The Chemical Interplay between Nitric Oxide and Mitochondrial Cytochrome c Oxidase

12 Pages·2017·0.79 MB·English

The Chemical Interplay between Nitric Oxide and
Mitochondrial Cytochrome c Oxidase: Reactions,
Effectors and Pathophysiology.
Paolo Sarti, Elena Forte, Alessandro Giuffrè, Daniela Mastronicola, Maria
Chiara Magnifico, Marzia Arese
To cite this version:
Paolo Sarti, Elena Forte, Alessandro Giuffrè, Daniela Mastronicola, Maria Chiara Magnifico, et
al.. The Chemical Interplay between Nitric Oxide and Mitochondrial Cytochrome c Oxidase: Re-
actions, Effectors and Pathophysiology.. International Journal of Cell Biology, 2012, 2012, pp.571067.
￿10.1155/2012/571067￿. ￿pasteur-00955447￿
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InternationalJournalofCellBiology
Volume2012,ArticleID571067,11pages
doi:10.1155/2012/571067
Review Article
The Chemical Interplay between Nitric Oxide and Mitochondrial
CytochromecOxidase: Reactions, Effectors and Pathophysiology
PaoloSarti,1,2ElenaForte,1AlessandroGiuffre`,2DanielaMastronicola,2
MariaChiaraMagnifico,1andMarziaArese1
1DepartmentofBiochemicalSciencesandIstitutoPasteur-FondazioneCenciBolognetti,SapienzaUniversityofRome,
PiazzaleAldoMoro5,00185Rome,Italy
2CNRInstituteofMolecularBiologyandPathology,PiazzaleAldoMoro5,00185Rome,Italy
CorrespondenceshouldbeaddressedtoPaoloSarti,[email protected]
Received17February2012;Accepted23March2012
AcademicEditor:JuanP.Bolan˜os
Copyright©2012PaoloSartietal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Nitricoxide(NO)reactswithComplexIandcytochromecoxidase(CcOX,ComplexIV),inducingdetrimentalorcytoprotective
effects.Twoalternativereactionpathways(PWs)havebeendescribedwherebyNOreactswithCcOX,producingeitherarelatively
labilenitrite-boundderivative(CcOX-NO −,PW1)oramorestablenitrosyl-derivative(CcOX-NO,PW2).Thetwoderivatives
2
are both inhibited, displaying different persistency and O competitiveness. In the mitochondrion, during turnover with O ,
2 2
one pathway prevails over the other one depending on NO, cytochrome c2+ and O concentration. High cytochrome c2+,
2
and low O proved to be crucial in favoring CcOX nitrosylation, whereas under-standard cell-culture conditions formation
2
of the nitrite derivative prevails. All together, these findings suggest that NO can modulate physiologically the mitochondrial
respiratory/OXPHOSefficiency,eventuallybeingconvertedtonitritebyCcOX,withoutcelldetrimentaleffects.Itisworthyto
pointoutthatnitrite,farfrombeingasimpleoxidationbyproduct,representsasourceofNOparticularlyimportantinviewofthe
NOcellhomeostasis,theNOproductiondependsontheNOsynthaseswhoseactivityiscontrolledbydifferentstimuli/effectors;
relevanttoitsbioavailability,NOisalsoproducedbyrecyclingcell/bodynitrite.Bioenergeticparameters,suchasmitochondrial
∆Ψ,lactate,andATPproduction,havebeenassayedinseveralcelllines,inthepresenceofendogenousorexogenousNOandthe
evidencecollectedsuggestsacrucialinterplaybetweenCcOXandNOwithimportantenergeticimplications.
1.Introduction hemea -Cu site[7,8].TheinhibitionofComplexIresults
3 B
from the reversible S-nitrosation of Cys39 exposed on the
It is nowadays established that nitrogen monoxide (NO), surface of the ND3 subunit [9, 10]. The functional effects
nitricoxideintheliterature,inhibitsmitochondrialrespira- on cell respiration depend on the complex targeted by NO
tion. The inhibition is induced by the reaction of NO with and on type of reaction. Inhibition of both Complex I and
someofthecomplexesoftherespiratorychain,accordingto CcOX is mostly reversible, becoming irreversible, however,
mechanisms studied over more than 20 years. The reaction depending on duration of the exposure to NO and on its
ofNOwithComplexIIIissluggish[1],whereasthereaction concentration[10,11].TheonsetofNOinhibitiononCom-
ofNOwithComplexIandComplexIV,thatis,cytochrome plex I is slow (minutes [10]), whereas on CcOX is very fast
c oxidase (CcOX), is rapid and to a large extent reversible. (milliseconds to seconds [12]). In this paper the attention
Both reactions lead to formation of derivatives responsible is focused on the interactions between NO and CcOX.
ofthemitochondrial nitrosativestressobservedindifferent The balance between the concentrations of cytochrome c2+
pathophysiologicalconditions,includingmainneurodegen- and O proved to be critical in inducing different CcOX
2
erations [2–6]. The functional groups of the mitochondrial inhibition patterns,spanning froma finelytuned control to
complexes reacting with NO include the metals at the cat- a severe, almost irreversible enzyme inactivation [13]. The
alyticactivesiteofCcOX,namely,theFeandCuionsofthe interplaybetweenCcOXandNOisbasedontheinhibition
2 InternationalJournalofCellBiology
exertedbyNOontheenzymethat,inturn,activelycontrols intermediates populated during a single turnover are indi-
theNOconcentrationatthemitochondrialsite[14]. cated,startingandendingwiththefullyoxidizedOspecies:
The redox active site of CcOX contains one heme a
3
and one CuB tightly coupled in the so-called binuclear site, e− e− O2 e− e−
O E R A P F O
wheretheO andNOchemistryaswellasthereactionwith
2
common ligands occur. The active site receives electrons
intra-molecularly from the reduced heme a and Cu ,
A Since first proposed as a unified picture based on experi-
forming together the electron accepting pole of CcOX,
ments carried out using purified CcOX [28], the enzyme
maintained physiologically reduced by cytochrome c. Also
adducts formed upon reacting with NO have been spec-
relevant to the reaction of NO with CcOX, the availability
troscopically identified as a nitrosyl-derivative (heme a 2+-
3
in the mitochondrion of reduced cytochrome c depends NO)orasanitrite-bound(hemea 3+-NO −)derivative,or
3 2
on the relative rate at which it is reduced by Complex III
amixtureofthesetwospecies,dependingonthesteady-state
and oxidized by O via CcOX. It is also worth mentioning
2 fractional accumulation of all the intermediates [29]. It is
that the absolute cytochrome c concentration may vary in
worthy to notice that the Fe and Cu ions in the active site
different cell lines and tissues [15]. The rate of reaction
undergo redox changes only upon reacting in the oxidized
of CcOX with O is close to diffusion limited (k ≈ 1 ×
2 state (i.e., Fe3+, Fe4+, Cu2+) with NO. During the reaction
108M−1s−1[16,17]),whereasthereactionwithcytochrome NOisoxidizedtoNO −,thatisreleasedinthemedium;the
cisslower,k≈1×106M−1s−1,theactualrateconstantvalue 2
wholeeventisidentifiedaspathway1(PW1):
being dependent on pH and ionic strength [18]. During
turnover, the reduction level of the CcOX redox sites, and
H+
particularly of the metals in the active site, depends on
(i) the actual concentration of reduced cytochrome c and CuB2+OH−Fe3++NO CuB+NO2−Fe3+
O (weighted for their relative K values) at the redox e−
co2mpetent sites and (ii) the internMal electron transfer rate CuB2+OH−Fe3++NO CuB+Fe2++NO2−
from the electron accepting pole (heme a-Cu ), where
A
Otherwise, if the active site is partially or fully reduced, an
cytochromecreacts,totheactive(hemea -Cu )site,where
3 B affinity-driven NO binding to these metals takes place; the
the O reaction takes place. At saturating concentration of
2
whole event is identified as pathway 2 (PW2) and occurs
the physiological substrates, the rate limiting step in the
withoutfurtherredoxevents:
CcOXcatalyticcycleistheinternalelectrontransfer[19–21].
Overandabovethedescriptionofthereactionmechanisms,
theaimofthisworkistostresstheideathatCcOXusesboth CuB+Fe2++NO CuB+Fe2+NO
O andNOasphysiologicalsubstrates[5,14,22,23]andto
2
review the experimental evidence pointing to a central role NOisveryreactivetowardsthefullyreducedRbinuclearsite.
oftheNOinterplaywithCcOXincellbioenergetics. Itbindstohemea32+ ataratesimilartothatofO2,thatis,
k =0.4−1×108M−1s−1[16,17],yieldingthehighaffinity
Fe2+ nitrosyl adduct, whose accumulation is observable
2.CcOXBindsReversiblyorOxidizesNOto
directly by spectroscopy or indirectly by NO amperometry
NitriteattheActiveSiteWhereO Binds [30,31],whenthefullyreducedCcOXindetergentsolution
2
is mixed with NO. Interestingly, in the presence of NO, all
In order to better understand the reciprocal interactions circumstancesfavoringtheelectrondonationtothecatalytic
betweenCcOXandNO,itmayhelpsummarizingtheinter- siteofCcOXorslowingdownitsoxidationbyO asduring
2
mediates populated by CcOX during turnover with physio- hypoxia (i.e., when the [O ] ≤ K of CcOX ) proved to
2 M,O2
logicalsubstrates.Duringthecatalyticcyclethefullyoxidized favorCcOXnitrosylation[32].Figure1showsschematically
(O)hemea -Cu siteacceptsafirstelectronfromCu /heme how accumulation of the turnover intermediates correlates
3 B A
a,leadingtoformationofapartially,single-electron,reduced withthebuildupofthenitrosylated(Cu + Fe2+NO)orthe
B
(E)species;subsequently,asecondelectronistransferredto nitrite-bound(Cu +NO −Fe3+)species.
B 2
the active site, and the fully reduced (R) species is formed. It is worth mentioning that, contrary to a few bacterial
Once in the R state, O binds rapidly generating the short- oxidases [34–36], mitochondrial CcOX cannot reduce to
2
lived(microseconds,at20◦C)compoundA,inwhichO is N O the NO bound at reduced heme a [30]. This implies
2 2 3
complexedtohemea 2+[24].Electronsarerapidlydelivered thatthefunctionalrecoveryoftheenzymeafterNObinding
3
toboundO ,andCompoundAconvertstoanominalperoxy necessarilylagsbehindthethermaldissociationofNOfrom
2
(P)complexwithbothhemea andCu oxidized;actually, the active site. The dissociation reaction is relatively slow
3 B
the experimental evidence suggests that the O–O (peroxy) (koff=3.9×10−3s−1at20◦C)andphotosensitive[28].Pho-
bondinthisPspeciesisalreadycleavedoff,showinghemea tosensitivity has been widely used by Sarti and coworkers
3
intheferryl(Fe4+=O)formandatyrosineresidueinaradical to gain insight, through amperometric measurements, into
state[25,26].Byacceptingathirdelectron,Pdecaysquickly themechanismofCcOXinhibitionbyNOinmitochondria
intoacanonicalferryl(F)intermediate[27],thateventually orwholecells[37],thatis,underconditionsunfavorableto
convertsbacktothefullyoxidizedOstateuponarrivalofone spectroscopy. Since the fully reduced binuclear site reacts
lastelectronfromCu /hemea.Thesequentialstepsandthe eagerly with both O and NO, the inhibition of CcOX via
A 2
InternationalJournalofCellBiology 3
O, P, F 4.TheRoleofCuBintheReactionwithNO
a32+NO CuB+ NO ThereactionofNOwithCuB inthefullyoxidizedCcOXto
formnitritewasfirstreportedbyBrudvigandcoworkersin
NO a33+NO2−CuB++ theearly80s[40].Lateronthisreactionwasreinvestigatedby
E, R Cooperetal.[41]andGiuffre`etal.[42],usingapulsed(fast)
preparation of CcOX. The pulsing procedure that in vitro
O2 consistsinpreliminaryreduction-reoxidationofCcOX[43],
Cyt c2+
removeschloridefromtheoxidizedactivesiteoftheenzyme
therebyallowingfastreactionwithNO[42];indeed,CcOXis
Figure1:Dual-pathwaymodelfortheinteractionofNOwithmito-
expectedlyinthepulsedstateinvivowhereCcOXturnover
chondrial cytochrome oxidase. The nature of the interaction
takes place continuously. During the reaction with the
between NO and CcOX depends on the catalytic intermediates
targeted,andthesearedifferentlypopulatedatdifferentconcentra- oxidizedCuB(k=2×105M−1s−1at20◦C),NOistransiently
oxidized to nitrosonium ion (NO+), which is subsequently
tionsofO andreducedcytochromec.Theoxidizedintermediates
2
O, P, F (see text) are overall more populated with increasing hydroxylated(orhydrated)tonitrite/nitrousacid.
O availability, and/or decreasing the concentration of reduced Thus, after the reaction, the enzyme displays nitrite
2
cytochromecinthemitochondrion:theseintermediatesreactwith bound to ferric heme a3 and is inhibited. The affinity of
NOgeneratinganitrite-inhibitedCcOX.ThereducedspeciesEand nitrite for the reduced heme a , however, is much lower
3
R(seetext)buildup,instead,upondecreasingO and/orincreasing thantheaffinityfortheoxidizedactivesite.Theintramolec-
2
the concentration of reduced cytochrome c: upon reacting with ular electron transfer to heme a -Cu , therefore, causes
3 B
NO, these intermediates generate a heme a32+-NO complex, in the prompt dissociation of nitrite and the subsequent full
competitionwithoxygen.
restoration of activity [29, 44]. Relevant to possible patho-
physiologicaleffectsofCcOXinhibitionbyNO,itisworthy
to notice that the nitrite dissociation upon reduction of
formationofanitrosyladductisexpectedtooccurincompe- heme a (k∼6×10−2s−1 at pH = 7.3, T = 20◦C [29]) is
3
titionwithO ,thatis,accordingtoPW2.Consistently,theO approximately one order of magnitude faster than the NO-
2 2
competitionismoreclearlyobservedwhentheconcentration dissociation from the nitrosylated site, accounting also for
ofthereducingsubstratesfavorsthereductionoftheenzyme theobservedproductionofnitritebyisolatedmitochondria
[29,32].Inanycase,theinhibitionofthenitrosylatedCcOX [45,46].
is reverted at the rate of the NO thermal dissociation from Ithasbeenproposedthatnitriteformationcouldfollow
reduced heme a3 [28]. It is worth noticing that, although an alternative route via reaction with O2 of the NO bound
theNOdissociation processismechanisticallyindependent tothefullyreducedCcOX[46].Accordingtothisproposal,
of O2 concentration, bulk O2 shortens the duration of asuperoxideanion(O2−)formsbythereactionofO2 with
inhibitionbyoxidizingfreeNOinsolution,thushampering reduced Cu and reacts with NO bound to reduced heme
B
NOrebindingtoCcOX. a toyieldperoxynitrite;peroxynitriteisreducedinturnby
3
the enzyme to nitrite, which is finally released in the bulk.
The hypothesis, though feasible and intriguing, was not
3.TheFully-andHalf-ReducedBinuclearSite
confirmedbyindependentexperimentsspecificallydesigned
The ability of the single electron reduced E species to bind to investigate the kinetics and the products of the reaction
NO was investigated using the K354M mutant of the Para- of fully reduced nitrosylated CcOX with O2 [50]. Using
coccus denitrificans CcOX [38]. In this mutant the internal myoglobin as an optical probe for free NO, the NO bound
electron transfer from the electron accepting pole to the to reduced heme a3 was shown to be displaced by excess
active site is severely impaired, so that the full reduction of O2 at the low rate of thermal dissociation, to be eventually
theactivesiteanditsreactionwithO isachievedveryslowly, releasedinthebulkassuch,andnotasnitrite[50].TheNO
2
that is, within several minutes. Under these conditions the dissociation from the heme iron takes minutes, also when
electron transferred intramolecularly from heme a/Cu assayed in mitochondria or intact cells, at 37◦C and in the
A
residesoneitherhemea orCu ,andtheresultingEspecies dark, that is, under conditions common in vivo in internal
3 B
can not react with O . Interestingly, however, E reacts organs and tissues. The slow recovery of function of the
2
promptly with NO generating the nitrosyl derivative. Thus, nitrosylatedCcOXiscompatiblewithamoreseverestateof
onecanconcludethat,unlikeO ,NObindstothebinuclear inhibitioncharacteristicofPW2.
2
active site even before its complete reduction [12, 31]. TheroleofCu intheCcOX-mediatedoxidationofNO
B
WhetherthereactionwithEplaysaroleinthemechanismof to nitrite was also addressed in experiments carried out
CcOXinhibitionbyNOduringturnoverisstillunclear,since usingtheE.colicytochromebd.ThisoxidaselacksCu and,
B
ithasbeenalsosuggestedthatatsteady-statethereactionof consistently, reacts with NO much more slowly (k = 1.5×
NOwithEisnotrequiredtoaccountforfastinhibition[32, 102M−1s−1 at 20◦C) than mitochondrial CcOX, without
39].RegardlessofwhetherthereactionofNOwitheitherE formingnitrite[51].Interestingly,theNOdissociationfrom
or R is predominant, it seems feasible to conclude that all the Cu -lacking cytochrome bd oxidase (from E. coli) is
B
conditions leading to reduction of the binuclear site in the muchfaster[52,53],pointingtoaspecificpropertyofheme
presenceofNOfavornitrosylationoftheenzyme. d [54] and/or to a role of Cu also in the NO dissociation
B
4 InternationalJournalofCellBiology
Table1:CytochromecoxidaseversusNO—kineticandthermodynamicparameters.
CcOXintermediate CcOXadductformed KI(nM) kon(M−1s−1)(T=20◦C) koff(s−1)(T=20◦C) O2-competition
E,R NitrosylatedCcOX-NO 0.2[32] 0.4–1×108[16,17] 3.9×10−3[28] yes
O,P,F NitriteboundCcOX-NO − 20[32] ∼1×105(O,P)∼1×104(F)[29,58] 6.0×10−2[29] no
2
fromtheactivesite.Asamatteroffact,thispeculiaritywas CcOX turnover intermediates [28, 29], whose dis-
suggested to confer to cytochrome bd-expressing bacteria a tribution depends in turn on the in situ availability
higher resistance to nitrosative stress [53, 55, 56], a hypo- of O and reduced cytochrome c; the concentration
2
thesissupportedbyinvitrostudiesonE.colideletionmut- of the latter ultimately depends on its absolute con-
antsofeachofthetwoalternativerespiratoryoxidases(cyto- centrationandontheelectronflowleveltroughthe
chromebdandcytochromebo )[55]. respiratorychain;
3
(iii) PW1 prevails under basal mitochondrial metabolic
5.CellsRespiringinthePresenceof conditions;
NOandUsingEndogenousSubstrates
(iv)PW2prevailsunderconditionsfavoringtheaccumu-
lationof EandR,thatis,whentheconcentrationof
The respiration of cells grown under standard conditions,
cytochromec2+attheCcOXsiteincreasesand/orthe
that is, in the presence of (unlimited) O and endogenous
2 O tensiondecreases;
reducing substrates, is inhibited by NO but without detec- 2
tableaccumulationofnitrosylatedCcOX[37,57].Asamat- (v)the accumulation of CcOX-NO or CcOX-NO2−
teroffact,thesestandardcultureconditionsfavortheoverall affects differently the mitochondrial bioavailability
accumulationoftheCcOXintermediatesP,FandO[29,41, of NO: the nitrosyl-derivative releases NO in the
42,58];thesearethespeciesresponsiblefortheNOoxidation medium as such, that is, still reactive, whereas the
to nitrite. Consistently, upon rapid and efficient scavenging nitrite-derivative releases nitrite to be further oxi-
of bulk NO, respiration is promptly recovered. It is worthy dizedtonitrate,eliminatedorrereducedtoNO.
to point out that nitrite, far from being a simple oxidation
TheNOconcentrationlevelinthecellvariesdepending
byproduct,representsasourceofNOparticularlyimportant
on the relative rate of its production, and degradation or
inviewoftheNOcellhomeostasis[59–62].Whentheoxygen
scavenging. Unless exogenously supplemented to the cells
tension decreases in tissues, not only respiration but also
(NO-donors),theenzymaticendogenousNOproductionis
the production of NO by nitric oxide synthases (NOSs) is
controlled via the activation/inhibition of the cell NO-
severely impaired, as the NOS uses O as cosubstrate [63].
2
synthases. Alternatively, as mentioned above, NO is gener-
Anoxia, however, induces tissue acidification, which pro-
atedbytheprotein-boundorfreemetalions(Fe2+,Cu+)cat-
motes the reduction of nitrite to NO, compensating for
alyzedreductionofNO −,areactionthatcommonlyoccurs
impairmentoftheNOS-dependentNOproduction[59,60, 2
in solution, at acidic pH [59, 60]. The NO bioavailability
64].Consistently,andapparentlyimportantforacardiovas-
cular response, low doses of nitrite (∼50nM) administered can be lowered, therefore, by specific cell-permeable NO-
synthase inhibitors or by NO scavengers, such as heme-
to ischemic, heart-arrested mice, early during resuscitation
proteinsorreducedglutathione[65].
procedures,wereshowntosignificantlyimprovesurvivalof
AspointedoutbyCooperandGiulivi[5],whentheNOS
thetreatedanimalscomparedtocontrols[61].
activity is inhibited, one may expect the O consumption
The CcOX NO-inhibition pathway prevailing in mito- 2
by respiring mitochondria to increase. This event, however,
chondriaundergivenmetabolicconditionsmightberespon-
hasbeenoftenbutnotalwaysobserved[5],probablyowing
sible for pathological responses of cells and tissues [57].
to the activation of alternative NO-releasing systems, such
Compelling experimental evidence has been collected sug-
asnitrosoglutathioneandS-nitrosatedproteinthiols,orthe
gesting that the O -uncompetitive nitrite inhibition path-
2 NO −reduction,allactiveregardlessofthepresenceofNOS
way (PW1) prevails under conditions of low electron flux 2
inhibitors.
throughtherespiratorychainandhighO ,whereastheO -
2 2
competitive nitrosyl pathway (PW2) takes over as the elec-
tronfluxincreasesandO concentrationdecreases[32,37]. 6.EffectorsandPathophysiology
2
The main features of the two pathways can be summa-
rizedasfollows: Overtheyears,theenzymaticNOreleasehasbeeninduced
in cultured cells, tissues, and organs, either using effectors
(i)both reactions lead to the rapid accumulation of a able to activate cell Ca2+ fluxes [66], thus stimulating the
CcOX inhibited species, characterized by different
constitutive NOS, or by enhancing the expression of the
stability,K ,andO competitiveness(Table1);
I 2 inducible isoform of NOS (iNOS) [67]. Morphine is the
(ii) one pathway prevails over the other one depending prototypeofafamilyofdrugsusedinanalgesiaandcancer
on the fractional accumulation of the NO-targeted pain treatment [68, 69]. Relevant to the NO chemistry,
InternationalJournalofCellBiology 5
(a) (b)
150
∗
6E+06 Ctr 100
a.u.) val %)
e ( (
nc 4E+06 Ox
ce N
es
or 50
u Nig
fl 2E+06
ed Morphine
1-r
C
J 0E+00
0 1000 2000 3000 0
Time (s) Ctr Morphine
(c) (d)
Figure2:Morphine-inducedmitochondrialmembranepotentialandNO changesinGliomacells.Fluorescencemicroscopy:controlcells
x
(a), 20nM morphine incubated for 24h (b). Mitochondrial membrane potential (∆Ψ) was probed using Rhodamine123; the dye is
electrophoreticallyaccumulatedbythecellmitochondria.Bulkfluorescence (c).Control(ctr)versusmorphine-treated(morphine)cells,
assayed in air-equilibrated medium and in the presence of 2µM ouabain and 0.4µM JC-1; after signal stabilization, 0.6µM nigericin is
addedandfluorescencechangesfollowedovertime.Additionofvalinomycinabolishesthemembranepotential.Excitationandemission
wavelength,575nmand590nm,respectively.Nitriteaccumulation(d).ThereleaseofNO (nitriteandnitrate)inthemediumandduring
x
incubationwithmorphinewasassessedspectrophotometricallybytheGriessreaction;resultsexpressedaspercentageofcontrolcells(ctr).
∗P<0,05.Modifiedfrom[33].
morphineactivatestheopioidandtheN-methyl-D-aspartate temperature(HaCaT)cells,maintainedinastandardculture
receptors of neuronal cells, triggering Ca2+ fluxes and NO medium,inthepresenceofnanomolar(orless)melatonin.
release[70,71].In2004,Mastronicolaetal.[33]confirmed After a few hours incubation compatible with a receptor-
that the persistence of nanomolar morphine in the cell mediated process [73], and with a timecourse compatible
cultureofgliomacellswasabletoinducetheaccumulation with the circadian melatonin biorhythm, the basal mRNA
ofnitrite/nitrateinthemedium.Interestingly,thecellmito- expressionleveloftheneuronalNOS(nNOS)inthecellswas
chondriadisplayedamembranepotentialdropoff,asprobed raised by a factor of ∼4 (Figure3(a)), returning, thereafter,
byasignificantdecreaseoftheintramitochondrialJC-1red- to basal level [48]. As shown in the same figure, within the
aggregates,whoseaccumulationrequireshighmitochondrial sametimescale,theauthorsobservedthat:(i)theproduction
∆Ψvalues(Figure2)[72].Thus,overthesametimescaleofa ofnitriteandnitrate(NO )wasincreased(Figure3(b))and
x
cellCa2+ transient(secondstominutes)theNOSactivation (ii) the mitochondrial membrane potential was decreased
can affect the mitochondrial potential [33]. More recently, (Figure3(c)). Consistently, the ATP production was
OXPHOS
Arese et al. [48] have shown a transient inhibition of the alsodecreasedandanincreaseofglycolyticATPandlactate
mitochondrialrespiratorychaininhumanadultlowcalcium wasdetected[48].Takentogether,allthesefindingssuggest
6 InternationalJournalofCellBiology
thatmediatedbythemelatoninreceptors,NOisreleasedand 600 15
CcOX is reversibly inhibited, with significant bioenergetic ∗∗
consequences. Since cells are not likely facing conditions ∗∗
cEomorpaRti,blweewictahnthienfaecrcuthmautlaintihoinbiotifonCchOaXs ionctceurmrreeddiavteias n (%) 400 60cells) 10
PW1. Interestingly, therefore, under physiological condi- o 1
ttoionhso,rwmitohninalt-hliekelimcoitnscoefnatrcaetlilocnusltuorfe,maefleawtohnoinursisexapbolesutroe e expressi ×moles/1
exert some inhibition on mitochondrial OXPHOS and to v
raise the ATPglycolytic/ATPOXPHOS ratio by a factor of ∼2 Relati 200 O(nx 5
(Figure3(d)) as expected on the basis of a compensatory N
physiologicalWarburgeffect[74].Alltogetherthesefindings
suggest that physiological concentrations of melatonin may
0 0
play a mitochondrial role and interestingly in a circadian
(a) (b)
context. Indeed, the hypothesis that the melatonin-driven
shift towards glycolysis might have a physiological role in ∗
1.5 200
thechemistryofthenightrest,thoughattractive,ispresently
fullyspeculative,andremainstobeinvestigated.
Basedontheeffectsofmelatoninandontheinformation %)
∗ (
cmoiltloecctheodndarbioau[t75th,e76N],OitiinshailbsiotiotenmopftinpgurtiofiesdpeCcuclOatXe oonr 50) 1 PHOS
1 X
how the mitochondrial state can affect the response to NO, × PO
particularlyunderconditionscompatiblewithalimited,and a.u. /AT 100
transientraiseofNOconcentration.Itisworthytoconsider ∆(F 0.5 olytic
that isolated state 3 mitochondria proved to be inhibited Pglyc
by NO more effectively than state 4 mitochondria [75, 76]. T
A
ThissuggeststhatthesensitivitytoNOinhibitionincreases
with the electron flux level of the respiratory chain, and
0 0
particularly with the turnover rate of CcOX; under these
conditionstheCcOXinhibitionisoxygencompetitive[32]. Ctr Ctr
In state 3 mitochondria, therefore, and in the presence of Melatonin Melatonin
suitable amounts of reduced cytochrome c, the fractional
(c) (d)
accumulation of the reduced (E and R) CcOX species is
expectedtoincrease;thesespeciesarepromptlynitrosylated
in the presence of NO. At low turnover rate, as in state Figure3:Melatonin-inducedchangesofthenNOSmRNAexpres-
4, the oxidized catalytic intermediates (O, P and F) are sioninHaCaTcells:effectonNOx productionandmitochondrial
membranepotential.(a)—Real-timePCRquantificationofnNOS
expectedtobemorepopulated[29],andtheNOinhibition
mRNA (β-actin gene used for normalization). (b)—Fluorometric
predominantly occurs following PW1. Both in state 3 and
determinationoftheNO releaseinthecellculturemedium.(c)—
state4,iftheNOconcentrationislow(e.g.,subnanomolar), x
Mitochondrial membrane potential evaluated as the fluorescence
thefractionofCcOXinhibitedislimited,andthedepression difference, ∆F, from the maximal (plateau) to the minimal level
of respiration is almost insignificant [77, 78], a finding
reached after addition of valinomycin (see also Figure2(c)).
consistent with an excess capacity of CcOX [79, 80]. When (d)—Contribution of oxidative phosphorylation and glycolysis
NO persists in the cell environment, as during a prolonged to ATP production, directly evaluated according to [47]. The
incubation with even low (nM) concentration of NO, and ATP /ATP ratio is indicative of the ability of a given
glycolytic OXPHOS
particularly if the turnover rate of CcOX is increased, a cell line to compensate with glycolysis an OXPHOS impairment
substantialinhibitionoftherespiratorychainispredictable (so-called, Warburg effect) [48, 49]. The release of NO induced
and synthesis of ATP decreases [81]. Under these by melatonin almost doubles the glycolytic contribution to ATP
OXPHOS
synthesisinHaCaTcells.Cellswereincubatedwith1nMmelatonin
conditions, glycolysis likely takes place to compensate for
for6h.(a),(b):∗∗P <0,01versusCTR;(c),(d):∗P <0,05versus
ATPloss[82].
CTR.Modifiedfrom[48].
7.HowDoestheNO/CcOXInterplay
TurnintoPathology
inhibited by NO. In this respect, it is worth considering
As just mentioned, the transient inhibition of mitochon- that neurons, astrocytes, lymphoid, keratinocytes cells, and
drial OXPHOS may induce a physiological, compensatory in general different cell lines may possess a different gly-
activation of glycolysis [74]. This original observation by colyticcompensatorycapacityofcopingwithOXPHOSNO-
WarburgwasrecentlyreproposedbyAlmeidaetal.[83],to inhibition[48,57,83].Alltheevidencesofarcollectedshows
rationalize the energetic changes of astrocytes and neurons that under standard cell culture conditions, a pulse of NO
InternationalJournalofCellBiology 7
leadstotheaccumulationoftheCcOX-NO −derivative[37], 8.TheDarkSideoftheInterplaybetween
2
which is able to immediately and fully recover its function, NOandCcOX
provided that free NO is scavenged in the mitochondrial
environment. On the contrary, when CcOX nitrosylation is Inconclusion,regardlessofthepathwayleadingtoinhibition
induced by (artificially) rising the electron flux level at the of CcOX, in the presence of NO, mitochondrial OXPHOS
CcOXsiteorbyallowingthecellstorespiretowardshypoxia is impaired to some extent. Impairment is due to the slow
([O ]≤ K ),therespiratorychainremainsinhibitedfor displacementofNOfromtheactivesiteortotheinvolvement
2 M,O2
longer times at the CcOX site [28, 29, 32, 84]. It is worth of the site in the NO oxidation to nitrite. The evidence so
recalling that indeed everything else being equal, the func- far collected suggests that, if NO remains available in the
tional recovery of CcOX-NO is approximately 10–20 times mitochondrial environment, the mitochondrial membrane
slower than recovery of CcOX-NO −. Thus, at least in a potential decreases, and glycolysis begins to contribute sig-
2
firstapproximation,itisfeasibletoproposethat,compared nificantly to ATP synthesis. Thus, it seems crucial that cells
toconditionspromotingtheformationoftheCcOX-nitrite responding to NO pulses are endowed with an efficient
adduct, conditions favoring CcOX nitrosylation are expect- glycolytic machinery able to compensate for the decreased
edly more dangerous for cells, since causing a 10–20 times aerobicATPproduction[82,83].
longer inhibition of the mitochondrial respiratory chain. Finally, let us consider for the sake of the argument a
Onemayindeedspeculatethatthecompensatoryglycolytic chronic hypoxia induced by an impaired microcirculation,
ATP synthesis might become insufficient, when CcOX is forinstanceinthebrain.Undertheseconditionscommonto
maintainednitrosylatedforlongertimes. many age-related neurodegenerations, one might expect an
In2008Mascietal.[57]characterizedthemitochondria increasedNOreleasetoenhancethebloodflowinresponse
NOinhibitionpatternofcellscollectedfrompatientsaffected tohypoxia.Inthisalreadypathologicalscenario,however,the
byAtaxiaTelangiectasia(AT).Thisisamultisystemicgenetic bloodflowandthusO2 concentrationmaynotincreasesig-
humandisordercharacterizedbyaconjunctivaltelangiecta- nificantly,owingtothevesselsclerosis;neuronscouldrather
sia and by a cerebellar degeneration leading to progressive become hypoxic and in the presence of an increased NO
ataxia[85,86].ThediseaseiscausedbymutationsoftheAT- concentration. These are the circumstances favouring PW2
mutatedgene(ATM),coding fora nuclear 350kDa protein (CcOXnitrosylation),evenmoresoiftherespiratorychain
that controls cell cycle and DNA damage repair [87–89]. concentration of reducing substrates is still large enough.
AT patients are characterised by a genetic instability and Under these conditions and in the absence of a suitable
vulnerability to radiation-induced oxidative stress [90–94]. glycolyticcompensation,theATPlevelscoulddecreasedra-
Comparedtocontrolcells,ATcellsdisplayadefectivereac- matically,leadingtocelldeath.
tiveoxygenspecies(ROS)scavengingcapacity[95,96],with
adecreasedbioavailabilityofreducedglutathione[96].
Abbreviations
RelevanttoapossiblepathologicalimplicationoftheNO
mitochondrial inhibition, AT patients show a bioenergetic
deficiency [97]. The mitochondrial functional characteri-
CcOX: Cytochromecoxidase
zation, and the NO inhibition pattern of lymphoid cells
CcOX-NO: Nitrosylcytochromecoxidase
collectedfromATpatients,provedtobesignificantlyaltered.
derivative
Based on the rate of respiration recovery from inhibition, CcOX-NO −: Nitrite-boundcytochromecoxidase
2
underotherwiseidenticalconditionsofsubstratesavailabil-
PW1: NOreactionpathwayleadingto
ity (O and reductants), the CcOX in AT cells underwent
2 nitrite-boundCcOX
nitrosylationtoasubstantiallyhigherextentthanincontrol
PW2: NOreactionpathwayleadingto
cells [57]. As expected, based on the higher stability of the nitrosylCcOX
nitrosyl-adduct compared to the nitrite-adduct, after NO OXPHOS: Oxidativephosphorylation
inhibition and subsequent removal of free NO, recovery ∆Ψ: Membraneelectricalpotential
of respiration of AT cells is slow, occurring at the rate of difference
the NO displacement from the reduced CcOX active site, O: FullyoxidizedCcOX
whereas control cells recover almost immediately (Figures E: CcOXwithsingle-electronreduced
4(a)and4(b)).AsamatteroffacttheinhibitionofATcells hemea -Cu
3 B
respiration was promptly removed upon shedding light R: CcOXwithfullyreducedheme
on the cells (photosensitivity of the nitrosyl-adduct!). This a -Cu
3 B
peculiarity of AT cells has been correlated to their 1.7 A: CcOXwithferrousoxygenatedheme
fold higher concentration of mitochondrial cytochrome c a
3
compared to control cells (Figure4(c)) [57]. The whole P: “Peroxy”CcOXintermediate
picture is consistent with the hypothesis that in AT cells, F: “Ferryl”CcOXintermediate
showing a lower ATPglycolytic/ATPOXPHOS ratio compared to NOS: Nitricoxidesynthase
controlcells(Figure4(d)),theformationofEandRandthus nNOS: NeuronalNOS
CcOXnitrosylationisfavoredowingtothehigheravailability NO : Nitrite-nitrate
x
ofreducedcytochromec[29,32]. AT: AtaxiaTelangiectasia
8 InternationalJournalofCellBiology
HbO2
NO
350
Reductants ) ∆t AT
Cells −1 15 NO
O(M)µ2125500 NOHbO2 consumption−17××min10cells 10 Vb ∆t Va Ctr
M) 2 O2 mol 5 In
50 µ 1 n
O ( Ctr (
N 0 AT
AT Ctr
−50 0
0 10 20 30 40 9 11 13 15 17 19 21
Time (min) Time (min)
(a) (b)
Cytochromec ATP
∗
2
150
%)
etric 1.5 (OS
Relative densitomintensity ()f 0.51 P/ATPOXPHglycolytic 15000 ∗
T
A
0 0
Ctr AT Ctr AT
(c) (d)
Figure4:OxygenconsumptionofAtaxiaTelangiectasia(AT)cells:theinhibitoryeffectofNO.(a)—O consumptionprofilesofATand
2
controllymphoblastoidcells,recordedinthedarkandinthepresenceofexcessascorbateandtetramethyl-p-phenylenediamine(TMPD).
InhibitionofrespirationwasinducedbyaddingasinglebolusofpureNOgassolution(seelowerNOprofiles).Inordertoassessthefraction
ofresidualinhibitedCcOX-NO,theinstantaneousratewasmeasuredat45safterHbO addition.(b)—Firstderivativeplots(integration
2
timet = 2s).RateofO consumptionbeforeadditionofNO(V ),andafteradditionofoxygenatedhemoglobin,HbO (V ),thatis,in
2 b 2 a
theabsenceoffreeNO.In:inhibitedstate(inthepresenceoffreeNO).The∆t valueisthetimenecessaryforcompleterecoveryofactivity
afteradditionofHbO .T =25C.(c)—Cytochromecimmunoblot.Cell-lysate(30µg/well)ofATpatientsandcontrols(ctr).(d)—Relative
2
contributionofOXPHOSandglycolysistoATPproductioninATandcontrolcells.Modifiedfrom[57].
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