Báo cáo khoa học: Correlation between functional and structural changes of reduced and oxidized trout hemoglobins I and IV at different pHs doc

Correlation between functional and structural changes of reduced and oxidized trout hemoglobins I and IV at different pHs A circular dichroism study Rosita Gabbianelli1, Giovanna Zolese2

Correlation between functional and structural changes of reduced and oxidized trout hemoglobins I and IV at different pHs

A circular dichroism study

Rosita Gabbianelli1, Giovanna Zolese2, Enrico Bertoli2and Giancarlo Falcioni1

1 Dipartimento di Biologia M.C.A., Universita` di Camerino, Camerino, Italy; 2 Istituto di Biochimica, Facolta` di Medicina,

Universita` Politecnica delle Marche, Ancona, Italy

Circular dichroism (CD) spectra of two major hemoglobin

components (Hb), HbI and HbIV, from Oncorhyncus

mykiss (formerly Salmo irideus) trout were evaluated in

the range 250–600 nm HbI is characterized by a complete

insensitivity to pH changes, while HbIV presents the Root

effect Both reduced [iron(II) or oxy] and oxidized (met)

forms of the two proteins were studied at different pHs, 7.8

and 6.0, to obtain information about the pH effects on

the structural features of these hemoglobins Data obtained

show that oxy and met-HbI are almost insensitive to pH

decrease, remaining in the R conformational state also at

low pH On the contrary, the pH decrease induces similar structural changes, characteristics of ligand dissociation and Rfi T transition, both in the reduced and in the oxidized HbIV The structural changes, monitored by CD, are compared with the peroxidative activity of iron(II)-Hb and met-Hb forms and with the superoxide anion scav-enger capacity of the proteins

Keywords: trout hemoglobin derivatives; hemoglobin per-oxidase activity; superoxide anion; circular dichroism; pH effect

The hemoglobin system of the Oncorhyncus mykiss

(for-merly Salmo irideus) trout is made up of four

electro-phoretically distinct components, two of which [trout

hemoglobin (Hb)I (
20%) and trout HbIV (60%)]

repre-sent quantitatively a large fraction of the whole pigment In

the last years, the properties of these two major components

(HbI and HbIV) have been investigated in considerable

detail under various experimental conditions [1,2] Their

structural and functional characterization has indicated

some striking differences between the two proteins that have

been related to their different physiological role [2] HbI is

characterized by the presence of cooperative phenomena

and complete absence of pH and organic phosphate effects,

while in HbIV, oxygen affinity and cooperativity depend on

pH and organic phosphates (Root effect) In air (pO2¼

155 mmHg) and at pH 7.8, iron(II) HbIV is entirely in the

oxygenated form, while at pH 6.0 it is almost completely in

the deoxy form, as a consequence of the Root effect [2] On

the contrary, iron(II) HbI is fully oxygenated at both pHs

Moreover, both hemoglobins are stable towards

dissoci-ation even in the ligated form; the value of the tetramer–

dimer dissociation constant is between 10 and 50 times less

than that of human hemoglobin measured under similar

conditions [2]

The main function of HbI is to assure the basic level of

O2 to active tissues, providing a normal oxygen supply

in emergency, while a basic role of HbIV is to release

O2against high hydrostatic pressure in the swim bladder Our previous study by circular dichroism (CD) demon-strated large structural differences in HbI and HbIV ([3] and references cited therein), which are likely related to their different physiological roles as gas carriers

Trout HbIV and HbI are also characterized by different peroxidative activities [4,5], which are dependent from the

pH of the medium and/or the iron oxidation state In fact, it

is known that the hemoglobin molecules present peroxida-tive properties [5–7], which may be important for the life span of red blood cells (RBCs), continuously exposed to extracellular and intracellular sources of reactive oxygen species (ROS), which are a potential cause of oxidative injury and could have a role in erythrocytes senescence [7] The toxicity of H2O2, a reactive oxygen species involved

in cellular injury under various pathophysiological condi-tions, is known to be enhanced in the presence of hemoglobin [8] H2O2 binds to and reacts with Hb, generating the highly reactive ferrylhemoglobin intermedi-ate, which in turn oxidizes the substrate [6] The final products of the reaction are superoxide radical and met-Hb However, our previous studies on human hemoglobin demonstrated that the presence of Hb reduces the level of superoxide in the medium [7] Met-Hb was shown to be more efficient in reducing the level of this radical with respect to oxyHb, and this difference was more marked at low pH values [7] It is known that, during reversible oxygen binding, Hb undergoes a slow autoxidation to met-Hb, producing superoxide, which is released into the heme pocket [9] The superoxide released in the heme pocket reacts with globin, producing a secondary radical [9]

Correspondence to R Gabbianelli, Dipartimento di Biologia M.C.A.,

Universita` di Camerino, Via Camerini 2–62032 Camerino (Mc), Italy.

Fax: + 39 073 7636216, Tel.: + 39 073 7403208,

E-mail: rosita.gabbianelli@unicam.it

Abbreviations: HbI, hemoglobin I; HbIV, hemoglobin IV; ROS,

reactive oxygen species; RBCs, red blood cells.

(Received 23 December 2003, revised 15 March 2004,

accepted 25 March 2004)

As heme’s interaction with globin appears to be

import-ant both for autoxidation [9] and H2O2 binding [6], a

systematic study of the heme–globin interaction, at different

pH and Hb oxidative state [iron(II)- or met-Hb], could be

important to increase the knowledge of the structural basis

of these interactions

Our previous CD study [3] demonstrated that the heme–

globin interaction in oxy-HbI and oxy-HbIV are quite

different The aim of the present paper will be to

charac-terize the heme–globin interaction in iron(II)- and

met-HbIV and HbI, at different pH values, by CD These studies

will be compared with the peroxidative and superoxide

anion scavenger activities of these proteins The CD

spectroscopy will be particularly useful because this

tech-nique permits the evaluation of the optical activity of heme

proteins [10,11], which result from different kinds of heme

interaction with the protein matrix

Three different wavelength regions (near-UV, far-UV

and visible) can offer different degrees of information These

CD regions are largely used to study the tertiary and

quaternary structure of heme proteins [10,11]

A direct comparison for differences in structure between

trout HbI and HbIV in solution will increase the knowledge

on the physiological roles of these Hbs and on the molecular

adaptation mechanisms of these aquatic organisms living

under particular environmental conditions

Materials and methods

All reagents were of analytical grade Preparation of trout

hemoglobin components were performed as previously

described [12] Iron(III) hemoproteins were obtained by the

addition of K3[FeCN)6] (molar ratio 2 : 1) to the

oxygen-ated derivative; excess oxidizing agent and ferrocyanide

were removed by gel filtration through a Sephadex G-25

column eluted with 50 mMTris/HCl pH 7.8 or 50 mMBis/

Tris, pH 6.0 The hemoglobin concentration was

deter-mined by the pyridine–hemochromogen method [13]

Circular dichroism

CD spectra were recorded on a Jasco spectropolarimeter

under nitrogen flux at 6C In the near UV and Soret region

the Hb concentration was 0.1 mgÆmL)1, while in the range

490–670 nm the concentration was 0.5 mgÆmL)1

Measure-ments were carried out in 1 cm path length quartz cuvettes

The molar ellipticity is always expressed, on a molar heme

basis, as degreeÆcm2Ædmol)1 Readings were carried out

against a reference cuvette containing the same components

without protein Data were acquired at 6C, in order to

increase Hb stability [14] and to mimic environmental living

conditions of the trout All spectra are the averages of four

experiments, where three different recording were

accumu-lated for each sample

Peroxidase activity assay

The assay for peroxidase activity was performed as reported

by Everse et al [6] using guaiacol as substrate Fifty

millimolar sodium phosphate/citrate buffer (0.9 mL) at

pH 5.4 and containing Hb and 10 mMguaiacol was used

The reaction was started by the addition of 157 mMHO

solution (0.1 mL) and monitored by absorbance changes at

470 nm The absorbance change was due to Hb-catalyzed oxidation of the substrate by hydrogen peroxide

Chemiluminescence measurements Chemiluminescence measurements were performed by lucigenin as chemiluminogenic probe, and superoxide radicals were produced by xanthine/xanthine oxidase sys-tem as previously described [15] Briefly, the chemilumines-cence (CL) was measured in automatic LB 953 (Berthold Co., Wildbad, Germany) in a reaction mixture containing 0.9 UÆmL)1xanthine oxidase, 40 lgÆmL)1of hemoprotein and 150 lmolÆL)1lucigenin in 1 mL of the chosen buffer The reaction was started by the injection of xanthine at the final concentration of 50 lM and followed for 60 s as previously described [16] Values obtained are expressed as counts per second (c.p.s.)

Results

Circular dichroism L-Band (260 nm region) and 270–300 nm region The 270–300 nm region of CD spectra was used to study changes

in the Hb quaternary structure at the a1b2interface [3,17] Within the near UV region (1250–300 nm) CD bands are due

to aromatic amino acids, S-S bridges and heme groups [11], and are poorly characterized Near this region, theL-band (centered around 260 nm) is considered to be sensitive to the interactions between the heme and the surrounding globin, being influenced by the attached ligand and thereby by the spin state of the iron atom [11] According to Perutz et al [18], the region around 285 nm is considered as indicative of the Rfi T transition: in the T-form, the ellipticity is more negative than in the R-form [10,11,19] The ellipticity change

in this band is independent from the ligand state of heme, but

it is indicative only of Rfi T transition [10]

CD spectra acquired in the near-UV and Soret regions (250–320 nm and 320–470 nm, respectively) for iron(II)-HbI and iron(II)-iron(II)-HbIV at both pHs and in air are shown in Figs 1 and 2 In the range 250–320 nm (Fig 1), CD spectra are baseline corrected to zero ellipticity at 320 nm, accord-ing to Henry et al [20] In the region 320–470 nm (Fig 2), spectra are corrected to zero ellipticity at 470 nm In line with our previous data [3] iron(II)-HbI (Figs 1B and 2B) and iron(II)-HbIV (Figs 1A and 2A) spectra in the range 250–470 nm show similar positive bands, resembling the dichroic characteristics of other oxy-hemoglobins [21] In our experimental conditions, the ellipticity of the L band (centered around 260 nm) is directly related to pH (Fig 1A,C) for both iron(II)-HbIV and met-HbIV Only small changes between iron(II)- and met-HbIV, at both pH values, are evident (Fig 1A,C) L-Band ellipticity is directly related to pH decrease also in iron(II)-HbI and met-HbI (Fig 1B,D), although this effect is more evident for met-HbI Following iron oxidation (Fig 1D) almost no changes

in this band were observed at pH 7.8 [comparing this value

to the L-band of iron(II)-HbI at the same pH]

In both iron(II)- and met-HbIV, the change from pH 7.8

to 6.0 induces a shift towards a more negative ellipticity (correlated to Rfi T transition) in the region of 285 nm

Fig 1 Near-UV CD spectra of iron(II)-HbIV (A), iron(II)-HbI (B),

met-HbIV (C) and met-HbI (D) in 50 m M Tris/HCl, pH 7.8, (solid line)

and 50 n Bis/Tris, pH 6.0 (broken line) Temperature ¼ 6 C.

Fig 2 Soret CD spectra of iron(II)-HbIV (A), iron(II)-HbI (B), met-HbIV (C) and met-HbI (D) in 50 m M Tris/HCl, pH 7.8 (solid line) and

50 n Bis/Tris, pH 6.0 (broken line) Temperature ¼ 6 C.

(Fig 1A,C) For iron(II)-HbIV, this behavior is in

agree-ment with the presence of the Root effect, where the

pH decrease induces deoxygenation and transition to the

T-form However, met-HbIV, compared with

iron(II)-HbIV at the same pH, always shows a slightly more

negative ellipticity (Fig 1A,C) In trout iron(II)-HbI, the

pH decrease induces hardly any changes in the negative

ellipticity value at 285 nm (Fig 1B); while in met-HbI, at

pH 7.8 (Fig 1D) a slight increase in the negative ellipticity

compared with the same protein at pH 6.0 is evident On the

other hand, met-HbI at pH 6.0 shows a similar ellipticity to

iron(II)-HbI at both pHs

Visible region The Soret region for both iron(II) and

met-HbIV at pH 6.0 and 7.8 are shown in Fig 2A,C Changes

in the CD Soret region (near 400 nm) were related to the

interaction of the heme prosthetic group with the

surround-ing aromatic residues and to modifications in the spatial

orientation of these amino acids with respect to heme [22]

These modifications affect porphyrin transitions and p–p*

transitions in the surrounding aromatic residues However,

the protein-induced heme distortions from planarity and the

contributions of polarizable groups (near the heme) have

been recently postulated to participate to the ellipticity in the

Soret region [23]

According to some authors ([11,23] and references cited

therein) a blue shift in the Soret band is a consequence of

Rfi Ttransitionanditreflectstheinteractionbetweena1and

b1subunits It may be due to tertiary structural changes in

regions including aromatic residues and it may be involved in

the interactions between these subunits [24] However, this

bandissensitivealsotoliganddetachment(deligation)[17,19]

In the Soret region at pH 7.8, HbIV in the

oxygenated-form is characterized by a band at 418 nm, in agreement

with our previous data [3] A similar band is present in other

oxy-hemoglobins, such as human HbA [10,25] A significant

red shift (to about 433 nm) and a decreased intensity in this

peak was measured in both iron(II)- (Fig 2A) and

met-HbIV (Fig 2C), as a consequence of pH decrease, although

the superimposition of two different bands in met-HbIV at

pH 6.0 is evident (Fig 2C) Compared with the iron(II)

form at the same pH, met-HbIV shows a small blue shift in

the Soret band (from 418 to 416 nm at pH 7.8, while the

position of the peak measured at pH 6.0 is more difficult to

calculate, due to its form) and an increased positive

ellipticity (Fig 2A,C) In iron(II)-HbI (Fig 2B), the Soret

band is also localized at 418 nm, in line with previous results

[3] In this protein, the pH lowering induces a slight decrease

in the Soret band ellipticity, without wavelength shift

(Fig 2B) Met-HbI (Fig 2D), compared with the

iron(II)-form, shows a small blue shift and an increased ellipticity in

this band, at both pH values

The two major peaks in the region 500–600 nm (Q0and

Qm) give indications on the constraints at the heme site and

reflect the symmetric properties of the heme-iron bound

material [11] In particular, they are correlated to the

asymmetry of the proximal bond In fact, a decreased

symmetry leads to an enhanced intensity in the Q0 band

[25,26] The splitting of these bands has been regarded as a

lowering of the heme symmetry in HbA-CO [17,27], due to

nonperpendicular iron–ligand bond above the xy plane of

the heme group

Fig 3 Circular dichroismspectra in the visible region of iron(II)-HbIV (A), iron(II)-HbI (B), met-HbIV (C) and met-HbI (D) in 50 m M Tris/ HCl pH 7.8 (solid line) and 50 n M Bis/Tris, pH 6.0 (broken line) Temperature ¼ 6 C.

Both iron(II)-HbI (Fig 3B) and iron(II)-HbIV (Fig 3A)

at pH 7.8 (oxygenated), show very sharp Q0 bands with

similar intensities, while Qmshows a lower ellipticity in HbI

The spectra obtained are similar to those observed in many

vertebrate hemoglobins and myoglobins [10,17,21,25] The

Q bands of iron(II)-HbIV (Fig 3A) and -HbI (Fig 3B) at

pH 7.8 show some variations in relative intensity, suggesting

small differences in the constraints at the heme pocket In

HbI, the spectrum is only slightly modified by pH lowering

(Fig 3B), while HbIV at pH 6.0 (Fig 3A) shows a band

centered around 545 nm and a shoulder around 572 nm

(Fig 3A) This spectrum is quite similar to that of deoxy

human HbA [19], suggesting similar changes at the heme

site in HbA and trout HbIV

Following oxidation, met-HbIV (Fig 3C) shows spectra

similar to iron(II)-HbIV at both pHs tested These data

indicate only small modifications at the heme site On the

contrary, when met-HbI is compared with iron(II)-HbI, it

shows (Fig 3D) a slightly decreased intensity in the Q0band

(around 572 nm) with respect to Qmband (around 545 nm),

suggesting a modified symmetry in the proximal bond This

behavior is similar at both pHs

Peroxidase activity

The peroxidase activity was followed by monitoring the

increase in absorbance at 470 nm Guaiacol is a

methoxy-phenol that oxidizes to a radical, followed by dimerization

[6] Figure 4 shows the peroxidase activity of iron(II)- and

met-forms of HbI and HbIV It is evident that the enzymatic

activity decreases according to the order iron(II)-HbIV >

met-HbIV > met-HbI > iron(II)-HbI

Chemiluminescence measurements

Chemiluminescence (CL) measurements were performed by

using lucigenin as chemiluminogenic probe for superoxide

radical produced by xanthine/xanthine oxidase system The

reaction of lucigenin with superoxide radical gives rise to chemiluminescence whose level indicates the presence of superoxide in the medium under study Table 1 shows results obtained on met- and iron(II)-Hbs at two different

pH values, 7.8 and 6.0 The first parameter reported in Table 1 is the duration of the reaction, which was very different in the two buffers used At pH 7.8, the reaction time was always about 15 s, while at pH 6 this time was

40 s The maximum peak value was 1.640 ± 0.001 (· 107) c.p.s for Tris and 2.453 ± 0.0020 (· 105) c.p.s for Bis/Tris buffer, respectively The presence of 40 lgÆmL)1 of Hb [iron(II)- or met-derivatives of both hemoglobins] does not significantly reduce the CL level when the experiments were performed in 50 mMTris/HCl pH 7.8 The CL reduction was no larger than 5% in each sample When the experiments were performed in 50 mMBis/Tris pH 6, the

CL reduction was significantly different for HbI and HbIV: iron(II)-HbI and met-HbI induce a 23.2 and 50.1% reduction of the peak intensity, respectively, when com-pared with the peak of the buffer Iron(II)-HbIV and met-HbIV induce a 35.8 and 62.1% reduction of the peak intensity, respectively

Discussion

In red blood cells, Hb can readily generate or interact with free radicals [28] In fact, during spontaneous Hb autoxi-dation to met-Hb, the superoxide anion is produced Most superoxide is reduced by superoxide dismutase to H2O2 Catalase and glutathione peroxidase eliminate H2O2 Hemoglobin also presents peroxidative properties (hydro-gen peroxide removal activity), which was recognized more than 30 years ago and could be important for the cellular lifespan [5,6]

The mechanism of hydrogen peroxide removal by iron(II)-Hb (oxyHb and deoxyHb) and met-Hb results in the formation of ferrylhemoglobin (ferrylHb) and oxo-ferrylhemoglobin (oxoferrylHb), respectively Both are strong oxidizing agents, which can be source of cellular and tissue damage [8] A recent work [8] suggested that ferryl-Hb takes an electron from a second molecule of H2O2and is reduced to met-Hb, while the H2O2 is oxidized to super-oxide anion:

HbðIIÞ þ H2O2! HbFeðIVÞ ¼

Oþ H2O2! HbFeðIIIÞ þ O2 ! heme degradation Ferryl-Hb [formed by iron(II)-Hb] can be also capable of withdraw an electron from a suitable substrate, resulting in the formation of metHb and a substrate radical [6] The superoxide anion produced at the heme pocket is thought

to easily react with porphyrin molecule, resulting in heme degradation and iron release [8] However, H2O2-induced heme degradation products were demonstrated with iron(II)-Hb, but not with met-Hb [8,29] The inability to produce heme degradation products by met-Hb with H2O2, was explained by the reaction of oxoferrylHb with H2O2 with the production of met-Hb and oxygen instead of superoxide anion [8] Our data performed, at pH 5.4, in the presence of H2O2 and guaiacol (as reductant) indicate a larger peroxidase activity of HbIV, with respect to HbI, in

Fig 4 Peroxidase activity of different hemoglobins using guaiacol as

substrate, monitored by the increase in optical density at 470 nm For

experimental details see Materials and methods (j), iron(II)-HbI; (h),

met-HbI; (r), iron(II)-HbIV; (e), met-HbIV.

line with a previous work [5] However, these results show a

decrease of the peroxidase activity in the order:

iron(II)-HbIV > met-iron(II)-HbIV > met-HbI > iron(II)-HbI The larger

peroxidative activity of iron(II)-HbIV with respect to

met-HbIV is in line with previous data obtained with human

HbA derivatives [7] The unexpected HbI results could be

explained suggesting a restricted accessibility to guaiacol for

the heme pocket of HbI In fact, it is known that, although

H2O2binds directly to the heme iron, the guaiacol can have

different kind of interactions with Hb, because it could be

too voluminous to penetrate in the heme pocket [6] It is

known that one important difference between the two trout

Hbs is present on the a-subunit, where the distal Val residue

(E11) (present in both a and b pockets of human Hb) is

substituted in HbIV by a Thr, and in HbI by an Ile Because

the Val(E11) residue is known to affect the ligand

accessi-bility to heme pocket, its substitution with the bulky

hydrophobic side chain of Ile could impose a certain steric

hindrance to the a-pocket On the other hand, the polar Thr

could likely facilitate the access of guaiacol

(o-methoxyphe-nol) to a-pocket by binding it with a hydrogen bond The

b-pocket of HbI is mainly affected by the interaction of

Tyr(F1)85b with Ala70b and Ala74b [30] As these residues

are maintained in HbIV, similar b-pocket features are

expected in both proteins However, other amino acid

substitutions could affect pockets structure, e.g the change

of Cys(F9)93b of human Hb with Ser in HbIV and Ala in

HbI; or the change of His(HC3)146b with Phe in HbI are

known to affect the physiological behavior of the proteins

[31] However, these residues may affect also the b-pocket

structure [30,31] According to Perutz and Brunori [31], the

polar Ser in HbIV prefers an external position to the pocket

On the contrary, an internal position could by hypothesized

for the hydrophobic Ala of HbI Moreover, the residue b66,

present in the b-pocket [32] is a Val for HbI and a Thr for

HbIV, so that also in this case the polarity of the pocket is

increased for HbIV Although it is impossible to simply

foresee the effect of these changes on the pocket structure,

they could suggest a general lower accessibility of a quite

bulky and polar molecule a guaiacol to HbI heme pockets

A structural modification of the heme pocket could be

hypothesized also to explain the larger peroxidase activity shown by met-HbI, compared with iron(II)-HbI (Fig 4), in the presence of substrate (guaiacol)

The lower reactivity of HbI towards reactive oxygen species is confirmed also by the reaction with the superoxide anion obtained by the xanthine/xanthine oxidase reaction, both at pH 7.8 and pH 6, although a larger activity of both Hbs is evident at the acidic pH, in line with previous data obtained with human HbA [7,33] Also in this case the larger reactivity shown by HbIV, when compared with HbI

in the same iron oxidative state, could suggest a lower accessibility of the HbI heme pocket to superoxide anion Moreover, a different pocket accessibility to the relatively small and charged superoxide could be related also to the pH-dependent reactivity of the proteins

Differences between HbI and HbIV in the reactions with

H2O2 and superoxide anion are likely to be related to possible different features of the heme–globin interaction, which can be modified in iron(II)- and met-Hbs and as consequence of pH changes As the comparison between the structural characteristics of HbI and HbIV, at two different

pH values, had been never studied, we have performed these studies by CD, whose spectra acquired in near UV and visible regions are particularly sensitive to heme surround-ing In line with a previous work [3], the near-UV and visible regions of HbI and HbIV (oxygenated forms, pH 7.8) are characterized by quite different spectra, more evident at the

a1b2interface, as suggested by the CD spectra in the region 270–290 nm (Fig 1)

Data presented in this work demonstrate that CD spectra (Figs 1–3), acquired under different conditions of pH and oxidation, show different patterns of modifications for the two trout hemoglobin components (HbI and HbIV), which are likely to be related to their different functional properties [2] HbIV spectral modifications induced by a pH decrease, are quite similar to those reported in the literature for human HbA under deoxygenation [17,18] (Figs 1–3) HbIV deoxygenation is evident by the L-band which responds strongly to spin state [20,21] because it shows a large positive ellipticity for low-spin Hbs and a very reduced contribution for high-spin Hbs Deoxygenation is evident

Table 1 Lucigenin-amplified chemiluminescence of the xanthine/xanthine oxidase reaction in the presence of 40 lgÆmL)1of oxy and met derivatives of both hemoglobins The system contained 0.9 U of xanthine oxidase per mL and 150 l M lucigenin; the reaction was started by injecting xanthine at the final concentration of 50 mmol in 50 m M Tris pH 7.8 or 50 m M Bis/Tris pH 6.0 Values obtained are expressed in count per second (c.p.s.).

Sample

Duration (s)

T half rise

(s)

T max

(s)

T half fall (s)

Peak value (c.p.s.)

CL reduction (%)

50 m M Tris pH 7.8 15 < 2.40 6.60 (1.640 ± 0.001) · 107

Fe(II)-HbIV 15 < 2.40 7.20 (1.599 ± 0.006) · 10 7

2.5 Met-HbIV 15 < 2.40 7.20 (1.555 ± 0.005) · 10 7 5.1 Fe(II)-HbI 15 < 2.40 7.20 (1.610 ± 0.005) · 10 7 1.8 Met-HbI 15 < 2.40 7.18 (1.599 ± 0.005) · 10 7

2.5

50 m M Bis/Tris pH 6.0 40 < 3.00 25.80 (2.453 ± 0.002) · 10 5

Fe(II)-HbIV 40 < 1.20 22.80 (1.575 ± 0.010) · 10 5 * 35.8 Met-HbIV 40 < 1.80 24.60 (0.929 ± 0.010) · 10 5

* 62.1 Fe(II)-HbI 40 < 3.00 24.00 (1.884 ± 0.010) · 10 5 * 23.2 Met-HbI 40 < 5.40 26.40 (1.207 ± 0.010) · 10 5 * 50.1

*P < 0.05.

also by the large red shift of the Soret band (433 nm at

pH 6.0 and 418 nm at pH 7.8), in line with the reported

spectral characteristics of human deoxy-HbA [17,18] This

red shift is most likely not linked to changes in the

quaternary structure, as a similar deoxygenation-induced

shift was also observed in the Soret peak of monomeric

Lucina pectinata Hb [25] Moreover, although the data

reported here seem not to be in line with previous data by

Perutz et al [18], which show that the Soret band in the

T-form is slightly blue shifted and higher in intensity than

the R-form, the measured effect in the Soret band is likely

due to the superimposition of deoxygenation and the

proton-dependent Rfi T transition of HbIV This

possi-bility could be consistent with the results indicating that in

deoxygenated human Hb, the difference CD spectra shows

a maximum in the Soret peak at 437 nm for the R-form,

and at 433 nm for the T-form [24]

The near-UV and visible CD spectral features are very

similar in HbIV and in human HbA However, the CD

Soret band of human deoxy-HbA shows an increased

ellipticity when compared with the oxygenated derivative

[17,34] The spectrum of deoxy-HbIV (pH 6.0) follows an

opposite behavior (Fig 2A) A previous work performed

on human pathologic Hbs related the decrease in the Soret

band ellipticity to a decreased cooperativity [35] This

possible interpretation of the unexpected decrease in the

Soret band ellipticity in deoxy-HbIV is in agreement with

data indicating that an acid pH causes a reduction in

cooperativity (together with a marked reduction in ligand

affinity) in a fish hemoglobin, exhibiting the Root effect

[1,2,34]

Comparison between iron(II)-HbIV and met-HbIV at

pH 7.8 in the regions of the L-band (about 260 nm), 280–

290 nm and the Soret band indicate quite similar structural

features between these two ligated Hbs showing a similar

R structure The small, but evident differences between the

spectra of iron(II)-HbIV and met-HbIV pH 7.8 around

285 nm (Fig 1A,C) may be related to larger values of the

equilibrium constant L¼ [T]/[R] for met-HbIV (in terms of

a two-state concerted model)

The CD spectra obtained at pH 6.0 for met-HbIV

indicate a shift to the T-form, high spin iron, a mixed

population of six and five coordinated molecules (as

suggested by the Soret band) and a decreased cooperativity

(as suggested by the decreased ellipticity of the Soret band)

These results on HbIV are in line with a previous work on

human HbA by Perutz et al [14], which suggested that the

high-spin ligands as H2O (which occupies the sixth

coordi-nation state in met-Hbs) favor the transition to the T state

more than the low-spin ligands

HbI at pH 7.8 and pH 6.0 is reported to be completely

oxygenated and cooperative [2] As expected, the CD

spectra recorded at pH 6.0 show the characteristic bands of

a ligated Hb, at each wavelength range tested The small

spectral changes induced by low pH on iron(II)-HbI

(decreased intensity, either negative or positive, of each

band), in the region 250–470 nm could be due to a possible

protein destabilization, which may affect the quaternary

structure of the protein and, as a consequence, the a1b2and

a1b1contacts

Oxidation does not induce important structural

modifi-cations in the HbI spectra, although a slightly more negative

ellipticity in the 285 nm region, at pH 7.8, can indicate a larger value of the equilibrium constant L¼ [T]/[R] However, the modified ratio Q0/Qm (visible region) indicates

a slightly different symmetry at the axial bond with the proximal histidine, as a consequence of iron oxidation Comparison with HbIV spectra suggests the possibility that the modified ratio Q0/Qm could be due to a partial change to the unligated form following oxidation, at both pH values

At pH 6.0, the low ellipticity value of the L-band (centered around 260 nm) for met-HbI suggests a high spin iron This is in agreement with the presence, at low pH, of the high-spin ligand, water, bound to iron(III) subunits [36] According to Perutz et al [14] in the absence of organic phosphates, the R structure is dominant in all Hbs in which the irons are six-coordinated, but the spin state can modify the allosteric equilibrium between the two structures, shifting the equilibrium constant L¼ [T]/[R] to a higher value However, this is not the case of met-HbI at pH 6.0, as indicated by the CD spectra obtained which show no changes indicative of Rfi T transition in the region of

285 nm and no blue shift in the Soret band

Conclusions

Data presented in this work demonstrate different structural features for the heme–globin interaction in HbIV and HbI, which could be related to their different activities towards ROS However, CD data did not give any indication about the different reactivity shown by both Hbs towards superoxide anion at pH 7.8 and 6 For this reason it is suggested that the larger reaction with the superoxide at pH

6 could be linked to the pH-induced decrease of negative charge density on dissociable groups on the proteins Differences in HbI and HbIV behavior are likely related

to the known differences [36,37] in number and position of amino acids with charged lateral groups

The larger activity of met-HbI, with respect to iron(II)-HbI, in the presence of the guaiacol (Fig 4) could be due to the inhibition of Rfi T transition in met-HbI We suggest that the persistence of R form could hinder the structural changes, induced by the low pH, which could affect the interaction of the guaiacol large molecule at the heme pocket

A modification of the heme pocket structure is suggested also

by the modified ratio Q0/Qm (visible region), indicating a changed symmetry at the axial bond with the proximal histidine This modified symmetry seems to be related to a partial dissociation of ligand by met-HbI, which could permit a binding of more H2O2molecules with heme iron and, as a consequence, a higher rate of peroxidative reaction

by the R-form of met-HbI pH 5.4 with respect to the R-form

of iron(II)-HbI at pH 5.4 This hypothesis is also suggested

by the comparison with data obtained for HbIV: at low pH the iron(II) form is completely deoxygenated (as confirmed

by CD data, in particular by Soret band), and met-HbIV is only partially delegated (see Soret band) For HbIV the peroxidative reaction follows the order iron(II)-HbIV > met-HbIV, suggesting that in the case of this protein, the deligation also affects the rate of the reaction

We stress that our CD data give new information about ligation features and/or Rfi T transition of HbIV and HbI: HbIV appears to dissociate ligands, as a consequence

of pH also decreases in the oxidized form, even if the shift to

the non ligated form is incomplete Moreover, met-HbIV

spectra (at pH 6.0) show an Rfi T transition, which is

related to the change from a low- to high-spin iron This

behavior is in line with data indicating that tetramers with

high-spin ligand water of iron(III) subunits, show the most

T-state behavior [38]

CD spectra show that iron(II)- and iron(III)-HbI

struc-tural features are almost insensitive to pH decrease, as the

protein remains in the ligated form This result is expected in

iron(II)-HbI, which remains in the R-form and low-spin

iron However, a particularly high stability of the R-form is

evident for met-HbI at pH 6.0, also in the presence of

high-spin iron, in contrast with the expected transition to the

T form

In spite of the efficiency of the antioxidant defense

system, the trout RBCs can be exposed to a considerable

flux of reactive oxygen species, implying a red cell oxidative

stress Our data could suggest that trout HbI, which shows a

reduced sensitivity to reactive oxygen species (such as

superoxide anion and H2O2), with respect to HbIV, could

have a role in the maintenance of normal oxygen supply

during ROS production

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