Low spin states of microperoxidases produced by inter- and intra-peptide chain sixth ligands: Effect...

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Journal of Inorganic Biochemistry 100 (2006) 226–238

InorganicBiochemistry

Low spin states of microperoxidases produced by inter- andintra-peptide chain sixth ligands: Effect of pH and the oligopeptide type

Alessandra Riposati a, Tatiana Prieto b, Claudio S. Shida b, Iseli L. Nantes b,*,Otaciro R. Nascimento a,*

a Departamento de Fısica e Informatica, Instituto de Fısica de Sao Carlos, Universidade de Sao Paulo-USP Sao Carlos - SP, Brazilb Centro Interdisciplinar de Investigacao Bioquımica – CIIB, Universidade de Mogi das Cruzes – UMC, Av. Dr Cndido Xavier de Almeida,

Mogi das Cruzes – SP, Brazil

Received 22 March 2005; received in revised form 8 November 2005; accepted 10 November 2005Available online 3 January 2006

Abstract

The low spin states of microperoxidases (MP)-8, -9 and -9 N-acetylated (N-Ac) were characterized using UV–visible, circular dichro-ism, and electron paramagnetic resonance spectroscopies over the 6.0–12.0 pH range. The first MP-8 alkaline transition (pKa = 8.53)produced hemepeptide aggregates in the low spin state in which a water molecule was replaced by the peptide chain N-terminal groupof a neighboring MP-8 molecule. Higher pH led to the deprotonation of the MP-8 histidine imidazole ring (pKa = 10.37) at the fifthcoordination position. This MP-8 species was in equilibrium with a high spin state aggregate in which OH� replaced histidinate, the his-tidinate becoming the heme iron sixth ligand in a neighboring MP-8 molecule. In a similar way to the N-AcMP-8, the low spin state of N-AcMP-9 was produced by the deprotonation of the water molecule (pKa = 9.6) situated at the sixth coordination position of the hemeiron. Up to pH 8.5, the low spin states of MP-9 were aggregates in which the a-amino group of Lys13 replaced water at the sixth coor-dination position of a neighboring MP-9 molecule. Above pH 8.5, the e-amino groups of Lys13 established intra-chain coordination andimpaired the formation of aggregates. Such intra-chain interaction in MP9 was supported by molecular dynamics simulation. These MP-9 monomers might also exhibit OH� or histidinate at the fifth coordination position.� 2005 Elsevier Inc. All rights reserved.

Keywords: MP-9; EPR; Heme iron; CD

1. Introduction

Microperoxidases (MPs) are hemepeptides obtained byproteolytic digestion of cytochrome c [1–4]. These peptidescan be prepared with different numbers of amino acid res-idues comprising the polypeptide chain attached to theheme group [5,6]. In a similar way to cytochrome c, inthe MP structure the heme moiety remains covalentlylinked to Cys-14 and Cys-17 by thioether bridges and his-tidine is maintained as the fifth heme iron ligand but not

0162-0134/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2005.11.016

* Corresponding authors. Fax: +55 11 4798 7102 (I.L. Nantes); fax: +5516 3371 5381 (O.R. Nascimento).

E-mail addresses: [email protected] (I.L. Nantes), [email protected](O.R. Nascimento).

methionine at the sixth coordination position. The absenceof a heme iron ligand with methionine sulfur at the sixthcoordination position allows this site to be occupied by anumber of different ligands [7], since in normal aqueoussolution is coordinated with water molecule (Fig. 1). Dueto these structural characteristics, microperoxidases areconsidered a realistic model for the study of the redoxmechanism of hemoproteins such as cytochromes [8],hemoglobin [9], myoglobin [5] and peroxidases [10–13].

Microperoxidases do not form l-oxo dimmers [14] and,in aqueous solution, are in the monomer form only at lowconcentrations.

Several studies have been made concerning the coordi-nation sphere of the heme iron present in microperoxidases[15–18]. Considering that many of the physical and spectro-

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Fe3+

11

GLn

Lys13

Cys14

Ala15

Gln16

Cys17His18

Thr19Val20

21Glu

O

O-

O

N

N N

N

S

S

CH3CH3

CH3

OH2

N

N

CH3

CH3

CH3

H

O-

Mp11: Val-11 to Glu-21Mp9: Lys-13 to Glu-21Mp8: Cys-14 tp Glu-21

Fig. 1. Three of the hemepeptides from cytochrome c. Microperoxidase-11 (MP11) retains cytochrome c residues 11–21, microperoxidase-9 (MP9)retains cytochrome c residues residues 13–21 and microperoxidase-8(MP8) retains cytochrome c residues residues 14–21.

A. Riposati et al. / Journal of Inorganic Biochemistry 100 (2006) 226–238 227

scopic studies of microperoxidases require high concentra-tions, the problem of aggregation has been circumventedby using alcohol–water mixtures [19,20], micellar solutions[21,22] and by N-acetylation [23–29].

Yang and Sauer [30] published the first EPR (electronparamagnetic resonance) and magnetic susceptibility studyof MP-8 with an N-acetylated amino terminal group (N-AcMP-8). These authors showed that this chemical modifi-cation prevents dimerization by blocking intermolecularcoordination. Wang and Van Wart [31] reported Ramanresonance and optical study of N-AcMP-8 claiming thatthe acetylation of N-terminal Cys14 residue abolishes itsaggregation in neutral aqueous solution. At neutral pHand room temperature [31], the heme iron of N-AcMP-8exhibits His18 and water, respectively, as proximal distalligands and also exists as a thermal equilibrium of the inter-mediate (S = 3/2) and the high-spin (S = 5/2) states. Thebehavior of MP-11 bis(N-acetylated) has also been wellcharacterized [32]. According to Peterson and co-workers[33], the treatment of MP-11 with acetic anhydride resultsin the acetylation of the Lys13 e- and a-amine groups.The bis(N-acetylated) MP-11 exhibits a water molecule atthe sixth coordination position which can be displaced byexogenous ligands such as azide. The replacement of waterby another ligand at the sixth coordination position isaccompanied by changes in the heme iron’s magneticproperties.

From the above literature survey, it is evident that thereis not much information about the low spin state of MPsproduced by coordination of MP heme iron with the e-or a-amino group from another hemepeptide molecule.Furthermore, the behavior in aqueous solution of N-acety-lated MP-9 remains to be studied.

In this work, for the first time, we present a systematiccomparative study of N-Acetyl heme nondecapeptide (N-AcMP-9), MP-8, and MP-9 behavior in aqueous solutionas a function of pH. The characteristics of these speciesof MP were investigated using EPR, CD (circular dichro-ism), and UV–vis (ultra-violet and visible) spectroscopies,and MD (molecular dynamic) simulations.

2. Materials and methods

MP-8 and MP-9 were purchased from Sigma anddiluted in purified water (Millipore system). The bufferwas prepared with 30 mM of both sodium phosphate andsodium carbonate. N-AcMP-9 was prepared from MP-9following the previously described method [34]. Acetyla-tion was accomplished by dissolving powdered MP-9(�3 mg) in 740 lL of 0.2 M sodium carbonate buffer, atpH 9.3, prior to cooling the solution to 4 �C and injecting500 M excess of acetic anhydride (Merck). The reactionmixture was allowed to heat to 30 �C, at the heating rateof the thermostatic bath, and maintained at this tempera-ture for 3 h. Production of acetic acid during acetylationshifted the pH to �4, thereby inducing precipitation ofN-AcMP-9. Saturated ammonium sulfate solution wasadded dropwise to maintain the precipitate. Thereafter,the mixture was centrifuged at 6000 rpm for 40 min. Thesupernatant was removed, and the remaining solid was dis-solved in dilute NaOH (�10�5 M). The solution wasdesalted in an ultrafiltration cell (25 mL) using a YM1 Mil-lipore membrane. The concentration of N-AcMP-9 wasdetermined by optical absorption using the molar absorp-tion coefficient e397 nm = 1.56 · 105 M�1 cm�1 [31]. To ver-ify the efficiency of the acetylation process, analyticalHPLC was performed, by an BioRad HPLC-2800, with aWatters C18 (250 · 4.6 mm) analytical column. Samples(20 lL) were injected onto the column at equilibrium withthe initial solvent composition of 80% A (50 mM phos-phate buffer at pH 6.75). Elution was effected at1 mL min�1 with a linear gradient resulting in 30% (A)and 70% B (acetonitrile) at t = 25 min.

Absorption spectra of all samples were recorded on aMultispec 1501 Shimadzu spectrophotometer at room tem-perature. Optical titration of MP-8, N-AcMP-9 and MP-9were performed with samples dissolved in 30 mM sodiumphosphate and sodium carbonate buffer. The pH of solu-tions was measured with a Beckman 250 pH meter.

Circular dichroism (CD) measurements at the wave-length interval 300–500 nm were performed at ambienttemperature with a Jasco J-720 spectropolarimeter. AllCD spectra were subtracted from their respective baselines.To analyze the CD spectra, the convex constraint analysis(CCA) method proposed by Perczel and co-workers [35]was applied in order to estimate the number of species inthe equilibrium as a function of pH. This is a generalmethod of spectra decomposition based on the simplexalgorithm that calculates a variable number p of base func-tions fi (pure spectra corresponding to different species),and the coefficients Cij, which will give the recovered func-tions fj (experimental spectra), as shown in

fj ¼Xp

i¼1

Cij � fi. ð1Þ

Three restrictions on these coefficients are considered: (1)the sum of coefficients is unity for each value of j,

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Fig. 2. pH and concentration effects on the UV–vis spectra of MP-8. (A)UV–visible spectra of 50 lMMP-8 obtained at pH 7.0 (thin solid line), pH10.0 (dashed line) and pH 12.0 (dotted line) in a quartz cuvette with 1 mmoptical path. (B) UV–visible spectra of MP-8 obtained with 10, 25 and50 lM of the hemepeptide at pH 12.0 (thin solid line indicated by thearrow, respectively). The dotted line represents the 0.5 lM MP-8 spectraobtained at pH 12.0. The spectra obtained at high MP-8 concentrationswere normalized according the Soret band peak exhibited at 10 lM. TheMP-8 spectrum obtained at 0.5 lM was normalized and multiplied by afactor of 0.98 for clarity. The inset shows the concentration dependence onthe absorbance intensity of MP-8 Soret band peaking at 401 nm obtainedat pH 12.0 over the concentration range of 0–1.25 lM. The fit linearobtained for the points over the concentration range of 0–0.75 lM wasextrapolated to 1.25 lM using tools available in the Microcal Origin 6.0program. The spectra were obtained at room temperature in 30 mMsodium phosphate and carbonate buffer adjusted to different pH values.

228 A. Riposati et al. / Journal of Inorganic Biochemistry 100 (2006) 226–238

PCij ¼ 1, where the sum is for i from 1 to p,

j = 1,2, . . . ,N, . . ., and N is the number of experimentalfunctions in the analysis; (2) all coefficients are positives,that is, Cij > 0; (3) the points corresponding to Cij obey asimplex in Euclidian space of dimension p with the minimalvolume. A more detailed discussion of the use of this pro-cedure is given in Refs. [36–39].

EPR continuous wave spectra were recorded using anX-band spectrometer (Bruker Elexsys line E-580) equippedwith a standard rectangular cavity. The temperature of�4 K was maintained by a low temperature accessory(Helitran Oxford Systems). The sample was placed in aquartz tube and frozen in liquid nitrogen prior to beingintroduced into the microwave cavity to record the spectra.Blank samples, containing only the buffer at each pH, werealso recorded, and the data subtracted from that of thesample containing MP-8, N-AcMP-9 and MP-9, takinginto account the matching of microwave frequency. Exper-imental parameters were as follows: microwave frequency,9.5 GHz, microwave power, 8 mW, magnetic field scanranges 40–540 mT, and modulation amplitude, 1 mT.EPR spectra were analyzed using QPOW program [40–42] in order to estimate the number of species in the equi-librium as a function of pH. This software is widely used tosimulate EPR powder spectra including both Gaussian andLorentzian line shapes.

MP9 MD simulation with explicit water [43,44] solventwas carried out using the GROMACS package [45], withGROMOS96 force field [46]. The initial coordinates forMP9 were taken from the corresponding fragment ofhuman cytochrome c 1AKK, from a Protein Data Bank.The long range electrostatic potential was calculatedthrough the reaction field method [47] with e = 54 and14 A cut-off radius. For the short range potential calcula-tions 12 A was used as the cut-off distance. Temperatureand pressure were kept constant at 295 K and 1 bar usingthe Berendsen heat and pressure coupling algorithm [48].A typical simulation of an MP-9 molecule, depending onthe initial coordinates and microperoxidase protonatestate, has about 3800 water molecules and few Na+ ionswhich maintains no net charge in the system. Different runswere carried out using MP9 with both the Lys13 N-termi-nal and e-amino group either protonated or deprotonated.

3. Results and discussion

3.1. Effect of pH on UV–vis spectra of microperoxidases

The pH and concentration dependence of the electronicspectra of MP-8 in an aqueous multicomponent buffer isshown in Fig. 2A and B, respectively. Fig. 2A shows a dropof the MP-8 Soret (�400 nm) band accompanied by hyper-chromism of the N band (�340 nm) when pH is increasedfrom 7.0 to 12.0. The electronic spectra that are includedhere take in account the high concentration of sample, topromote more intensive aggregation. These spectralchanges are consistent with aggregation of MP-8 molecules

probably due to the establishment of an inter-chain coordi-nation between the heme iron of one MP-8 molecule andthe cysteine amino group (N-terminal) of a neighboringMP-8 molecule. This amino group replaced a water mole-cule that, at low pH values, should be the heme iron ligandat the sixth coordination position.

The change of the sixth heme iron ligand, produced bythe increase of pH, led to the conversion of the heme ironfrom a high to low spin state. Higher pH also led to spec-tral changes in the visible region bands, suggesting achange of the axial ligands of the heme iron. S = 5/2 Q


and CT bands were red shifted. For N-AcMP-8, higher pHled to the conversion of the heme iron from a high to lowspin state due to deprotonation of a water molecule, whichis located at the sixth coordination position [34]. However,in the case of MP-8 (Fig. 2A), the equilibrium between S=5/2 and S = 1/2, observed around pH 10.0, should be pro-moted by the replacement of water as the sixth ligand bythe deprotonated N-terminal group of a neighboring MP-8 molecule. This proposal is congruent with the hyperchro-mism of the N-band and hypochromism of the Soret bandboth being indicative of MP-8 aggregation [16]. However,the spectral changes exhibited with the increase of the pHfrom 10 to 12 suggest that MP-8 aggregates are favored.Fig. 2B compares the spectra of MP-8 samples obtainedat pH 12 and containing 0.5 (dotted line), 10, 25 and50 lM (solid lines as indicated by the arrows) of the heme-peptide. The spectrum of 0.5 lM MP-8 corresponds to themonomeric form of the hemepeptide exhibiting OH� at thesixth coordination position. This proposition is groundedon the fact that, at pH 12, MP-8 Soret band obeys Beer’slaw below 1 lM (Fig. 2B, inset). At pH 12, the blue shiftexhibited by the Soret band with the increase of MP-8 con-centrations (solid lines as indicated by the arrows), suggeststhat another MP-8 high spin state was also favored in suchconditions.

Electronic spectral changes for N-AcMP-9, similar tothose described for N-AcMP-8 [34], suggest the heme ironconversion from a high to low spin state without signal ofaggregation (Fig. 3).

The pH dependence of the electronic spectra of MP-9 inan aqueous multicomponent buffer is shown in Fig. 4A.Differently to MP-8 (Fig. 2A), at high pH, MP-9 electronicspectral changes suggest conversion from a high to low spinstate but not accompanied by aggregation (Fig. 4A). InFig. 4A, the red shift of Soret and CT bands associatedwith the intensification of Q0 and Qv bands that were also

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N-AcMP-9 pH 7N-AcMP-9 pH 10N-AcMP-9 pH 12

Fig. 3. pH effect on the UV–vis spectra of N-AcMP-9. UV–visible spectraof 50 lM N-AcMP-9 obtained at pH 7.0 (thin solid line), pH 10.0 (dashedline) and pH 12.0 (dotted line) in a quartz cuvette with 1 mm optical path.The spectra were obtained at room temperature in 30 mM sodiumphosphate and carbonate buffer adjusted to different pH values.

red shifted, are compatible with the formation of the lowspin state, but the hypochromism and hyperchromism ofthe respective N and Soret bands are indicative of a non-aggregate state of MP-9 at high pH values.

N-AcMP-8 [34] and N-AcMP-9 could attain the lowspin state at approximately pH 10.0 only by the presenceof OH� at the sixth coordination position, and so didnot exhibit aggregation. In the case of MP-9, consideringthat the e- and a-amino groups of Lys13 were availablefor coordination at high pH values, an unaggregate lowspin state could be attained only by an intra-chain coordi-nation. According this proposition, completely differentspectra were obtained for 0.5 and 50 lM MP-9 at pH 7.0(Fig. 4B) and almost identical spectra were obtained for0.5 and 50 lM MP-9 at pH 10.0 (Fig. 4C). At high pH, dif-ferently of MP-8 (Fig. 2B), MP-9 did not exhibit Beer’s lawdeviation that occurred when the MP-9 spectra at the sameconcentrations were obtained at pH 7.0 (inset Fig. 4B).Considering that MP-9 could exhibit intra-chain coordina-tion, different MPs structures were expected for low spinstates obtained at high pH values and should be prone todetection by CD (Fig. 5A–C).

3.2. Effect of pH on the CD spectra of microperoxidases

Fig. 5A shows that, at pH 7.0 and 10.0, MP-8 exhibitsdifferent CD spectra but with a complex and symmetricSoret Cotton effect, respectively. At pH 12.0, two overlap-ping positive bands composed the CD spectrum. It isimportant to note that, the spectra were measured varyingpH between 6 and 13 in steps of 0.5 (not shown).

N-AcMP-9 exhibited CD spectra approaching Gaussianfunctions in the whole investigated pH range (Fig. 5B). Ared shift of the CD Soret band accompanied the increaseof the pH. MP-9, at pH 7.0 exhibited a CD spectrum sim-ilar to MP-8 (Fig. 5C). The MP-9 CD and UV–vis spectraobtained at pH 7.0, suggest that similar aggregates shouldbe present in this condition. However, at higher pH values,MP-9 CD spectra differed dramatically from those exhib-ited by MP-8.

Circular dichroism spectra of hemeproteins at the400 nm region are strongly dependent on the microenviron-ment provided by the peptide associated to the heme group[49]. For cytochrome c the trough at 420 nm relates to theheme iron-Met80 coordination [50,51]. Therefore, formicroperoxidases this trough could reflect the sixth hemeiron ligand. In fact, in previous studies, interactionsbetween heme octapeptide units have been correlated withan increase in the complexity of the Soret–Cotton effect[16].

Considering that the CD spectra of microperoxidasesobtained at different pH values could be a mixture of spec-tra coming from different species co-existing in differentproportions, it was important to carry out a decompositionprocess in order to obtain the CD spectra of the differentcontributing species (see Section 2). In the decompositionprocess it is important to minimize the error by determin-

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Fig. 4. pH and concentration effects on the UV–vis spectra of MP-9. (A) UV–visible spectra of 50 lM MP-9 obtained at pH 7.0 (thin solid line), pH 10.0(dashed line) and pH 12.0 (dotted line) in a quartz cuvette with 1 mm optical path. (B) UV–visible spectra of MP-9 obtained with 0.5 (thick solid line) and50 lM (thin solid line) of the hemepeptide at pH 7.0. For the low and high MP-9 concentrations the optical lengths were 10.0 and 1.0 mm, respectively (C)UV–visible spectra of MP-9 obtained with 0.5 (thick solid line) and 50 lM (thin solid line) of the hemepeptide at pH 10.0. For the low and high MP-9concentrations the optical lengths were 10.0 and 5.0 mm, respectively. In panels B (pH 7.0) and C (pH 10), the insets show the concentration dependenceon the absorbance intensity of MP-9 Soret band peaking at 396 and 405 nm, respectively over the concentration range of 0–1.25 lM. For both the insets,the fit linear obtained for the points over the concentration range of 0–0.75 lM was extrapolated to 1.25 lM using tools available in the Microcal Origin6.0 program. The spectra were obtained at room temperature in 30 mM sodium phosphate and carbonate buffer adjusted to different pH values.


ing the number of pure spectral components (p) that moreprecisely describe the complete set of CD spectra obtainedover the studied pH range. Considering that p is one of theinput parameters, the magnitude of p must be presumed bya rough estimation. In this work, the estimation of p mag-nitude was based on the UV–vis (Fig. 2A) and EPR(Fig. 8A) results that suggested, respectively, the existenceof two and three pH-dependent MP-8 species. Therefore,a series of calculations were performed by assuming p val-ues = two, three and four, whose respective standard devi-ations (r)p values were: 0.712, 0.138 and 0.119 [35].Although r4 value was lower than r3 value, the four spec-tral components obtained for p = 4 were a linear combina-tion of the three pure CD spectra obtained for p = 3.Therefore, the most precise description of the MP-8 CDspectra obtained at different pH values was obtained withthree spectral components (p = 3). Similar procedure wasadopted to determine the N-AcMP-9 and MP-9 species.

Fig. 6A (left panel) shows three MP-8 CD spectra calcu-lated according to Eq. (1) as a pure components (fi). Thestandard deviation in this case was r3 = 0.138. The rightpanel of Fig. 6A shows the calculated relative spectral con-tribution (Cij) of the three MP-8 species obtained at differ-ent pH values with an interval of 0.5 pH units. Over the pHrange 6.0–7.5 the composite MP-8 experimental spectraresulted from �65% contribution of species I (Fig. 6Adashed lines) and �35% of species II (Fig. 6A, solid lines).The calculated CD spectrum for species I matched with theCD spectrum of 1.7 lM MP-8 aggregated at pH 7.0 withrespectively positive and negative maximal wavelength at382 and 408 nm as shown in ref. [16]. This MP-8 aggregateresulted from interactions between heme moieties in astacked conformation. The calculated MP-8 species II spec-trum that attained the maximal spectral contribution atapproximately pH 9.5 did not match with any MP-8 aggre-gate CD spectra [16] at pH 7.0, which was expected since

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Fig. 6. Spectra of (A) MP-8, (B) N-AcMP-9 and (C) MP-9 speciesinvolved in the equilibrium and relative spectral contributions of species asa function of pH. The thick solid line represents the conversion of theaquo to hydroxide species of N-AcMP-8 [34].

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Fig. 5. pH dependence of the CD spectra of (A) MP-8 (B) N-AcMP-9 and(C) MP-9. The CD spectra of 100 lM hemeptide were obtained at pH 7.0(thin solid line), pH 10.0 (dashed line) and pH 12.0 (dotted line). Thespectra were carried out at room temperature in 30 mM sodium phosphateand carbonate buffer.


this aggregate should result from the coordination of adeprotonated N-terminal group of one MP-8 moleculewith the heme iron of a neighboring one.

The pH curve for the disappearance of species I run par-allel but it does not match with the pH curve for the N-AcMP-8 conversion from a high to low spin state due tothe deprotonation of the water molecule placed at the sixthcoordination position. It suggests that a similar conversionshould be occurring but exhibiting different pKa values(8.53 and 9.50, respectively), since the sixth heme iron

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357

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Fig. 7. Concentration dependence of the N band absorbance (357 nm) ofMP-9 at pH 7.6 and 9.0 in 30 mM sodium phosphate and carbonatebuffer. The inset shows the CD spectra of MP-9 as a function ofconcentration at pH 9.0. The concentrations used were 2 (thin solid line),10 (dashed line) and 250 lM (thick solid line).


ligands, that are replacing a water molecule, are different.The pKa = 8.53 is lower than that expected for the a-aminogroup of cysteine (the N-terminal group of MP-8) which is10.28 [52]. The reduction of the intrinsic pKa value for thecoordinated amino group was expected since the metal ion(Fe3+) competes with protons for this group.

The second alkaline transition with pKa = 10.37 corre-sponds to the conversion of MP-8 species II to MP-8 spe-cies III, that at a first glance could be assigned to thereplacement of the N-terminal group of the neighboringhemepeptide by OH�. In this case, a larger proportion ofan MP-8 monomer form could be expected. However, datafrom previous studies demonstrates that heme octapeptide[16] and undecapeptide [43] aggregates formed around pH7.0, exhibit large complex CD bands due to interactingheme moieties while their monomer forms exhibit simplebands of lesser amplitude. Fig. 6A, left panel shows thatspecies III exhibited a simple Gaussian CD spectrumpeaking around 406 nm that might indicate the absenceof aggregates. However, the comparative UV–vis spectrumof species III obtained at low and high concentrations(Fig. 2B) indicated that the aggregates are still present inhigh concentrations since Soret band hypochromism andN-band hyperchromism occurred in these conditions andMP-8 spectra obtained at different concentrations did notobey Beer’s law in concentrations above 1 lM (Fig. 2B).As showed below, the EPR results obtained with MP-8 atpH 12 supplied additional data to propose the MP-8ligands in the aggregates formed at high pH.

Fig. 6B shows the decomposition of N-AcMP-9 CDspectra obtained over the pH range 6.0–13.0 In this case,the more precise description of the N-AcMP-9 CD spectraobtained at different pH values was obtained with twospectral components (p = 2) and the standard deviationobtained was r2 = 0.501. The predominant CD spectrumover the pH range 6.0–9.0 (Fig. 6B, dashed line) shouldcorrespond to the N-AcMP-9 high spin state in the mono-mer form since the spectrum of N-AcMP-9 species Iapproaches a simple Gaussian function peaking at397 nm. N-AcMP-9 species II should exhibit OH� occupy-ing the heme iron sixth coordination position (Fig. 3B,thick solid line). Interestingly the pKa = 9.6 for the conver-sion between N-AcMP-9 species matched exactly with thepKa value determined for the conversion of the equivalentspecies of N-AcMP-8 [34].

Fig. 6C shows the decomposition of MP-9 CD spectraobtained over the pH range 6.0–13.0 (left panel) with therelative contributing species at different pH values (rightpanel). In this case, the more precise description of theMP-9 CD spectra obtained at different pH values wasobtained with five spectral components (p = 5) and thestandard deviation obtained was r5 = 0.190. Fig. 6C (leftpanel) shows that the CD spectra of five MP-9 species, cal-culated according to Eq. (1), contribute in different propor-tions to the composite MP-9 CD spectra over the pH range6.0–13.0. The right panel of Fig. 6C shows the calculatedrelative spectral contribution of the five MP-9 species at

different pH values. At pH 6.0, MP-9 experimental CDspectra resulted from the almost 100% contribution of aspecies I (Fig. 6C dotted lines). This species exhibited acomplex Cotton effect with bands at 378 and 407 nm andcould be attributed to the corresponding MP-9 species Iwith H20 as the sixth heme iron ligand. The conversionof MP-9 species I to species II occurred at pKa = 7.0. Con-sidering that the a-amino group of Lys, in this case, the N-terminal group of MP-9, presents an intrinsic pKa = 8.95, itis reasonable to consider that species II was formed when awater molecule was replaced by the N-terminal group of aneighboring MP-9 molecule. In this case the pKa shouldalso decrease due to proximity to the metal ion.

A complex CD spectrum accompanied the conversion ofMP-9 species II to III. The spectrum seemed to result fromthe increasing contribution of positive bands peakingaround 410 nm overlapped with a Cotton effect. The anal-ysis of the corresponding UV–vis spectra (Fig. 4A) suggeststhat species III was not an aggregated MP-9 form since theN-band almost disappeared and the Soret band exhibitedhyperchromism. The pKa = 8.5 for the MP-9 species II toIII transition was compatible with the decrease of theintrinsic pKa value (10.53) for the Lys e-amino group. Con-sidering that this amino group was the sixth ligand in MP-9species III, the absence of aggregation could only beexplained by intra-chain coordination. A new species pre-dominated above pH 9.4 (Fig. 6C, dotted dashed line). Thisvalue was close to the pKa for water deprotonation(Fig. 6C, thick solid line), suggesting the presence ofOH� as a heme iron ligand in this species. Above pH12.5, a fifth MP-9 species was recorded and could involvethe presence of histidinate as the fifth heme iron ligand,since this pKa value matched that previously reported for

1000 2000 3000 4000 5000

IIIIII

IIII

I

pH 7

Inte

nsity

(a.

u.)

Magnetic Field (G)

pH10

pH 9

pH 11

IpH 12

′

1000 2000 3000 4000 5000

Inte

nsity

(a.

u.)

Magnetic Field (G)

I

pH 9

pH 10

pH 7

pH 11

III

II

pH 12

II

III

1000 2000 3000 4000 5000

IV

IIIIII

Magnetic Field (G)

pH 7II

III

IV

Inte

nsity

(a.

u.)

pH 9

pH 10

pH 11

pH 12V V

A

B

C

Fig. 8. pH dependence of the EPR spectrum of (A) MP-8 (300 lM), (B)N-AcMP-9 (200 lM) and (C) MP-9 (300 lM) at 4 K temperature in30 mM sodium phosphate and carbonate buffer.

Table 1The g values of microperoxidases species at different pH values

Samples g-Values

Spin 5/2 Spin 1/2

Species I Species II Species III Species IV Species V

MP-8 6.030 3.180 2.8755.780 2.140 2.2402.005 1.340 1.685

N-AcMP-9 5.990 3.380 2.9905.780 2.070 2.2062.005 1.500 1.590

MP-9 6.051 3.193 2.982 2.919 2.7155.775 2.125 2.258 2.230 2.2342.010 1.380 1.800 1.725 1.905


histidine coordinate with Co(II) [34] and was close to thatdetermined for a similar N-AcMP-8 species.

If the assumption that MP-9 species III was formed bythe establishment of an intra-chain coordination was

correct, the influence of concentration on MP-9 aggrega-tion should be diminished by increasing the pH from 7.6(the optimal pH for species II) to 9.0 (the optimal pH forspecies III).

Fig. 7 shows that the formation of MP-9 aggregatesmeasured the increasing the N band at 357 nm as a func-tion of the hemepeptide concentration exhibited a slope35% lower when obtained at pH 9.0. This result shows that,as expected, it was more difficult for the N-terminal groupof a neighboring MP-9 molecule to replace the intra-chainligand (Lys13 e-amino group), that is the competing hemeiron ligand at pH 9.0, than a water molecule, that is thecompeting heme iron ligand at pH 7.6. The increase ofthe N band in the MP-9 UV–vis spectrum at high concen-trations suggested an increase of the species II/species IIIratio at these conditions. The insert in Fig. 7 shows clearlythat the MP-9 CD spectrum obtained at pH 9.0 withincreasing hemepeptide concentrations concomitantlyexhibited a more significant contribution of MP-9 speciesII spectrum (compare with Figs. 5C and 6C).

3.3. Effect of pH on the EPR spectra of microperoxidases

Considering that the heme Fe(III) in microperoxidases isa paramagnetic species, EPR measurements were used tobetter investigate the spin state of the contributing speciesof MPs at different pH values (Fig. 8A–C). EPR measure-ments require more concentrated samples of microperoxid-ases then optical spectroscopy to obtain high signal/noiseratio. In order to establish the minimal microperoxidaseconcentration necessary to obtain the best quality of theEPR signal, measurements were performed with 100, 200(not shown) and 300 lM. The highest quality of the signalwas obtained with 300 lMmicroperoxidases. However, thecomparison of the EPR spectra obtained with the three dif-ferent concentrations revealed an increase of the contribu-tion of the S = 1/2 species signal relative to S = 5/2 speciessignal. This result indicates clearly that the aggregation wasdependent of the microperoxidase concentration. The EPRspectrum of MP-8 obtained by spectral simulation (Table1) at pH 7.0 presented the high spin state with quasi-axial

pH > 10

pH ≥ 7

high spin state EPR signal

NH2

Cys

NH

2

Cys

His

Fe

OH

-

Fe

OH

-

OH

-Fe

His

OH-H

isHis-

Cys

OH

-

Fe

-H

is

Cys

NH2

NH

2

NH2Cys

Fe

OH

-

OH

-

Species IIILow spin states,

aggregate.

Species I’

High spin state,aggregate

Species II Aggregate and low spin state.

The extremity contributes to the

Species I Monomer form, high

spin state

Cys

Cys

Fe

HisFe NH

2

Cys

NH2

Cys

Fe

Fe NH3+

His

H2O

Cys

Fe

NH2

OH-

His-

NH2

His

H2O His

Scheme 1. Species distribution as a function of pH for MP-8 in an aqueous multicomponent buffer system.

pH >11

FeFe

H2O

C CH3

O

HisHis

OH-

N

His-

O

CH3CFe

Species I Monomer form, high

spin state

Species II.

Species III. Monomer form, low spin

state.

Monomer form, low spin

N

OH-

pH > 8CH3

O

CN

Scheme 2. Species distribution as a function of pH for N-AcMP-9 in an aqueous multicomponent buffer system.


symmetry (species I, g1 = 6.030, g2 = 5.780 andg3 = 2.005), as the predominant form of the heme-peptide.At this pH value a significant amount of the low spin state

with rhombic symmetry (species II) was also present withg1 = 3.180, g2 = 2.140 and g3 = 1.340, as shown in Table1. The increase of the pH led to the decrease of the EPR


signal from the high spin state with a concomitant increaseof the EPR signal of the low spin state.

Above pH 10.0 new high and low spin states appeared(Fig. 8A). Wang and co-workers have previously describedthis new high spin state [10] for N-AcMP-8 and attributedit to a pentacoordinated heme iron where OH� replacedHis18 at the fifth coordination position and this histidineresidue replaces water at the sixth coordination positionof a neighboring MP-8 molecule. The replacement of a his-tidine and an amino group as the respective fifth and sixthheme iron ligands by a hydroxide ion and histidine (ormore probably histidinate) may be expected to convertthe MP-8 aggregate to a high spin state (species I 0). Similarresults have been obtained with the N-AcMP8/3-CNPy (6-

NH3

Species IV Monomer form, low

spin state.

His

Lys

CysFe

OH-

NH2

NH2

+

NH3+

pH ≥ 7

H2O

His

Fe Cys

NH2

Fe Cy

Ly

HO

N

Lys

Species I Monomer form,.high spin

state

Scheme 3. Species distribution as a function of pH for M

coordinate 3-cyanopyridine complex of N-AcMP-8) system[34]. The EPR spectrum of MP-8 above pH 11.0 reveals thepredominance of a low spin state MP-8 aggregate (speciesIII, g1 = 2.875, g2 = 2.240 and g3 = 1.685). This speciesshould be attributed to the permanence of the N-terminalgroup as the sixth heme iron ligand and OH� at the fifthcoordination position. In both types of MP-8 aggregatesan extremity with the porphyrin ring exhibiting a hydrox-ide ion at the two sites of heme iron axial coordination isexpected (see Scheme 1). The apparent absence of theEPR signal from this bis-OH� coordinated species remainsto be elucidated. It is important to note that the longer theaggregates the lesser the contribution of the signal comingfrom their extremities. These assumptions are congruent

pH = 11

Species VMonomer form, low

spin state.

Monomer form, low spin state.

Lys

CysFe

His-

NH2

NH2

s

s

NH

2

NH3+

His

Fe Cys Lys

2

HisFe

Cys

Lys NH3

+

NH2

HisFe

Cys

Lys

NH3+

NH2

H2

Species II. Aggregated form, low spin state.The extremities contribute to the

high spin state EPR signal.

Species III

P-9 in an aqueous multicomponent buffer system.

Fig. 9. Snapshot showing a typical conformation of Mp9 with the sixthcoordination between heme iron (green ball) and Lys-13 Ne (blue atomjust above iron), after 8 ns MD simulation. The water molecules are notshown for clarity and disulfide bonds are indicated in yellow. The figurewas generated using VMD molecular visualization package according toHumphrey et al. [60].


with the presence of MP-8 aggregation at high pH values,since in both proposed models, OH� would replace theintra-chain ligand (histidine) without impairing the occur-rence of the inter-chain coordination.

The EPR spectrum of N-AcMP-9 (Fig. 8B) obtained byspectral simulation at pH 7.0 presented high spin state withaxial symmetry (species I, g1 = 5.990, g2 = 5.780 andg3 = 2.005, Table 1), as the predominant form of theheme-peptide. The increase of pH led to the conversionof the high spin state to an N-AcMP-9 low spin state withg1 = 3.380, g2 = 2.070 and g3 = 1.500 (species II, Table 1)and was compatible with the deprotonation of a water mol-ecule situated at the sixth coordination position. At pH 2.0,a new species appeared exhibiting g1 = 2.990, g2 = 2.206and g3 = 1.590 (species III, Table 1), obtained by spectralsimulation (Fig. 8B). This EPR signal should be assignedto the ionization of the coordinated histidine residue form-ing the S = 1/2 imidazolate complex.

Fig. 8C shows the EPR signal of MP-9 species obtainedat different pH values. Five MP-9 species were detectedcompatible with the CD spectra decomposition for thishemepeptide. At pH 7.0, besides the high spin state (speciesI, g1 = 6.051, g2 = 5.775 and g3 = 2.010, obtained by spec-tral simulation, Table 1) significant amounts of two S = 1/2 species were also present (species II, g1 = 3.193,g2 = 2.125 and g3 = 1.380 and species III, g1 = 2.982,g2 = 2.258 and g3 = 1.800), probably produced by inter-chain and intra-chain coordination, with predominanceof the first, since aggregation was still present in this condi-tion. Increasing pH led to disappearance of the MP-9 highspin state and the appearance of two new low spin states(species IV, g1 = 2.919, g2 = 2.230 and g3 = 1.725 and spe-cies V, g1 = 2.715, g2 = 2.234 and g3 = 1.905). These spe-cies could exhibit OH� or imidazolate at the fifthcoordination position, while the intra-chain Lys13 e-aminogroup remained at the sixth coordination position.

It is possible to see the conversion from the species II tospecies III, by comparing the EPR spectra obtained at pH 9and 11 (Fig. 8C). The spectral simulation permitted toobtain more exactly the g values of these species. Althoughsmall differences have been obtained for the g values ofthese species, it is well known that small differences in g val-ues could represent notable changes heme iron mcro-envi-ronment. Scheme 1–3 illustrate the proposal for MP-8, N-AcMP-9 and MP-9 species at different pH values,respectively.

3.4. Modeling of MP-9 intra-chain coordinated structure by

MD simulations

MP-9 MD simulations were carried out by using GRO-MOS force field, in order to verify the hypothesis of theoccurrence of the intra-chain Lys13 e-amino group atthe heme iron sixth coordination position. Reliability ofthe GROMOS force field to simulate ferric porphyrinstructure can be attested by the well-known modeling workspublished by the research groups of Shelnutt and Woody

[53–59]. Furthermore we checked the validity of the methodby reproducing adequately the structure of a number ofhigh- and low-spin ferric porphyrins, the coordinates ofwhich were obtained from the Protein Data Bank (datanot shown). Simulations as long as 20 ns of MP-9 haveshown that the distance between Lys13 Ne to heme ironcould be less than 4.0 A, sometimes as short as 3.2–3.4 A(data not shown). MP-9 could assume these short distancesbetween Lys13 Ne and heme iron only if the Cys14 disulfatebond (yellow bonds in Fig. 9) is, taking porphyrin ring planeas reference, flipped to the opposite side of Cys17 andHis18.For such Ne-Fe distances, the sixth coordination is not pos-sible, but by assuming a rotation of the Lys Cd–Ce planetowards the e amino group, the distance achieved (2.0–2.1 A) could be enough to establish the intra-chain coordi-nation. For two of these obtained conformations and exhib-iting rotation of the Lys Cd–Ce plane towards the e aminogroup, the sixth coordination was imposed, assuming thesame force field parameters of the MP-9 penta-coordinatedstructure. After energy minimization, 10 ns MD simulationwere carried out for the two MP-9 rotated conformationsthat remained structurally stable during all simulationsand did not present stress in any part of the molecule (datanot shown). A typical conformation of the Lys Ne-ironcoordinated MP-9 after 8 ns MD simulation is shown inFig. 9. The MP-9 conformations obtained by MDsimulations indicate that the intra-chain coordinatedstructure of the hemepeptide is structurally feasible and sta-ble as suggested by the spectroscopical results showedpreviously.


4. Conclusions

This study showed that non-acetylated microperoxid-ases -8 and -9 could form low spin state aggregates bythe establishment of inter-chain coordination encompass-ing a deprotonated amino group of N-terminal amino acidresidues. MP-9 exhibited the ability to establish intra-chaincoordination with the e-amino group of its Lys13 residueand thus exists predominantly in the monomer form abovepH 8.5. The following results corroborated the proposedintra-chain coordinated MP-9 structure: (i) The MP-9UV–visible spectra obtained at high concentrations andhigh pH values did not differ significantly of the MP-9UV–visible spectra obtained at low concentrations at thesame pH values. In both conditions (high and low concen-trations) MP-9 spectra did not exhibit hyperchromism of Nband and hypochromism of Soret band; (ii) Oppositeresults were achieved when the MP-9 spectra were carriedout at pH 7.0. In this pH condition, completely differentUV–visible spectra were obtained at low and high concen-trations and the spectrum obtained at high concentrationswas, as expected, clearly indicative of the occurrence ofMP-9 aggregation; (iii) At high pH values MP-9 obeysBeer’s law in concentrations in which a deviation wasobserved at pH 7.0 and at pH 12 for MP-8; (iv) EPR andMP-9 CD spectra obtained at high pH values differ com-pletely of that obtained for MP-8 in the same conditions.Only for MP-9, the deconvolution of CD spectra obtainedat different pH values shows the contribution of five differ-ent species compatible with the occurrence of inter andintra-chain coordination.

The results presented here will contribute to the study ofthe heme iron coordination sphere of microperoxidases.

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