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Superoxide radical protects liposome-contained cytochrome c against oxidative damage promoted by peroxynitrite and free radicals

Superoxide radical protects liposome-contained cytochrome c against oxidative damage promoted by peroxynitrite and free radicals
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  Original Contribution Superoxide radical protects liposome-contained cytochrome  c   against oxidativedamage promoted by peroxynitrite and free radicals Camila M. Mano a , Marcelo P. Barros b , Priscila A. Faria c , Tatiana Prieto c , Fábio H. Dyszy a ,Otaciro R. Nascimento d , Iseli L. Nantes c, ⁎ , Etelvino J.H. Bechara a,e, ⁎ a Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil b Programa de Química Ambiental, Ciências Biológicas e da Saúde, Universidade Cruzeiro do Sul, São Paulo, Brazil c Centro Interdisciplinar de Investigação Bioquímica, Universidade Mogi das Cruzes, Mogi das Cruzes, Brazil d Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil e Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, Diadema, Brazil A   b s t r a c ta r t i c l e i n f o  Article history: Received 26 December 2008Revised 13 May 2009Accepted 18 June 2009Available online 25 June 2009 Keywords: Cytochrome  c  SuperoxideNitric oxidePeroxynitritePeroxidase activityPhosphatidylcholinePhosphatidylglycerol The effects of nitrosative species on cyt  c   structure and peroxidase activity were investigated here in thepresence of O 2 • - and anionic and zwitterionic vesicles. Nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, using cyt  c   heme iron and/or molecular oxygen as electronacceptor. Far- and near-UV CD spectra of SIN1-treated cyt  c   revealed respectively a slight decrease of  α -helixcontent (from 39 to 34%) and changes in the tryptophan structure accompanied by increased  󿬂 uorescence.The Soret CD spectra displayed a signi 󿬁 cant decrease of the positive signal at 403 nm. EPR spectra revealedthe presence of a low-spin cyt  c   form ( S  =1/2) with  g  1 =2.736,  g  2 =2.465, and  g  3 =2.058 after incubationwith SIN1. These data suggest that the concomitant presence of NO • and O 2 • - generated from dissolved oxygen,in a system containing cyt  c   and liposomes, promotes chemical and conformational modi 󿬁 cations in cyt  c  ,resulting in a hypothetical bis-histidine hexacoordinated heme iron. We also show that, paradoxically, O 2 • - prevents not only membrane lipoperoxidation by peroxide-derived radicals but also oxidation of cyt  c   itself due to the ability of O 2 • - to reduce heme iron. Finally, lipoperoxidation measurements showed that, although itis a more ef  󿬁 cient peroxidase, SIN1-treated cyt  c   is not more effective than native cyt  c   in promoting damageto anionic liposomes in the presence of   tert  -ButylOOH, probably due to loss of af  󿬁 nity with negativelycharged lipids.© 2009 Elsevier Inc. All rights reserved. Introduction Cytochrome  c   (cyt  c  ) is a hemeprotein peripherally located on theexternal side of the inner mitochondrial membrane, which acts as amobile electron carrierof the respiratorychain. In response tospeci 󿬁 csignals, cyt  c   can detach from the inner mitochondrial membrane andtrigger apoptosis in cytosol [1]. Besides these two functions, cyt  c   alsoexhibits peroxidase activity. However, because cyt  c   bears hexacoor-dinated heme iron in the native form, it reacts very slowly withperoxides (0.2 M -1 s -1 at pH 7.0) in comparison with pentacoordi-nated hemeproteins such as myoglobin (10 2 M -1 s -1 ) and horseradishperoxidase ( ∼ 10 7 M -1 s -1 ) [2]. However, the peroxidase activity of cyt c   can be increased in response to changes in microenvironmentalconditions such as the interaction with (anionic) lipid bilayers. Theseconditions lead to loss of the heme iron sixth coordination positionwith the sulfur atom of Met 80  or the replacement of Met 80  by otheramino acid lateral chains [3 – 5]. Therefore, by affecting the sixthcoordination position and symmetry of the heme iron, negativelycharged membranes strongly in 󿬂 uence cyt  c   oxidase/peroxidaseactivity on several substrates, including  tert- butyl hydroperoxide,diphenylacetaldehyde, and methylacetoacetone. The binding of cyt  c  to phospholipid bilayers encompasses electrostatic and hydrophobicinteractions with the lipid acyl chains [6,7]. According to the type of  lipidacylchainandtheheadgroupchargepresentinthebilayer,threespin states of cyt  c   were observed in different proportions: the nativecyt  c   low-spin state with rhombic symmetry (spin 1/2  g  //=3.07 and  g  ⊥ =2.23),the high-spinstate(spin5/2,  g  //=6.0 and  g  ⊥ =2.0),anda low-spin state with less rhombic symmetry (spin 1/2  g  1 =2.902,  g  2 =2.225, and  g  3 =1.510) [4]. The latter cyt  c   species was called analternative low-spin species (ALSScyt c  ), whose spectroscopic andfunctional features were recently characterized [8]. The proportion of  Free Radical Biology & Medicine 47 (2009) 841 – 849  Abbreviations:  ALSScyt c  , alternative low-spin species; C 11 -BODIPY  581/591 , 4,4-di 󿬂 uoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoicacid; CD, circular dichroism; cyt  c, , cytochrome  c  ; DEPC, diethylpyrocarbonate; EPR,electron paramagnetic resonance; PC, phosphatidylcholine; PG, phosphatidylglycerol;SIN1, 3-morpholinesydnonymine; TNM, tetranitromethane. ⁎  Corresponding authors. E.J.H. Bechara is to be contacted at Instituto de Química,Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, Butantã, 05508-900 São Paulo,SP, Brazil. I.L. Nantes, Centro Interdisciplinar de Investigação Bioquímica, UniversidadeMogi das Cruzes, Av. Cândido Xavier de Almeida Souza, 200, Centro Cívico, 08780-911Mogi das Cruzes, SP, Brazil. E-mailaddresses:, 0891-5849/$  –  see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2009.06.028 Contents lists available at ScienceDirect Free Radical Biology & Medicine  journal homepage:  spin states of cyt  c   bound to lipid bilayers at pH 7.4 is also dependenton the lipid/protein ratio and is modulated by the pH of the medium[4 – 6,9 – 11].According to Kagan et al., the peroxidase activity of cyt  c   oncardiolipin should be involved in its detachment from the innermitochondrial membrane to reach the cytosol and trigger apoptosis[12,13]. It is also proposed that cardiolipin peroxide does not interactwith cyt  c   [7,13,14] and could promote the release of proapoptotic factors, such as Smac/Diablo and adenilate kinase-2 [12]. Also in apoptosis, the cyt  c  -mediated oxidation of phosphatidylserine in theplasmatic membrane is apparently related to the phospholipidexternalization, revealing a cellular signal for macrophage phagocy-tosis and initiation of the apoptotic cell clearance [15 – 17].Lipid-derived carbonyl compounds and peroxides (LOOH) aresubstratesofcyt c  thatleadtotheformationof freeradicalderivatives,which could contribute to the propagation of the oxidative processesin the biological membranes to which cyt  c   is bound. The reaction of cyt c   withlipid-derivedcarbonylcompoundsresultsin theproductionof triplet products able to generate O 2 ( 1 Δ g ) by energy transfer tomolecular oxygen [18,19]. In turn, O 2 ( 1 Δ g ) contributes to the increaseof LOOH content, which is a target [20] for cyt  c   attack [21]. On the other hand, experiments performed with low-temperature EPR havedemonstrated that O 2 ( 1 Δ g ) causes an increase of the high-spin signalof Fe 3+ cyt  c  , indicating a disruption of the Met 80  coordination withheme iron [20].It has been reported recently that the prooxidant peroxidaseactivity of cyt  c   can be prevented by superoxide ion (O 2 • - ). Cyt  c   canef  󿬁 ciently oxidize O 2 • - to oxygen, thus acting as a true antioxidant byscavenging O 2 • - without producing secondary and potentially harmfulROS [22]. Also, the reduction of cyt  c   heme iron by O 2 • - impairsperoxidase activity in hydrogen peroxide and prevents progressiveROS generation. Paradoxically, the presence of SOD1 in the mitochon-drial intermembrane space should have a prooxidant effect, since itmay compete with cyt  c   for O 2 • - and generate hydrogen peroxide [23].The consumption of O 2 • - changes the equilibrium of ferrous and ferricyt c towardthe oxidized form. Fe 3+ cyt  c   is convertedtohigh valencespecies by hydrogen peroxide with concomitant production of hydroxyl radical. The oxidative attack on the membrane leads to theproduction of LOOH molecules, which could then react with Fe 3+ andhigh valence species of cyt  c  , generating ROS, with consequent cellinjury [22].Cytochrome  c   is known to participate in the molecular/cellularevents underlying both oxidative and nitrosative stress. In turn, NO • has been suggested as an ef  󿬁 cient antioxidant due to its high rate of peroxyl and alcoxyl radicals scavenging, and its ability to reduce highvalence species (Compound I and Compound II) formed during theperoxidase catalytic cycle of hemeproteins [24,25], but it is also a key factor in eliciting nitr(osyl)ative modi 󿬁 cations in proteins and lipids.The ability of NO • to inhibit mitochondrial respiration leads to theoverproduction of O 2 • - and other ROS, particularly peroxynitrite(ONOO - ) formed from the reaction of NO • and O 2 • - [26]. As for theparticipation of cyt  c   in nitrosative stress, ONOO - favors its peroxidaseactivity due to the following mechanisms: (i) conversion of nativeFe 2+ cyt  c   to Fe 3+ [27]; (ii) oxidation of native Fe 3+ cyt  c   toCompound II with concomitant generation of the nitrogen dioxideradical ( • NO 2 ) [28], and (iii) conversion of native cyt  c   to a nitratedform (probably a Tyr 67  residue) with enhanced peroxidase activity[3,27]. The participation of cyt  c   in oxidative and nitrosative stressalso damages hemeprotein [20], including impairment of theproapoptotic activity [29] that could be circumvented by theassociation with unsaturated lipid bilayers [3a,b].In the present study, the effects of NO • and ONOO - on the cyt  c  structure modulated by the presence of O 2 • - and lipid bilayers wereinvestigated on the basis of the SIN1 decomposition mechanism. Inthe presence of an electron acceptor, SIN1 decomposes at 37°C by 󿬁 rst-order kinetics to generate NO • [30]. Under aerobic conditions,molecular oxygen is the main electron acceptor and generates O 2 • - inequimolar concentrations with NO • . Under these circumstances, theprompt reaction of O 2 • - with NO • generates ONOO - [34]. Using UV-visible absorption, circular dichroism (CD), and low-temperatureelectron paramagnetic resonance (EPR) techniques, we study herethe effects of NO • and ONOO - on cyt  c   structure and peroxidaseactivity in the presence of O 2 • - and anionic and zwitterionic vesicles.Superoxide anion radical was shown to protect cyt c and thephospholipids from oxidative damage induced by  tert- butyl hydro-peroxide  (tert  -ButOOH). A cyt  c   species whose rhombicity was lowerthan that of native cyt  c   was detected, exhibiting higher peroxidaseactivity and attributable to a hypothetical bis-histidine hexacoordi-nated heme iron. Materials and methods Chemicals All the chemicals, including cyt  c   (horse heart, type III), werepurchased from Sigma-Aldrich (St. Louis, MO, USA), with the exceptionof   󿬂 uorescent probe 4,4-di 󿬂 uoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C 11 -BODIPY  581/591 )(Molecular Probes, Eugene, OR, USA), the HPLC grade solvents  n -hexane, chloroform, methanol, ethanol, and hydrogen peroxide 30%(Perhydrol), which were supplied by Merck Co. (Darmstadt, Germany).All stock solutions and buffers were treated with Chelex-100 and100  μ  M EDTA added to the reaction mixtures to further minimizepossible metal contaminant effects. Preparation of unilamellar liposomes Liposomes were prepared from stock solutions in chloroform of egg yolk phosphatidylcholine (PC) and phosphatidylglycerol (PG).Aliquots of PC and PG were brie 󿬂 y mixed and the solvent wasevaporated by  󿬂 ushing with N 2  to allow for the formation of ahomogeneous dry  󿬁 lm. Two mixtures were prepared with a  󿬁 nalphospholipid concentration of 5 mM: (i) PC vesicles (PC); and (ii)vesicles containing PC with 10% of PG (PCPG 10% ).The lipid  󿬁 lm was stored in the dark under vacuum to eliminatetraces of chloroform. Multilamellar vesicles were prepared by mixing50 mM sodium phosphate buffer (50 mM phosphate buffer, pH 7.4)with the lipid  󿬁 lm, followed by an ultrasonic water bath for 5 min at35 °C. Next, unilamellar liposomes were prepared at room tempera-ture by extrusion from the previous multilamellar suspension in aMini-Extruder (Avanti Polar Lipids, Inc., Alabaster, AL, USA) through0.1- μ  mmeshpolycarbonate membranes.Theaveragediametersof theextruded unilamellar liposomes PCL and PCPGL  10%  were, respectively,(191.5±1.7) and (189.1±3.6) nm, as indicated by dynamic lightscattering using a ZetaPlus-ZetaPotential Analyzer (BrookhavenInstruments Corporation, Holtsville, NY). Fresh vesicle solutionswere prepared before the experiments. Peroxynitrite and nitric oxide production Cytochrome  c   (0.15 mM) was modi 󿬁 ed in aqueous media by theincubation of 0.15 mM 3-morpholinosydnonimine (SIN1) for 82 minat37°C.InordertofavortheproductionofNO • overthatof theONOO - ,the incubation of 0.15 mM cyt  c   with 0.15 mM SIN1 was alsoconducted underhypoxia bypurging the 50 mM phosphate buffer, pH7.4,withoxygen-freenitrogengasfor10min.Someexperimentswereperformed with the addition of 50  μ  M (diluted sample) or 15 mM(concentrated sample) of   tert  -butyl hydroperoxide ( t  -ButOOH) inorder to induce the peroxidase activity of cyt  c  . 842  C.M. Mano et al. / Free Radical Biology & Medicine 47 (2009) 841 – 849  Carboxyethoxylation of cytochrome c by diethylpyrocarbonate (DEPC) The carboxyethoxylation of cyt  c   was performed as previouslydescribed, using samples of 500  μ  M of the protein in 10 mM acetatebuffer, pH 6.5. Cyt  c   was reactedwith 15-fold molarexcess of DEPC for1 min and then dialyzed for 4 h [8]. UV-Vis spectrophotometry Absorption spectra of the samples (in 1 mL quartz cuvettes) weretraced on a Varian Carry 50 Bio spectrophotometer at 37°C. Structuralchanges in cyt  c   during reactions with SIN1 and hydroperoxides(LOOH) were accompanied by monitoring the bleaching of the cyt  c  Soret band (409 nm), the increase of its 550 nm band, and thedisappearance of the 695 nm charge transfer band, indicatingdisruption of the hexacoordinate form of heme iron [3]. Circular dichroism spectrometry Solutions of cyt  c   (10  μ  M) in phosphate buffer 20 mM, pH 7.4,before and/or after 82 min of incubation with SIN1 (10  μ  M) at 37°C,were irradiated with circularly polarized light. The spectra werecollected at room temperature within 190 – 630 nm (far-UV, near-UV,and Soret bands) in a Jasco J-720 spectropolarimeter. All the spectrawere corrected by subtracting their corresponding backgrounds. Thespectra were acquired with 0.5 nm/min resolution, applying anaverage of 12 scans per spectrum. CD-based secondary structuredeconvolution of cyt  c   was performed by online available software( ∼ andrade/k2d/). Low-temperature electron paramagnetic resonance spectrometry EPR continuous wave spectra were recorded in a standardrectangular cavity-equipped X-band spectrometer (Bruker Elexsysline E-580). The temperature of   ∼  11 K was maintained by liquidhelium (Helitran Oxford Systems). The sample was placed in a quartztube and frozen in liquid nitrogen prior to being introduced into themicrowave cavity for spectra recording. The set experimentalparameters were microwave frequency, 9.5 GHz; microwave power,5.05 mW; magnetic  󿬁 eld scan range of 35 – 425 mT; and modulationamplitude, 1 mT. EPR spectra were analyzed using the EasySpinprogram [31], which is frequently employed to simulate EPR powderspectra, including both Gaussian and Lorentzian line shapes [32]. Fluorescence spectra measurements Tryptophan  󿬂 uorescence measurements were carried out in a F-2500 Hitachi  󿬂 uorescence spectrophotometer at room temperatureusing excitation at 292 nm; slit, 5.0 nm; scan range, 300 to 420 nm. Lipoperoxidation  󿬂 uorescence assays The  󿬂 uorescent compound C 11 -BODIPY  581/591 displays the  󿬂 uor-ophore moiety attached to a hydrophobic anchor (undecanoic acid,C 11 ), which allows the probe to evidence peroxyl radicals — majorintermediates of the propagation step of lipid oxidation — close to themembrane – water interface [33]. To reach a  󿬁 nal concentration of 5 nM (from a stock solution in DMSO), the C 11 -BODIPY  581/591 probewas mixed with the dried lipid  󿬁 lms and the solvent was rapidlyevaporated by heating the  󿬂 asks under vacuum. Fluorescencemeasurements were taken in a 1-cm quartz cuvette using a HitachiF-4010spectro 󿬂 uorometerequippedwithastirrerandatemperature-controlled cell holder (set to 37°C). The  󿬂 uorescence kinetics wasrecorded for 8200 s at the 600-nm emission wavelength withexcitation at 580 nm. Results Structural studies of cytochrome c by UV-Vis, CD, and low-temperatureEPR Cytochrome  c   (150  μ  M) was incubated for 82 min with anequimolar amount of SIN1 and the spectral changes were accom-panied byelectron absorption spectrometry (Fig.1 and inset). The cyt c   UV-visible spectrum obtained after the treatment with SIN1 couldcorrespond either to a nitrated or to a nitrosylated cyt  c  . However,SIN1-treated cyt  c   exhibited neither the spectral characteristics of nitrated cyt  c   produced by the treatment with ONOO- and tetra-nitromethane (TNM) characterized by Cassina et al. [27] nor those of nitrosylated cyt  c   with the heme iron coordinated with NO • characterized by Sharpe and Cooper [36] and by Schonhoff et al.[37]. Fig. 1 shows the spectrum of Fe 3+ native cyt  c   in phosphatebuffer, pH 7.4, obtained in a 0.1-cm cuvette before (black line) andafter (gray line) incubation with SIN1 for 82 min at 37°C. In the 250 – 300 nm region of the spectrum, the increase of the 281 bandaccompanied by a blue shift to 278 nm is evident. These spectralchanges are expected from the decomposition of SIN1, whichgenerates a product known as SIN1C [34]. The cyt  c   Soret presented1nmblueshiftandQbanddidnotpresentanysigni 󿬁 cantchangeafterincubation with SIN1.When the reaction was accompanied at 695 nm in a 1-cm opticallength cuvette, a signi 󿬁 cant decrease of the charge transfer band wasrevealed (Fig. 1, inset), suggesting the displacement of Met 80  as theheme iron ligand in the sixth coordination position [35]. The absence of a concomitant Soret band blue shift indicates that that cyt  c  remained in the low-spin state due to the replacement of Met 80  byanother strong  󿬁 eld ligand. In the study of Cassina et al. [27], the UV visible spectrum of nitrated cyt  c   reveals a blue shift of 2 – 3 nm in theSoret band, a shoulder at 605 – 615 nm, and the loss of the chargetransfer 695 nm band. Differently, the SIN1-treated cyt  c   did notexhibit Soret band blue shift or any band or shoulder appearance at605 – 615nm,althoughthedisappearanceofthe695nmbandwasalsoobserved here. The signi 󿬁 cant structural differences between thespeciesdescribedbyCassinaetal.(ONOO - andTNM-treatedcyt c  )andSIN1-treated cyt  c   were reinforced by the capacity of SIN1-treated cyt c   to be reduced by ascorbate at the same rate as the native form (notshown).Regarding the formation of nitrosylated cyt  c  , the coordination of NO • with heme iron of ferricytochrome  c   can be observed by theappearance of a band peaking at 562 nm [36,37], absent in the SIN1- treated cyt  c   (Fig.1A). Fig. 1.  UV-visible spectra of cyt  c   treated with SIN1. Spectral changes of 150  μ  M cyt  c  obtainedbefore(blackline)andafter(grayline)82minincubationwith150 μ  MSIN1at37°C. The inset corresponds to absorbance decay at 695 nm during incubation of cyt  c  with SIN1.843 C.M. Mano et al. / Free Radical Biology & Medicine 47 (2009) 841 – 849  To better characterize the effect of SIN1 on cyt  c   structure, CDspectrawerealsorunbeforeandaftertheincubationwiththereagent.Fig. 2A shows the far-UV CD spectra of cyt  c   before (black line) and82 min after the incubation with SIN1 (gray line). The far-UV spectralchanges are suggestive of a slight decrease in the  α -helix content(from 39 to 34%), according to data obtained by decomposition of thespectra by the k2d program [38].The corresponding near-UV CD (Fig. 2B) spectra of cyt  c   exhibiteddifferences that could be attributed to changes in the microenviron-mentofPheresidues(260nm)andintheTrp59(305nm)[39],duetoa different tertiary structure and/or nitr(osyl)ations of these aminoacid residues [40]. Consistently, SIN1-treated cyt  c   exhibited Trp59 󿬂 uorescence that should not be assigned to unfold since the 󿬂 uorescence spectrum of Trp59 was only slightly enhanced (notshown) as compared with the intense  󿬂 uorescence exhibited by theunfolded corresponding apoprotein [8].Circular dichroism spectral features of native and SIN1-modi 󿬁 edFe 3+ cyt c  inthevisibleregionarepresentedinFig.2C.Intheregionof the N band, the CD spectrum of native Fe 3+ cyt  c   (Fig. 2C, black line)exhibitstwonegativebands(330and374nm),whichincreasedwhencyt c  wastreatedwithSIN1byvirtueofanincreaseinanegativesignalcontributing in this spectral region (Fig. 2C gray line). This result issimilar to that observed for SDS-induced ALSScyt c   and suggestschanges in the sixth ligand of the heme iron [8]. The native Fe 3+ cyt  c  CD spectrum (Fig. 2C, black line) exhibits the Cotton effect (two CDbands of opposite signals whose energy is slightly split) in the Soretband, with the zero crossing located near the electronic absorptionmaximum. The treatment with SIN1 (Fig. 2C, gray line) resulted in adecrease of the positive Soret band signal. The spectral featuresobtained in this condition could represent the overlap of the CDspectrum of the remaining native cyt  c   with the CD spectrum of thespecies formed after the SIN1 effect. To estimate the spectral featuresof the modi 󿬁 ed cyt  c   species, the spectrum presented as the gray linewassubtractedfromthenativecyt c  spectrummultipliedbyafactorof 0.5 and the result is presented as the dashed line. The weak spectralfeatures in the region between the Soret and the Q bands (450 – 520 nm) have been assigned to charge transfer transitions betweenthe porphyrin and heme iron and could be sensitive to changes in theaxial ligands [8]. However, CD visible bands of SIN1-modi 󿬁 ed Fe 3+ cyt c   did not exhibit signi 󿬁 cant differences relative to the CD spectrum of the native form (not shown). The signi 󿬁 cant changes in the cyt  c   CDspectrum in the region of the N and Soret bands observed after theincubation with SIN1 are suggestive of alterations in the heme ironmicroenvironment and conformation [8]. Recently a better character-ization of the ONOO – treated cytochrome  c   including through CDspectra analysis was found by Abriata et al. [41]. The CD analysis of ONOO – treated cytochrome  c   reported by Abriata et al. revealed a cyt  c  species different from the SIN1-treated cyt  c  . In the present study, thetreatment of native cyt  c   with ONOO - perfectly reproduced the CDresults presented by Abriata et al. (not shown), therebycorroboratingthat ONOO – treated cytochrome  c   is a species different from the SIN1-treated cytochrome  c  . In this regard, identical spectral changes in theelectronicabsorptionspectrumwereobtainedforcytcincubatedwithspermine NONOate. Taken together, the above results (i) suggest NO • as the modi 󿬁 er agent of SIN1-treated cyt c, (ii) exclude the possibilityof direct reactionof SIN-1with cyt c, and (iii) reinforce the hypothesisthat peroxynitrite is not the modifying species responsible for thespectral changes observed in the present study.At this point it was important to have experimental support topostulate a cyt  c   amino acid residue as the sixth ligand of the hemeironafterproteintreatmentwithSIN1.Twotypesofaminoacidscouldmore probably replace Met 80  in the heme iron sixth coordination Fig. 2.  CD spectra of cyt  c   on incubationwith SIN1. (A) Far UV, (B) near UV, and (C) SoretCD spectra of 10  μ  M cyt  c  . (D) Soret CD spectraof carboxyethoxylated 10  μ  M cyt  c  . The blackline represents the cyt  c   spectrum before incubation and the gray line after 82 min incubation with 10  μ  M SIN1 at 37°C. The dashed line represents 50% of the native cyt  c   spectrumsubtracted from the spectrum of cyt  c   treated with SIN1.844  C.M. Mano et al. / Free Radical Biology & Medicine 47 (2009) 841 – 849  position: a histidine or a lysine residue. Previous works have shownthattheassociationofcyt c  withnegativelychargedinterfacesleadstothe replacement of Met 80  by a lysine residue, probably Lys 79 . Inparticular, in the case of the association of cyt  c   with SDS micelles(protein/micelle ratio 1:1) and with sodium bis(2-ethylhexyl)sulfosuccinate/hexane reverse micelles, the carbethoxylation of His 33  and His 26  by DEPC resulted in no change in the UV-visible andEPR spectra of the cyt  c   associated with the surfactants. This  󿬁 ndingindicatesthatthealternativelow-spinstateofcyt c  shouldnotpresenta histidine residue in the sixth coordination position of heme iron.Therefore, the UV visible and CD spectra of SIN1-treated native cyt  c  specieswerecomparedwiththoseobtainedforSIN1-treatedhistidinecarbethoxylated cyt  c  . The UV-visible spectrum of SIN1-treatedcarbethoxylated cyt  c   (not shown) was similar to that of native cyt  c  subjected to the same treatment (Fig. 1). Considering that the lysine ɛ -amino and histidine imidazole groups are both strong  󿬁 eld ligands,signi 󿬁 cant changes in the UV-visible are not expected by replacinghistidine with lysine residues as the sixth ligand of the heme iron.However, due to striking changes in the symmetry of the hemegroup, the CD spectrum should be extremely sensitive to thereplacement of histidine by lysine as the sixth ligand of the hemeiron. Fig. 2D shows the CD spectra of carbethoxylated cyt  c   before(black line) and after (gray line) the treatment with SIN1. The mostimportant difference observed by comparing the CD spectra of nativecyt  c   before and after SIN1 treatment with that obtained withcarbethoxylated cyt  c   is the change in the Soret band negative/positive peak ratios. For noncarbethoxylated cyt  c  , the treatmentwith SIN1 led to a 2-fold increase of the Soret band negative/positivepeak ratios (from 0.99 to 2), while for carbethoxylated cyt  c   theincrease was 1.4-fold. This result could re 󿬂 ect a lower contribution of nitrated cyt  c   to the CD composite spectrum, and a decrease of thenegative Soret band blue shift would be expected. However, theextent of the blue shift was similar in both native and carbethoxy-lated cyt  c   after the treatment with SIN1. Since a 100% yield of cyt  c  carbethoxylation and nitration by SIN1 is not expected, this resultsuggests that the composite spectrum obtained after the treatment of carbethoxylated cyt  c   with SIN1 contains the signals of three species:remaining (native) cyt  c  , with Met 80  in the sixth coordinationposition; nitrated histidine blocked cyt  c  , with a lysine residue in theheme iron sixth ligand; and nitrated cyt  c   with histidine replacingMet 80  in the sixth coordination position.Tobettercharacterizethechangesinthecoordinationsphereofcyt c   heme iron promoted by SIN1, these samples were also analyzed byEPR at liquid helium temperature. Fig. 3 shows the EPR spectra of 150  μ  M native cyt  c   (line a) and cyt  c   after 82 min incubation with anequimolar amount of SIN1. Fig. 3 (spectrum a) shows the EPR spectrum(X-band)of150 μ  Mcyt c  in50mMphosphatebuffer,pH7.4,at 11 K, a condition where cyt  c   is in its native form. Spectrum acorresponds to the well-known Fe(III) low-spin form with a rhombicsymmetry that displays signals at  g  1 =3.07 and  g  2 =2.23 and  g  3  =1.35. After 82 min incubation with SIN1, the EPR signal of the Fe(III)low-spinformdecreasedconcomitantlywiththeappearanceofalow-spin form signal ( S  =1/2,  g  1 =2.736,  g  2 =2.465 and  g  3 =2.058). Thesigni 󿬁 cant decline of changes in  g  1 ,  g  2 , and  g  3  values suggests theconversion of cyt  c   to a low-spin species with a much lower rhombicsymmetry. A similar but less drastic decrease in the cyt  c   rhombicity(ALSScyt c  )wasobserved previouslyinthepresenceofcardiolipinandDCP vesicles, SDS aqueous micelles, and AOT/hexane reverse micelles[4,5]. The remarkable approximation among  g   values observed forSIN1-treated cyt  c   reinforces the supposition of a histidine residue asthe amino acid that replaces Met 80  in the sixth coordination positionof the heme iron, since a bis-histidine coordinated heme iron displaysa more symmetric structure.Under the conditions in which the data in Figs. 1 and 3 wereobtained, two electron acceptors are available for SIN1 decomposi-tion that leads to NO • generation: Fe(III) cyt  c   heme iron andmolecular oxygen. The reduction of molecular oxygen by SIN1results in the production of O 2 • - , which, besides SIN1, can reduce cyt  c  heme iron. On the other hand, ONOO - , generated from the reactionof O 2 • - plus NO • , could reoxidize cyt  c   heme iron. However the attackof ONOO - on the fraction of cyt  c   that remained oxidized could leadto changes in the protein structure, with consequences in the hemeiron coordination sphere. These structural changes could lead toreplacement of Met 80  by a histidine residue, which would explainthe high degree of symmetry exhibited by this new species. Thecompetition of molecular oxygen with heme iron for electronabstraction from SIN1 became evident when 150  μ  M cyt  c   wasincubated with SIN1 under hypoxia (Fig. 4). Unlike the data in Fig. 1, the data in Fig. 4 reveal a signi 󿬁 cant reduction of heme iron aftertreatment with SIN1. According to UV-visible spectra of SIN1-treatedcyt  c   in which the 562 nm band was absent, the corresponding EPR spectrum did not reveal a clear signal of the presence of nitrosylatedcyt  c  . Fig. 3. Low-temperature EPR studies of cyt  c   treated with SIN1. EPR spectra of 150  μ  Mnative cyt  c   (line a) and cyt  c   after 82 min incubation with an equimolar amount of SIN1 (line b). Line c is the spectral simulation (using the EasySpin program) of theline b spectra using the well-known Fe(III) low-spin form with a rhombic symmetry,which displays signals at  g  1 =3.07 and  g  2 =2.23 and  g  3 =1.35, the sum of a newspecies of Fe(III) low-spin form signal ( S  =1/2,  g  1 =2.736,  g  2 =2.465 and  g  3 =2.058).Experimental measurement conditions are microwave frequency 9.4715 GHz, micro-wave power 5.05 mW, modulation frequency 100 kHz,  󿬁 eld modulation amplitude1.0 mT, conversion time 81.92 ms, time constant 20.48 ms, ampli 󿬁 er gain 45 dB, andsample temperature 11.0 K. Fig. 4.  UV-visible spectra of cyt  c   treated with SIN1. Cyt  c   (150  μ  M) obtained before(black line) and after (gray line) 82 min of incubation with 150  μ  M SIN1 at 37°C,prepared under nitrogen atmosphere. Changes in the 550-nm absorption band indicatereduction of cyt  c   Fe 3+ to its ferro form.845 C.M. Mano et al. / Free Radical Biology & Medicine 47 (2009) 841 – 849
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