations to the X-chromosome [8] answered a long standing ques- environments. The channels for O2 entry into their respective active tion that, they are in fact separately expressed enzymes in a tissue sites are not known. Both active sites contain a number of rigid specific manner and are bound to the mitochondrial outer mem- water molecules observable in diffraction data and probably also brane [9]. Human placenta mitochondria are known to contain pre- contain “mobile” water molecules which are not observable. dominately MAO A while human platelet mitochondria mainly Both MAO A and MAO B contain covalently bound 8--S- contain MAO B. Other tissues contain varying levels of both en- cysteinyl FAD cofactors that are absolutely required for catalysis zymes whose expression is dependent on developmental factors. In (Fig. 3). These flavins are reduced to their hydroquinone forms on the newborn, MAO A is initially expressed followed by the appear- oxidation of the amine substrate to the imine product. The reduced ance of MAO B [10]. On aging, MAO B levels are increased at flavin is subsequently reoxidized back to the oxidized form by reac- least 2-3-fold in neuronal tissue and MAO A levels are increased ~9 tion with O2 to form H2O2. Since ground state oxygen is in its trip- fold in cardiac tissue. As will be discussed below, this increased let state, current thought is that the initial step in the flavin reaction expression with age is thought to give rise to age-dependent neuro- is a one-electron transfer reaction to form a “radical pair” complex and cardio-degenerative diseases resulting from an increase in reac- of superoxide anion and neutral flavin semiquinone in a rate limit- tive oxygen species originating from the increased levels of H2O2. ing step which rapidly reacts to form either a flavin C (4a) peroxide An advance in the field of MAO A and MAO B came from intermediate or alternately, undergoes a proton-coupled electron development of expression and purification procedures that allowed transfer resulting in the formation of oxidized flavin and H2O2 [18]. the isolation of reagent quantities of MAO A and MAO B sepa- Oxidation-reduction potential measurement of the MAO B flavin rately without any contamination of one with the other [7,11]. Pre- shows an Eh value for the Flox/Flsq couple of +0.043 V and that for vious studies on tissues containing both enzymes had to rely on the Flsq/Flhq couple of + 0.037 V [19]. From these values, a 2- electron potential for the MAO B flavin is calculated to be + 0.04 isozyme specific inhibition as both isoenzymes could not be sepa- .- rately purified since they are ~70% identical in sequence and ex- V. The known potential for the single electron O2/O2 couple is - hibit similar monomeric molecular weights (~60 kDa). Soon after 0.137 V [20] which is considerably lower than that for the two- the successful expression and purification of MAO B, the enzyme electron O2/H2O2 couple of +0.28 V [21]. These values demonstrate the thermodynamic driving force for the production of H2O2 rather was crystallized and its structure determined [12] (Fig. 2A). In a .- short period of time, the 3-dimensional structure of human MAO A than O2 as the catalytic reaction product.

Fig. (2). Structures of human MAO B (PDB code# 2V5Z) (A.), human MAO A (PDB code# 2Z5X) (B.), and of rat MAO A (PDB code# 1O5W) (C.). All structures were constructed with Pymol from coordinates deposited in the Protein Data Base (PDB). Contribution of MAO-produced Hydrogen Peroxide to Mitochondrial Reactive Oxygen Current Pharmaceutical Design, 2014, Vol. 20, No. 2 157

Fig. (4). Cartoon depicting the topological orientations of MAO A and of MAO B in the rat liver mitochondrial outer membrane.

For this method of isolation to work, MAO A must be on the cytosolic face of the mitochondrial outer membrane since an antibody with an attached magnetic nanoparticle bead is expected to be too large to enter the intermembane space. To date, no similar experiments have been done with monoclonal antibodies specific for MAO B. At least in rat liver extracts, one would predict no reaction since MAO B would be on the intermembrane face and thus un- available. It remains for future work to determine whether the dif- ferential outer membrane orientations of MAO A and MAO B observed in rat liver are also apparent in mitochondria isolated from other tissues.

DELETERIOUS EFFECTS OF MAO-GENERATED H2O2

MAO A and MAO B: Although H2O2 generated in biological Fig. (3). Proposed reaction mechanism for the oxidation of the 2-electron organelles can be used for signaling targets, one of the main de- reduced MAO flavin cofactor with O2 to form H2O2 as reaction product. The structive effects is the non-specific oxidation of thiol groups in second order rate for the reaction of the protonated imine complex of re- proteins to either disulfides or to sulfenic acids. Another oxidative duced MAO B with O2 is shown with the arrow. reaction is the oxidation of methionine thioethers to their respective sulfoxides. An important defense mechanism to protect protein MITOCHONDRIAL MEMBRANE TOPOLOGIES OF targets from destructive H O oxidations is the enzyme glutathione MONOAMINE OXIDASES A AND B 2 2 peroxidase which utilizes reduced glutathione and H2O2 as sub- Given the similar structures of MAO A and MAO B, the as- strates resulting in oxidized glutathione and water as products. Glu- sumption has been that both enzymes are bound to the cytosolic tathione peroxidase is found in the intermembrane space of mito- face of the mitochondrial outer membrane. Early investigations of chondria as well as in the cytoplasm as are concentrations of re- this question using antibody inhibition and protease inactivation duced glutathione which are linked to the redox state of the cell via experiments [22, 23] provided conflicting data and conclusions. the NADPH-dependent flavoenzyme, glutathione reductase. Experiments demonstrating the formation of oxidized glutathione The superoxide anion generated in the mitochondrial electron on benzylamine oxidation by MAO B in intact rat liver mitochon- transport chain is efficiently dismutated to O2 and H2O2 by the Mn- dria are insensitive to catalase suggested that the active site of dependent superoxide dismutase which is also located in the inter- MAO B may be on the intermembrane face of the outer membrane membrane space as well as in the matrix. Apolar environments are rather than the cytosolic face [24]. Using a combination of MAO .- known to reduce the rate of O2 dismutation thus extending its life- inhibition by TEMPO-pargyline analogs (which exhibit an inability time and probability of participating in metal-catalyzed reactions to cross the mitochondrial outer membrane) and protease inactiva- with any H O present to form the destructive hydroxyl radical. tion experiments, Wang and Edmondson [25] demonstrated that 2 2 MAO B is located on the intermembrane face of the mitochondrial Thus, an increased level of MAO-generated H2O2 can result in outer membrane in rat liver mitochondria while MAO A is located considerable destructive processes either within the mitochondrion on the cytosolic face (Fig. 4). A similar topology is also observed or in the cytoplasm. Studies from Shih’s laboratory [28] found that for MAO A in human placental mitochondria [26]. Of interest, the the MAO –catalyzed oxidation of tyramine resulted in considerable topological orientations of MAO A and MAO B are opposite in oxidative damage to brain mitochondrial DNA. This oxidative intact mitochondria isolated from the Pichia pastoris expression damage apparently occurs through hydroxyl radical formation since system suggesting that an alteration in lipid content could give rise spin trapping experiments showed the formation of hydroxyl radi- to the differences observed with the mammalian systems. In a sepa- cals in response to MAO catalytic turnover. Inhibition of MAO A rate study, Tang et al. [27] provide confirmation for the cytosolic and MAO B catalytic activity by the non-specific inhibitor tranyl- topological orientation of MAO A in the mitochondrial outer mem- cypromine resulted in a protection from this oxidative damage. branes from HepG2 and in HeLa cell cultures using MAO A- These data suggest that the H2O2 product from MAO catalysis is specific monoclonal antibodies modified with magnetic bead converted to the reactive hydroxyl radical through Fenton chemistry nanoparticles which allowed the specific isolation of mitochondria. and results in the observed oxidative damage. Of interest, these workers found that H2O2 concentration originating from tyramine 158 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Dale E. Edmondson oxidation is 48-fold higher than the level originating from the elec- cerebral arteries. In the cerebral circulation, H2O2 is believed to tron transport chain (succinate oxidation in the presence of antimy- function as a vasodilator [35] although it is found to inhibit relaxa- cin A) [28]. tion of smooth muscle cells [36]. Conversely, in the rat basilar ar- MAO B. A considerable number of investigations have shown tery, the mitochondrial H2O2 originating from the MAO A oxida- MAO B levels in neuronal tissue to be elevated ~3 fold on aging in tion of serotonin has been shown to induce contraction [37]. This humans [29]. The consequence of this age-related elevation in mode of signaling is proposed to involve inhibition of the BKCa MAO B expression is a decreased level of amine neurotransmitter channel by H2O2 via oxidation of amino acid residues since inhibition was not immediately reversed on wash-out of the added perox- levels and an elevation of H2O2 production. Since substrate levels of amines such as tyramine are ingested in foods, the concentration ide. Thus, MAO inhibitors may exhibit anti-hypertensive properties of reducing amine substrates is expected to remain at stable levels. [38] although more work is required to determine whether both forms of MAO or only MAO A is functioning in the regulation of The increased level of H2O2 production in neuronal tissue containing higher MAO B levels is expected to result in oxidative damag- this vascular function. ing effects leading to neurodegenerative diseases (such as Parkin- MAO INHIBITORS AS PROTECTIVE AGENTS son’s Disease), disorders that appear to increase in an aging popula- tion. A survey of the literature on MAO shows that the major reason for the design of new and specific inhibitors is to function as neuro- In a model system to test this hypothesis, Andersen’s group protectants for the age-dependent increase in MAO B in neuronal [29] used genetically engineered dopaminergic PC12 cells to in- tissue and as cardioprotectants for the age-dependent increase of crease MAO B levels 2-3 fold. They found that increased H O 2 2 MAO A in the heart. The increased longevity of humans provides a production followed this increase in MAO B which resulted in de- reason for the development of these agents to benefit the quality of creased activity of Complex I activity as measured by a 60% reduc- life in the elderly. The hypothesis is that reduction of MAO- tion of NADH dehydrogenase activity. In addition, a decrease in generated H O should result in a protection against age-dependent activity of the -ketoglutarate dehydrogenase complex is observed 2 2 degeneration of these tissues. To date, the available data with the on MAO B elevation. This complex is important in respiration in MAO B inhibitors deprenyl and rasagiline provide supporting data that it provides the NADH reducing substrate for Complex I in the for this hypothesis as regards retardation in the advancement of electron transport chain. The reduced levels of activities observed Parkinson’s Disease. Too little is known regarding the etiology of for Complex I and -ketoglutarate dehydrogenase in this model cell neurodegenerative diseases at this point in time to unambiguously system mirror those levels observed in Parkinson’s patients and support this role for MAO B. provide strong evidence for the proposed importance of MAO B- generated H2O2 in the etiology of neurodegenerative diseases. It To date, no definitive studies have been done with MAO A should be noted that a study of patients diagnosed as having specific inhibitors with patients at early stages of coronary disease. Huntington’s Disease an autosomal dominant inherited mutation in One of the problems is MAO A inhibition is to design inhibitors not the HTT gene, shows a 3-fold increase in MAOB transcripts relative only specific for MAO A but also to function mainly in the periph- to healthy controls [30]. This increase in MAO B level of expres- eral tissue and not be transported into the neuronal tissue and inter- sion is expected to lead to increased mitochondrial H2O2 levels fere with neuronal function. General MAO inhibitors were used in resulting in oxidative damage which may account for the loss in the 1960’s in therapy for cardiovascular diseases and have been mitochondrial function and loss in motor and cognitive function reported to diminish the intensity and frequency of anginal pain, to associated with Huntington’s disease progression. exhibit a hypotensive effect on patients, and an elevation of mood [39]. These therapeutic advantages are now abandoned due to com- MAO A. The biological and biomedical roles of MAO A in plications from diet-related hypertension (the Cheese Effect) due to generation of H O and possible hydroxyl radical species have also 2 2 ingestion of tyramine-containing foods which could function as been documented in degenerative processes in the heart [30]. Upon false neurotransmitters. This side effect is observed with the use of aging, MAO A levels increase ~9-fold in rats and in humans [32]. these earlier MAO inhibitors (phenelzine and tranylcypromine) These studies also implicate mitochondrial H O in cell apoptopic 2 2 since they are non-specific and inhibit both enzymes by covalent responses to ischemia-reperfusion leading to myocardial injury and reaction with their respective active site flavin cofactors. An addi- dysfunction [32]. Serotonin (a MAO A substrate) is stored in plate- tional aspect on comparison of the acetylenic MAO inhibitors with lets and released into circulation upon their activation. Recent stud- the hydrazine class involves the differences in O utilization for ies have shown MAO A to be involved in the regulation of sero- 2 their respective inhibition of MAO A or MAO B. Unpublished tonin concentrations important for ventricular remodeling via acti- work in our laboratory has shown that no oxygen is consumed on vation of the serotonin 2A receptors [33]. In the aged heart, the inhibition of MAO B with deprenyl or with rasagiline or on inhibi- increased MAO A levels generate increased levels of H O leading 2 2 tion of MAO A with clorgyline (all members of the acetylenic class to cardiomyocyte apoptotic responses and tissue necrosis. Although of MAO inhibitors). In contrast, the inhibition of MAO A or of both MAO A and MAO B are apparently present in the heart, the MAO B with phenelzine requires ~40 moles of O per inactivation majority of investigations has focused on the role of MAO A as a 2 event suggesting that multiple enzyme turnovers occur in the inac- source of oxidative stress in the heart. Protective effects have been tivation process [40]. This large oxygen requirement for the hydra- observed using deprenyl (a MAO B specific inhibitor) and par- zine inhibition reaction could lead to “spikes” in the production of gyline (an inhibitor of either MAO A and MAO B ) thereby impli- ROS which could limit any protective effects. MAO inhibition by cating MAO B inhibition as also important in reducing the levels of acetylenic inhibitors , therefore, is not expected to exhibit this pos- oxidative damage products expected from membrane interactions sible side effect. with hydroxyl radicals in aging rat hearts [34]. Although MAO B levels do not appear to increase in the heart cell on aging, this SUMMARY isozyme may still play an important (still undefined role) in age- dependent heart disease. The topologies of MAO A and MAO B in The purpose of this review is to inform the interested reader the mitochondrial outer membrane of heart mitochondria have not that MAO A and MAO B should be considered as important been determined and could be an important consideration in future sources of H2O2 and hydroxyl radical in the mitochondrial- studies relating to MAO and cardiomyopathies. generated supply of ROS in addition to the mitochondrial electron transport chain as a source of superoxide. . One reasonable question Additional biological effects of H2O2 known to occur also in- is whether the levels of amine neurotransmitters present in the cell clude the circulation system. Both MAO A and MAO B have been are sufficient for MAO- generated H2O2 to rival the production by demonstrated to exist in blood vessels with higher MAO A levels in Contribution of MAO-produced Hydrogen Peroxide to Mitochondrial Reactive Oxygen Current Pharmaceutical Design, 2014, Vol. 20, No. 2 159 the electron transport chain. This question needs to be addressed in whether to topology of MAO A remains on the cytosolic face of the more detail in a tissue-specific investigation. It should be noted that mitochondrial outer membrane in all tissues. the ingestion of amines (such as tyramine) in foods is probably the major source of amine substrates for the production of reactive CONFLICT OF INTEREST oxygen species arising from both MAO A and MAO B catalytic The authors confirm that this article content has no conflicts of turnover. interest. The recent studies on the topology of MAO A and MAO B in the mitochondrial outer membrane deserve comment. To date, the ACKNOWLEDGEMENTS evidence has shown MAO A to be located on the cytosolic face of The author wishes to acknowledge the financial support of NIH the outer membrane in mitochondria isolated from rat liver, human grant GM-29433 and to acknowledge helpful discussions with Drs. placenta [25,26], and in cell cultures [27]. It remains for future A. Mattevi, C. Binda (University of Pavia) and with Dr. R. Orru investigations to determine if this topological orientation occurs in (Emory University) leading to the views presented in this manu- all tissues. Presumably, the MAO A-generated H2O2 would diffuse script. into the cytosol where it could be utilized in signaling functions or be deactivated by glutathione peroxidase or catalase. Recent studies REFERENCES have shown perinuclear mitochondrial clustering at the cell nucleus [1] Murphy MP. How mitochondria produce reactive oxygen species. in response to hypoxia as a requirement for hydrogen peroxide Biochem J 2009; 417: 1-13. induced transcriptional signaling [41]. Therefore, MAO A catalysis [2] Sena LA, Chandel NS. Physiological Roles of Mitochondrial Reac- could be an important component for this signaling mechanism. tive Oxygen Species. Molec Cell 2012; 48: 158-431. [3] Fowler JS, Volkow ND, Wang GJ, et al. Age-related increases in The notion that H2O2 can diffuse through membranes in an brain monoamine oxidase B in living healthy human subjects. Neu- unrestricted manner has recently been questioned since it exhibits a robiol Aging 1997; 18: 431-5. dipole moment similar to that of the water molecule [42]. In fact, [4] Pizzinat N, Copin N, Vindis C, Parini A, Cambon C. Reactive there is now evidence that Aquaporin-3 and Aquaporin-8 mediate oxygen species production by monoamine oxidases in intact cells. N-S Arch Pharmacol 1999; 359: 428-31. the transport of H2O2 across membranes for downstream signaling [42, 43]. Whether this mode of H O transport occurs for all [5] Murphy MP, Holmgren A, Larsson NG, et al. Unraveling the Bio- 2 2 logical Roles of Reactive Oxygen Species. Cell Metab 2011; 13: mechanisms of membrane transport is not known. Current evidence 361-6. shows that Aquaporin-8 is expressed in the inner membrane of the [6] Ramsay RR. Kinetic Mechanism of Monoamine Oxidase A. Bio- mitochondrion [44] which contrasts with other reports [45]. Using chemistry 1991; 30: 4624-9. si-RNA knockdown experiments, the loss of functional transport of [7] Newton-Vinson, P,Hubalek F, Edmondson DE. High Level Ex- H2O2 via Aquaporin-8 is observed resulting in mitochondrial depo- pression of Human Liver Monoamine Oxidase B in Pichia pastoris. larization via the mitochondrial permeability transition mechanism Prot Expr Purif 2000; 20: 334-45. and consequent cell death. No experiments of this type to assess the [8] Lan NC, Heinzmann C, Gal A, et al. Human monoamine oxidase role(s) of Aquaporin-8 on the transport of MAO A and/or MAO B – A and B genes map to Xp 11.23 and are deleted in a patient with generated H O have been performed but should prove of interest. It Norrie disease. Genomics 1989; 4: 552-9. 2 2 [9] Edmondson DE, Binda C, Wang J, Upadhyay A, Mattevi A. Mo- should also be noted that Aquaporins can transport ammonia [46] lecular and Mechanistic Properties of the Membrane-Bound Mito- (another product of MAO-catalyzed amine oxidation) through chondrial Monoamine Oxidases. Biochemistry 2009; 48: 4220-30. membranes and therefore provide another protective effect on mito- [10] Wang CC, Borchert A, Ugam-Klusek A, et al. Monoamine Oxidase chondrial function. A Expression is Vital for Embryonic Brain Development by Modu- lating Developmental Apoptosis. J Biol Chem 2011; 286 28322-30. An interesting aspect of MAO B generated H2O2 function is the finding that the topological location of MAO B is on the intermem- [11] Li M, Hubalek, F, Newton-Vinson, P. Edmondson DE. High-Level brane face of the mitochondrial outer membrane [25]. This state- Expression of Human Monoamine Oxidase A in Pichia pastoris: Comparison with the Enzyme Expressed in Saccharomyces cere- ment needs qualification in that this topology has only been demon- visiae. Prot Expr Purif 2002; 24: 152-62. strated in rat liver and future work is required to determine whether [12] Binda, C, Newton-Vinson P, Hubalek F, Edmondson DE, Mattevi this topology is also found in mitochondria from other tissues. In A. Structure of human monoamine oxidase B, a drug target for the the event, this topological orientation is found to exist in all tissues treatment of neurological disorders. Nat Struct Biol 2002; 9: 22-6. and it brings up some unique questions regarding the MAO B gen- [13] DeColibus L, Li M, Binda C, et al. Three-dimensional structure of eration of H2O2 and its release into the cytosol. The intermembrane human monoamine oxidase A (MAO A): relation to the structures space contains protective enzymes such as glutathione peroxidase of rat MAO A and human MAO B. Proc Natl Acad Sci USA 2005; and superoxide dismutase which could effectively provide protec- 102: 12684-9. tion against destructive ROS. The view that small molecules such [14] Son SY, Ma J, Kondou Y, Yoshimura M, Yamashita E, Tsukihara T. Structure of human monoamine oxidase A at 2.2 angstrom reso- as H2O2 diffuse through the pores in the outer membrane or through lution: the control of opening the entry for substrates/inhibitors. the membrane is probably not correct. Recent studies have shown Proc Natl Sci USA 2008; 105: 5739-44. that the intermembrane space exhibits a differing pH (0.2-0.7 units [15] Ma J, Yoshimura M, Yamashita E, Nakagawa A, Ito A, Tsukihara more acidic than the cytosol) and a redox potential (more oxidizing T. Structure of Rat Monoamine Oxidase A and Its Specific Recog- than the cytosol; based on ox/red glutathione levels) and suggests nitions for Substrates and Inhibitors. J Mol Biol 2004; 338: 103-14. that barriers exist to separate the two spaces [47,48]. The gating [16] Upadhyay A, Borbat PP, Wang J, Freed, JH, Edmondson DE. properties of the porins in the mitochondrial outer membrane (also Determination of the Oligomeric States of Human and Rat Mono- amine Oxidases in the Outer Mitochondrial Membrane and Octyl referred to as the Voltage-Dependent Anion Channel) are open or closed depending on the existing membrane potential. Since the -D-Glucopyranoside Micelles Using Pulsed Dipolar Electron Spin Resonance Spectroscopy. Biochemistry 2008; 47: 1554-66. volume of the intermembrane space constitutes ~5 % of the total [17] Borgese N, Brambillasca S, Colombo S. How tails guide tail- mitochondrial volume, the effective concentration of H2O2 from the anchored proteins to their destinations. Curr Opin Cell Biol 2007; combined products of MAO B catalysis and electron transport chain 19: 368-75. .- generation of O2 could become very high and overwhelm the pro- [18] Chaiyen P, Fraaije MW, Mattevi A. The enigmatic reaction of tective levels of reduced glutathione present. These considerations flavins with oxygen. Tr Biochem Sci 2012; 37: 373-80. would suggest that MAO B-generated H2O2 could be more destruc- [19] Newton-Vinson P, Edmondson DE. High level Expression, Struc- tive than that of MAO A. Future work will be required to provide tural, Kinetic, and Redox Characterization of Recombinant Human experimental verification of these suggestions as well as determine Liver Monoamine Oxidase B. In: Ghisla S, Kroneck P, Macheroux 160 Current Pharmaceutical Design, 2014, Vol. 20, No. 2 Dale E. Edmondson

P, Sund H, Eds. Flavins and Flavoproteins. Rudolf Weber. Berlin: [34] Di Lisa F, Kaludercic N, Carpi A, Menabo R, Giogio M. Mito- Agency for Scientific Publications 1999; pp.431-4. chondrial pathways for ROS formation and myocardial injury: the [20] Petlicki J, van de Ven TGM. The equilibrium between the oxida- relevance of p66She and monoamine oxidase. Basic Res Cardiol tion of hydrogen peroxide by oxygen and the dismutation of peoxyl 2009; 104: 131-9. or superoxide radicals in aqueous solutions in contact with oxygen. [35] Modrick MI, Didion SP, Lynch CM, Dayal S, Lenz SR, Faraci FM. J Chem Soc Faraday Trans 1998; 94: 2763-7. Role of hydrogen peroxide and the impact of glutathione peroxi- [21] George, P. The Fitness of Oxygen. In: King TE, Mason HS, Morri- dase-1 in regulation of cerebral vascular tone. J. Cereb Blood Flow son M, Eds. Oxidases and Related Redox Systems. J Wiley New Metab 2009; 29: 1130-7. York 1965; pp. 15-23. [36] Hayabuchi Y, Nakaya Y, Matsuoka S, Kuroda Y. Hydrogen perox- [22] Russell SM, Davey J, Mayer RJ. The vectorial orientation of hu- ide-induced vascular relaxation in porcine coronary arteries is me- man monoamine oxidase in mitochondrial outer membrane. Bio- diated by Ca+2- activated K+ channels. Heart Vessels 1998; 13: 9- chem J 1979; 181: 7-14. 17. [23] Buckman TD, Sutphin MS, Eiduson S, Proteases as probes of mi- [37] Poon CCW, Seto SW, Shan-Au AL, et al. Mitochondrial mono- tochondrial monoamine oxidase topography in situ. Molec Pharma- amine oxidase-A-mediated hydrogen peroxide generation enhances col 1984; 25: 165-70. 5-hydroxytryptamine-induced contraction of rat basilar artery. Br J [24] Werner P, Cohen G. Intramitochondrial formation of oxidized Pharmacol 2010; 161: 1086-98. glutathione during the oxidation of benzylamine by monoamine [38] MacCauley RB Monoamine oxidase and the pharmacology of oxidase. FEBS Lett 1991; 280: 44-6. monoamine oxidase inhibitors. In: Palmer GC. Ed. Neuropharma- [25] Wang J, Edmondson DE. Topological Probes of Monoamine Oxi- cology of Central Nervous System and Behavioural Disorders. dases A and B in Rat Liver Mitochondria: Inhibition by TEMPO- Academic Press. New York 1980; pp. 1130-7. Substituted Pargyline Analogues and Inactivation by Proteolysis. [39] Griffith GC. Amine Oxidase Inhibitors: Their Current Place in the Biochemistry 2011; 50: 2499-505. Therapy of Cardiovascular Diseases. Circulation 1960; 22: 1156- [26] Upadhyay AK, Edmondson DE. Development of Spin-Labeled 65. Pargyline Analogues as Specific Inhibitors of Human Monoamine [40] Binda C, Li M, Hubalek F, Mattevi A, Edmondson DE. Structural Oxidases A and B. Biochemistry 2009; 48: 3928-35. and Mechanistic Studies of Arylalkylhydrazine Inhibition of Hu- [27] Tang B, Zhao, L, Liang R, Zhang Y, Wang L. Magnetic nanoparti- man Monoamine Oxidases A and B. Biochemistry 2008; 47: 5616- cles: An improved method for mitochondrial isolation. Molec Med 25. Repts 2012; 5: 1271-6. [41] Al-Mehdi A-B, Pastukh VM, Swiger BM, et al. Perinuclear Mito- [28] Hauptman N, Grimsby J, Shih JC, Cadenas E. The Metabolism of chondrial Clustering Creates an Oxidant-Rich Nuclear Domain Re- Tyramine by Monoamine Oxidase A/B Causes Oxidative Damage quired for Hypoxia-Induced Transcription. Science Signal 2012; 5 to Mitochondrial DNA. Arch Biochem Biophys 1996; 335: 295- (231) ra47 [DOI: 10.1126/scisignal.2002712]. 304. [42] Bienert GP, Moller ALB, Kristiansen KA, et al. Specific Aq- [29] Kumar MJ, Nicholls DG, Andersen JK. Oxidative -Ketoglutarate uaporins Facilitate the Diffusion of Hydrogen Peroxide across Dehydrogenase Inhibition via Subtle Elevations in Monoamine Membranes. J Biol Chem 2007; 282: 1183-92. Oxidase B Levels Results in Loss of Spare Respiratory Capacity. [43] Miller EW, Dickinson BC, Chang CJ. Aquaporin-3 mediates hy- Implications for Parkinson’s Disease. J Biol Chem 2003; 278: drogen peroxide uptake to regulate downstream intracellular signal- 46432-9. ing. Proc Natl Acad Sci USA 2010; 107: 15681-6. [30] Krzyszton-Russjan J, Zielonka D, Jackiewicz J, et al. A study of [44] Marchissio MJ, Eleazar, D, Frances, A., carnovale, CE, Marinelli molecular changes relating to energy metabolism and cellular stress RA. Mitochondrial aquaporin-8 knockdown in human hepatoma in people with Huntington’s disease: looking for biomarkers. J. HepG2 cells causes ROS-induced mitochondrial depolarization and Bioenerg Biomembr published online Springerlink.com: October loss of viability. Toxicol Appl Pharmacol 2012; 264: 246-54. 2012. DOI 10.1007/s10863-012-9479-3. [45] Yang B, Zhao D, Verkman AS. Evidence against Functionally [31] Bianchi P, Kunduzova O, Masini E, et al. Oxidative stress by Significant Aquaporin Expression in Mitochondria. J Biol Chem Monoamine Oxidase Mediates Receptor-Independent Cardiomyo- 2006; 281: 16202-6. cyte Apoptosis by Serotonin and Postischemic Myocardial Injury. [46] Ishibashi K, Kondo S, Hara S, Morishita Y. The evolutionary as- Circulation 2005; 112: 3297-305. pects of aquaporin family. Am J Physiol Regul Integr Comp [32] Maurel, A, Hernandez C, Kunduzova O, et al. Age-dependent Physiol 2011; 300: R566-76. increase in hydrogen peroxide production by cardiac monoamine [47] Herrmann JM, Reimer J. The Intermembrane Space of Mitochon- oxidase A in rats. Am J Physiol Heart Circ Physiol 2003; 284: dria. Antiox Redox Signal 2010; 13: 1341-58. H1460-H1467. [48] Hu J, Dong L, Outten CE. The Redox Environment in the Mito- [33] Lairez O, Calise D, Bianchi P, et al. Genetic deletion of MAO-A chondrial Intermembrane Space is Maintained Separately from the promotes serotonin-dependent ventricular hypertrophy by pressure Cytosol and Matrix. J Biol Chem 2008; 29126-34. overload. J. Molec Cell Cardiol 2009; 46: 587-95.

Received: March 11, 2013 Accepted: May 15, 2013