Báo cáo khoa học: The oxidation process of Antarctic fish hemoglobins pot

The oxidation process of Antarctic fish hemoglobins Luigi Vitagliano 1, *, Giovanna Bonomi 2, *, Antonio Riccio 3 , Guido di Prisco 3 , Giulietta Smulevich 4 and Lelio Mazzarella 1,2 1 Istituto di Biostrutture e Bioimmagini, CNR, Napoli; 2 Dipartimento di Chimica, Universita ` degli Studi di Napoli ‘Federico II’, Complesso Universitario di M.S. Angelo, Napoli; 3 Istituto di Biochimica delle Proteine, CNR, Napoli, Italy; 4 Dipartimento di Chimica, Universita ` degli Studi di Firenze, Polo Scientifico, Sesto Fiorentino, Italy Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation pro- cess, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectro- scopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differ- ences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemo- globins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemi- chrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly trans- formed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquomet and the hemichrome. In order to assign the heme region state of the a and b chains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an a(aquomet)- b(bishistidyl-hemichrome) state. This demonstrates that the a and b chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrome state [Riccio, A.,Vitagliano,L.,diPrisco,G.,Zagari,A.&Mazzarella, L. (2002) Proc. Natl Acad. Sci. USA 99, 9801–9806], the quaternary structures of these a(aquomet)-b(bishistidyl- hemichrome) forms are intermediate between the physiolo- gical R and T hemoglobin states. Together, these structures provide information on the general features of this inter- mediate state. Keywords: Antarctic fish; hemichrome; hemoglobin; hexa- coordination; oxidation. Hemoglobins (Hbs) are members of the globin superfamily devoted to the transport of oxygen to cells [1]. Except for the Antarctic fish belonging to the icefish family, these proteins are present in all vertebrates. In these organisms, Hbs are typically tetrameric proteins consisting of two pairs of identical a and b chains. While sharing a common general mechanism of action, Hbs extracted from different verte- brates have acquired specific functional properties in response to major evolutionary pressures. In Antarctic fish the evolutionary process of cold adaptation has produced unique hematological characteristics [2–5]. In fact, the blood of Antarctic fish contains fewer erythrocytes and less Hb than fish of temperate water so far studied. Furthermore, as constancy characterizes the conditions of the Antarctic marine environment, the blood of these fish is endowed with a markedly reduced Hb multiplicity. However, the charac- terization of Antarctic fish Hbs (AFHbs) has shown that they retain most of the structural and functional properties typical of Hbs of fish living in temperate environments. As found in other fish Hbs, the activity of AFHbs may be differently modulated by external effectors. Indeed, although most AFHbs display the Root effect [3], namely low oxygen affinity with loss of co-operativity at low physiological pH, the major Hb of Trematomus newnesi (Hb1Tn) does not show this effect [6]. Interestingly, this Hb exhibits very high sequence identity (95%) with Hb of Trematomus bernacchii (HbTb) which, conversely, exhibits a strong Root effect [7]. We have recently shown that, in contrast with human and other mammalian Hbs, Hb1Tn rapidly forms low-spin hexacoordinated oxidized species (hemichromes) when exposed to air [8]. In addition, we have determined the crystal structure of one of the intermediates of the oxidation process of this Hb [9]. This intermediate is characterized by a different binding state of the a and b chains. A CO molecule is bound to the a heme iron, whereas a bishistidyl complex is observed at the b heme. This structure, the first Correspondence to L. Mazzarella, Dipartimento di Chimica, Univer- sita ` degli Studi di Napoli ÔFederico IIÕ, Complesso Universitario di M.S. Angelo, via Cinthia, I-80126 Napoli, Italy. Fax: + 39 081 674090, Tel.: + 39 081 674279, E-mail: mazzarella@chemistry.unina.it Abbreviations: Hb, hemoglobin; AFHb, Antarctic fish Hb; HbTb, Trematomus bernacchii Hb; Hb1Tn, major Hb component of Trematomus newnesi; Hb2Tn, minor Hb component of Trematomus newnesi; HbCTn, cathodic Hb of Trematomus newnesi;HbGa, Gymnodraco acuticeps Hb; HbTbCO, carbomonoxy form of HbTb; aeHbTbOx and fcHbTbOx, structures of HbTb oxidized by air and ferricyanide, respectively. *Note: These authors contributed equally to this work. (Received 23 October 2003, revised 22 January 2004, accepted 24 February 2004) Eur. J. Biochem. 271, 1651–1659 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04054.x example of a tetrameric Hb in the hemichrome state, has demonstrated that the iron coordination by distal His, usually associated with denaturating states, may be toler- ated in a native-like Hb structure [9]. Furthermore, the analyses [9] of the quaternary structure and the critical interface a 1 b 2 have revealed that this partial hemichrome state has an intermediate structure between the relaxed (R) and tense (T) Hb functional states [10,11]. We here report extensive spectroscopic and crystallo- graphic analyses of the oxidation process of AFHbs. In particular, we show that oxidation through hemichrome formation is a common mechanism of five AFHbs extracted from three Antarctic fishes of the dominant, largely endemic Notothenioidei suborder. In this framework, we demon- strate that these hexacoordinated states may be successfully reduced to deoxy and carbomonoxy forms. Interestingly, the crystal structure of two oxidized forms of HbTb provides novel information on the different oxidation pathway of the a and b chains of AFHbs and on the accessible quaternary structures of tetrameric Hbs. Experimental procedures Spectroscopic analyses AFHb oxidation pathways were followed by UV-visible spectrophotometric analysis. In particular, T. bernacchii Hb (HbTb), Gymnodraco acuticeps Hb (HbGa) and the three Hb components (major, minor and cathodic, hereafter denoted Hb1Tn, Hb2Tn and HbCTn, respectively) of T. newnesi were considered. The proteins were purified following the procedures developed by di Prisco and coworkers [6,7,12] and oxidized by exposing their carbo- monoxy forms to air in 60 m M Tris/HCl (pH 7.6) or 60 m M potassium phosphate (pH 6.0) buffers at 20 °C. The oxidation process was initiated by exposing a cuvette containing 600 lL of protein to air. The proteins were also oxidized by using ferricyanide. These met-Hb derivatives were prepared by oxidation of the carbomonoxy forms using excess potassium hexacyanoferrate (III) in 60 m M Tris/HCl (pH 7.6) or 50 m M CAPS (3-cyclohexylamino- 1-propanesulfonic acid)/NaOH (pH 10.0) at 20 °C followed by gel filtration on a Sephadex G-25 column previously equilibrated and eluted with 60 m M Tris/HCl (pH 7.6) or CAPS/NaOH (pH 10.0) buffers to remove the oxidant. The oxy form of HbTb was obtained by exposing a CO- bound Hb solution tostrong light under an intense flux of O 2 . To check the reversibility of the oxidation process, the met-Hb forms were chemically reduced by adding 2–3 lL sodium dithionite (20 mgÆmL )1 )to50lL Hb solution. For comparative purposes all the experiments were repeated on human Hb and on sea bass hemolysate. This hemolysate contains five Hb components as a result of the combination of four different globins [13]. Denaturation was identified by the overall decrease in intensity of the electronic absorption spectrum and the increase in the intensity ratio of the aromatic band versus the Soret band. It is worth mentioning that the increase in the aromatic band in the Soret region was followed by a significant precipitation of the protein. However, the presence of the precipitate did not prevent the crystallization of the oxidized forms (see below). UV-visible electronic absorption spectra were recorded with a Jasco 560 spectrophotometer (Jasco Corporation, Tokyo, Japan) at room temperature. Crystallographic studies The oxidized forms of HbTb used for the crystallographic experiments were prepared using two different procedures. In the first one, HbTbCO was exposed to air and subsequently crystallized. The free interface diffusion tech- nique was used by pouring the protein (final concentration 5mgÆmL )1 )in60m M Tris/HCl (pH 7.6) on a solution containing 14% (w/v) MPEG 5000 (Fluka) into a capillary sealed in air at 20 °C. Single crystals suitable for X-ray analyses were obtained after 1 week. Diffraction data were recorded at 2.4 A ˚ resolution on these crystals 35 days after their appearance. A Nonius DIP2030b imaging plate mounted on a Nonius FR591 rotating anode (Nonius BV, Delft, the Netherlands) was used for data collection. Results and statistics of data processing, carried out using the program DENZO [14], are reported in Table 1. In contrast with Hb1Tn, for which the crystals of the carbomonoxy [15] and the air-exposed [8,9] forms are nearly isomorphous, the crystals of this air- exposed form of HbTb, hereafter referred to as aeHbTbOx, are not isomorphous to the crystals of HbTbCO [7]. The crystals are monoclinic (space group C2), with cell dimen- sion, a ¼ 108.52 A ˚ , b ¼ 65.09 A ˚ , c ¼ 55.75 A ˚ and b ¼ 113.48°.Anab dimer constitutes the asymmetric unit. In addition, crystallization trials were also set up for the oxidized form of HbTb prepared by using ferricyanide (fcHbTbOx), as reported in the previous section. Crystals suitable for X-ray analysis were obtained using conditions very similar to those adopted for aeHbTbOx. The final protein and MPEG5000 concentrations were 6.0 mgÆmL )1 and 12% (w/v), respectively. In these experiments the capillaries were sealed under CO. Crystals appeared after 1 week and were used for data collection 3 months later. Diffraction data were collected at 2.5 A ˚ resolution by using a Nonius rotating anode/imaging plate system. The crystals are triclinic (space group P1) with cell dimen- sions a ¼ 55.99 A ˚ , b ¼ 62.98 A ˚ , c ¼ 63.50 A ˚ , a ¼ 77.1°, b ¼ 69.8° and c ¼ 84.2°.Ana 2 b 2 tetramer constitutes the Table 1. Data collection statistics. R-merge ¼ S hkl S i |I i – <I>|/|I i |. aeHbTbOx fcHbTbOx Crystal data a(A ˚ ) 108.52 55.99 b(A ˚ ) 65.09 62.98 c(A ˚ ) 55.75 63.50 a (°) 90.0 77.1 b (°) 113.5 69.8 c (°) 90.0 84.2 Space group C2 P1 Data processing Resolution range (A ˚ ) 20.0–2.4 25–2.5 Number of observations 47657 38318 Number of unique reflections 14020 23515 Completeness (%) 99.5 87.2 R-merge (%) 9.9 10.6 1652 L. Vitagliano et al.(Eur. J. Biochem. 271) Ó FEBS 2004 asymmetric unit. Statistics of the data collection are reportedinTable1. Both structures (aeHbTbOx and fcHbTbOx) were solved by molecular replacement using the program AMORE [16] and the structure of Hb1Tn(hemi) (1LA6 Protein Data Bank code) [9] as a starting model. Straightforward solutions were obtained using the ab dimerassearch model. The overall position of the molecule was initially refined by a rigid body minimization. Subsequently, the individual chains were refined as distinct rigid units. The rigid body refinement cycles were followed by atomic positional refinements and B factor optimizations by using the program CNS [17]. Each refinement run was followed by manual intervention using the molecular graphic program O [18] to correct minor errors in the position of the side chains. In both structures the electron density maps corresponding to the heme regions showed that a water molecule was bound to the heme iron of the a chains, whereas in the b chain there was a clear indication of the formation of a bishistidyl complex. The bond distance between the heme iron and the N e2 atom the Fe of the bishistidyl complex was restrained and refined to 2.0 A ˚ . In the final steps of the refinement, water molecules were identified and included in the refining models. A detailed description of the refinement statistics of the two structures is reported in Table 2. The atomic coordinates of aeHbTbOx and fcHbTbOx have been deposited in the Protein Data Bank, with entry codes 1S5X and 1S5Y, respectively. Comparative analyses of AFHb structures To analyze the structural variations associated with hemi- chrome formation, the structures of aeHbTbOx and fcHbTbOx were compared with those of the HbTbCO (PDB code 1PBX) [7] and deoxy HbTb (HbTb-deoxy) (PDB code 1HBH) [19], which were used as reference structures to evaluate the position of these two structures along the R fi T transition pathway. Furthermore, to measure the structural variability of Hbs in partial hemi- chrome states, aeHbTbOx and fcHbTbOx were also compared with Hb1Tn(hemi) [9]. Specifically, this task was achieved by evaluating root mean square deviations and by generating difference distance matrices. The differ- ence distance matrix is indeed a sensitive probe for investigating structural differences between two models [20]. In this procedure, distances between pairs of Ca atoms aremeasuredineachmodel.Thedifferencesinthe corresponding Ca distances between the two models are then evaluated and used as elements of the matrix. Results Spectroscopic studies of the oxidation process of AFHbs Oxidation of AFHbs exposed to air. The oxidation process of five AFHbs (Hb1Tn, Hb2Tn, HbCTn, HbTb, and HbGa) was initially followed by exposing the carbomonoxy forms to air. As an example, the entire oxidation process of HbTb at pH 7.6 is reported in Fig. 1. The spectrum of the oxy form is reported for comparison. The spectrum of the CO complex is characterized by the presence of Soret (418 nm) and Q bands (538 and 567 nm) which are very similar to those of human HbA-CO [21]. On exposure to air, the spectrum starts to change. After 18 h, the Soret band broadens and blue-shifts to 414 nm, and the a band red- shifts to 575 nm. These spectral changes are consistent with the formation of the oxy form (Fig. 1, top spectrum). [A rapid formation of the oxy form is also observed when Hb1Tn is exposed to air. This observation suggests that the structure of Hb1Tn exposed to air, previously reported as an a(CO)/b(hemichrome) [9], probably corresponds to an a(O 2 )/b(hemichrome) state.] Concomitantly, a weak shoulder at 630 nm becomes evident. This band, assigned to Table 2. Refinement statistics. R-factor ¼ S hkl (||F hkl obs| ) k|F hkl calc||)/ S hkl |F hkl obs|. R-free ¼ S h (||F obs | ) k|F calc ||)/S h |F obs | where h is a sta- tistical subset (5%) of data. aeHbTbOx fcHbTbOx Resolution range (A ˚ ) 20.0–2.4 25.0–2.5 R-factor 0.190 0.199 R-free 0.233 0.247 Number of protein atoms 2153 4306 Number of heme groups 2 4 Number of water molecules 27 82 Root mean square deviations from ideal values Bond lengths (A ˚ ) 0.011 0.010 Bond angles (°) 1.4 1.4 Dihedral angles (°) 17.2 17.4 Improper angles (°) 0.93 0.94 Fig. 1. Electronic absorption spectra of air-exposed T. bernacchii Hb. The spectra were recorded at 20 °Cin60 m M Tris/HCl (pH 7.6) with a protein concentration of 0.4 mgÆmL )1 . The first five spectra from the bottom to the top were recorded after exposing HbTbCO to air for 0, 18, 49, 71, and 140 h. The top spectrum was recorded on the oxy form of HbTb. The spectrum of HbTbCO exposed to air for 140 h was recorded on a sample containing HbTb at a concentration of 1.2 mgÆmL )1 . The Soret band of the latter spectrum was not recorded because the protein was too concentrated. The first four spectra from the bottom to the top of the Soret region correspond to HbTbCO exposed to air for 71, 49, 18, 0 h. The region between 450 and 700 nm was expanded eightfold. Ó FEBS 2004 Oxidation of Antarctic fish hemoglobins (Eur. J. Biochem. 271) 1653 the CT1 band of a hexacoordinated (6c) high-spin (HS) form, is typical of a species with a water molecule coordinated to the heme [21,22]. After 49 h the Soret band further downshifts to 408 nm, and the Q bands broaden. After 71 h, just before denaturation, the formation of a new species characterized by a Soret band at 407 nm and Q bands at 530 and 565 nm is observed. In a parallel experiment, carried out on a protein three times more concentrated, the formation of this state was also observed after 71 h of exposure of the protein to air, and became clearly evident in the spectrum recorded after 140 h, when the oxy form had almost disappeared. These maxima are typical of a 6c low-spin (LS) heme with an endogenous ligand coordinated to the sixth position of the heme iron (hemichrome). Interestingly, all AFHbs form low-spin hexacoordinated hemichrome states, characterized by the occurrence of peaks in the visible region at 530 and 565 nm (Fig. 2). The slight red-shift observed for the Hb2Tn, Hb1Tn, and HbTb is due to the presence of the oxy form (Fig. 1). Notably, all spectra are characterized by the simultaneous presence of an aquomet 6cHS species (as judged by the presence of the weak CT1 band at  630 nm). However, the time evolution of the different species depends on the concentration of the protein (data not shown) and it slightly varies among the five AFHbs. It is worth noting, for example, that the appearance of hemichrome is faster in Hb1TnthaninHbTb. Hemichrome formation has also been detected for AFHbs in media containing high concentrations of MPEG 5000 (12% w/v) and salt (0.5 M ammonium sulfate) (data not shown). The oxidation pathway exhibited by AFHbs is very different from that reported for other vertebrate Hbs, including those extracted from fish living in temperate waters so far studied [23,24]. For comparative purposes the oxidation processes of human Hb and sea bass hemolysate were analyzed under the same experimental conditions. No evidence of hemichrome formation was detected on exposure of their CO complexes to air. Even after 185 h, before denaturation, human Hb only shows the formation of the oxy form. In the same time period the sea bass Hb spectrum is characterized by the coexistence of the oxy form with a very low amount of aquo 6c HS species (weak CT1 peak at 630 nm, data not shown). Finally, the influence of the Hb quaternary structure on hemichrome formation was analyzed by exposing HbTb to air at pH 6.0. As this Hb is endowed with a strong Root effect [7], the HbTb R/T equilibrium is shifted toward the T state at acidic pH. Indeed, the T state form of HbTb was crystallized by Fermi and coworkers by simply lowering the pH of the carbomonoxy form of the protein to 6.0 [19]. In the early stages of the oxidation process (2–9 h, Fig. 3) the exposure of HbTbCO to air leads to the formation of the deoxy form, as suggested by the appearance of shoulders at 434 and 556 nm. This species evolves towards the formation of the hemichrome state (24–30 h). The overall oxidation process of HbTbCO at pH 6.0 is faster than at pH 7.6. However, the relative intensity of the bands in the visible region suggests that the amount of the 6c HS grows at pH 6.0 at the expense of the hemichrome. Therefore, it appears that the latter form is favored at higher pH, as found for the 6c LS hydroxo in human Hb [21,22]. Chemical oxidation of AFHbs. The oxidation process of AFHbs in air requires many hours and the proteins denature before reaching complete oxidation. Therefore, to obtain the final completely oxidized form, chemical oxidation of AFHbs by potassium ferricyanide was also studied. Figure 4 compares the met forms of the various AFHbs at pH 7.6 with those of sea bass and human Hbs in the visible region. For the latter protein, the spectrum obtained at pH 10.0 is also reported. The electronic absorption spectra of human and sea bass Hbs at pH 7.6 are characterized by bands at 498, 541, 576, and 630 nm, and a shoulder at 600 nm, indicative of a dominant 6c HS aquomet state in equilibrium with a hydroxy 6c coordina- tion (HS and LS states) [22,25]. At alkaline pH, the spectrum of human Hb shows the presence of only the hydroxy forms (bands at 541, 576, and 600 nm) [22]. Fig. 2. Electronic absorption spectra of air-exposed derivatives of the five AFHbs. The spectra were recorded after exposure of the carbo- monoxy forms to air at pH 7.6. The spectra were recorded after 69 h for Hb2Tn and HbC, 75 h for Hb1Tn, 92 h for HbGa, and 140 h for HbTb. The concentration of HbTb, Hb1Tn and HbC was 1.2 mgÆmL )1 , whereas the concentration of Hb2Tn and HbGa was 0.7 mgÆmL )1 . Fig. 3. Electronic absorption spectra of air-exposed T. bernacchii Hb at pH 6.0. The spectra were recorded after exposure of the carbomonoxy forms of the Hbs to air at pH 6.0, as indicated. The concentration of HbTb was 0.45 mgÆmL )1 . The region between 450 and 700 nm was expanded eightfold. 1654 L. Vitagliano et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Although at pH 7.6 the aquomet form is present in all Hbs under investigation, the spectra of human and sea bass Hbs do not show the presence of the hemichrome state (characterized by the bands at 530 and 565 nm), which is the dominant species in the spectra of all AFHbs. It should be mentioned, however, that minor differences are observed among AFHb spectra. Indeed, in addition to the hemi- chrome 6c LS form and the 6c HS aquomet species present in the spectra of HbCTn, HbGa, and HbTb, the peaks at 541 and 576 nm in the spectrum of Hb2Tn also indicate the formation of a 6c LS hydroxymet form. The possibility that these bands may be due to the presence of the oxy form of Hb2Tn may be ruled out by considering that the protein had been treated with excess ferricyanide. The effect of temperature on the oxidation process of Hb1Tn and HbTb was analyzed by oxidizing these Hbs with ferricyanide at 4 °C. The spectra obtained in these experiments are virtually identical with those obtained at 20 °C (data not shown). Chemical reduction, using sodium dithionite, of oxidized HbTb leads to the formation of the deoxygenated form which, subsequently in CO, evolves toward the formation of HbTbCO (Fig. 5). The low intensity ratio of the aromatic versus the Soret band indicates that this HbTbCO form holds a folded structure. These observations suggest that this hemichrome form, although intermediate along the unfolding pathway of these proteins, retains a well-defined structure. This result is corroborated by the crystallographic analyses reported below. Crystallographic studies on the oxidized forms of HbTb Overall quality of the structures. The structure of aeHbT- bOx was refined to an R-factor of 19.0% (R-free 23.3%) using diffraction data in the resolution range 20.0–2.4 A ˚ . The final model includes 27 water molecules. The structure of fcHbTbOx was refined to an R-factor of 19.9% (R-free 24.7%) using diffraction data in the resolution range 20.0– 2.6 A ˚ . Eighty-two water molecules were included in the final model. In both structures, the electron density is well defined for both the main chain and the side chain of most of the residues. As frequently reported in R state Hbs, the regions corresponding to the CD loop (residues 45–52) and the C-terminus (residues 145–146) of the b subunit are com- pletely disordered. The stereochemical parameters of the refined structure (Table 2) are in close agreement with those obtained for well-refined protein structures at the same resolution. Although aeHbTbOx and fcHbTbOx reveal significant differences at the quaternary-structure level, the tertiary structures and iron-binding states are virtually identical in these two oxidized forms of HbTb (see below). Structure of the heme regions. In both aeHbTbOx and fcHbTbOx, differences in the heme structures of the a and b subunits were evident from inspection of the first electron density maps. In particular, analysis of the heme region of the a subunits shows that a water molecule is bound to the heme iron (Fig. 6A). Given the pH of the crystallization medium (pH 7.6), the electron density of the ligand of the a iron could also correspond to a hydroxide ion. This possibility can be, however, ruled out by taking into account the UV spectrum of air-exposed HbTb (Fig. 1), which does not indicate the formation of a detectable amount of the hydroxymet species. A completely different picture emerges from analysis of the electron density maps corresponding to the b heme (Fig. 6B). The iron atom coordinates both the proximal Fig. 4. Electronic absorption spectra of the met-Hb derivatives obtained by treating the five AFHbs with excess potassium ferricyanide at pH 7.6. For comparative purposes the spectra of human Hb (HbA at pH 7.6 and 10.0) and sea bass hemolysate (EMSP, pH 7.6) are also included. The concentration of the Hbs, measured before oxidation, was 5.0 mgÆmL )1 . Fig. 5. Chemical reduction of the met form of T. be rnacchii Hb. Upper trace: HbTbCO treated with excess potassium ferricyanide. Lower trace: carbomonoxy form obtained by reduction of the met form with excess sodium dithionite. The spectra of the Soret region were recorded at a protein concentration of 1 mgÆmL )1 , whereas the concentration used to collect the spectra in the region 450–650 nm was 8 mgÆmL )1 . Ó FEBS 2004 Oxidation of Antarctic fish hemoglobins (Eur. J. Biochem. 271) 1655 (92b) and distal (63b) histidine residues. Combining these observations with the above spectroscopic results, the present structure can be confidently assigned to an a(aqu- omet)b(hemichrome) form. In both structures, analysis of the heme coordination geometry of the bishistidyl form shows that the N e2 –Fe–N e2 angle deviates significantly from linearity. This finding is in agreement with the geometrical features of the bishistidyl complex in Hb1Tn(hemi) [9]. The nonlinearity of the N e2 –Fe–N e2 angle in these structures may be ascribed to the strain imposed by the protein matrix. Tertiary and quaternary structure of aeHbTbOx and fcHbTbOx. Despite the different functional properties of Hb1Tn and HbTb and the different binding state of the a-heme iron of Hb1Tn(hemi) [9] and aeHbTbOx/fcHbT- bOx, the formation of the bishistidyl complex produces similar structural modifications in these two AFHbs. In fact, the coordination of distal His by the b-heme iron in both aeHbTbOx and fcHbTbOx is associated with a scissors-like motion of helices E and F. As found in Hb1Tn(hemi), the distance between the C a atoms of distal and proximal His is 12.5 A ˚ . The value of this distance is usually larger than 14.0 A ˚ in both ligand-bound and deoxygenated tetrameric Hbs [9]. The formation of the bishistidyl complex also requires a significant shift of the heme group. Indeed, as shown in Fig. 7, in the oxidized forms of HbTb the heme group moves toward the exterior of the protein by  1A ˚ . Fig. 6. Electron-density Fo-Fc omit maps of the heme regions of aeHbTbOx. (A) a heme; (B) b heme. The maps were contoured at 3.3 r. A portion of the helices E and F are also shown to illustrate their orientation. Fig. 7. Heme shift on hemichrome formation at the b chains of HbTb. The EF regions of aeHbTbOx (black) and HbTbCO (gray) are shown after superimposition of the structurally conserved core composed of helices B, G and H. 1656 L. Vitagliano et al.(Eur. J. Biochem. 271) Ó FEBS 2004 The variations detected at the level of the tertiary structure propagate to the quaternary structure through the a 1 b 2 interface. The displacements of helix F and FG corner of the b subunit, which are necessary for hemichrome formation, are not compatible with the R state of HbTb. Therefore, the protein acquires a novel quaternary structure which is intermediate between the canonical T and R states. Indeed, the root mean square deviations resulting from the superimposition of the aeHbTbOx tetramer on the structures of HbTbCO and deoxy-HbTb are 1.50 and 1.65 A ˚ , respectively. Similar deviations are found for fcHbTbOx. Even more intriguing is the analysis of the difference distance matrices of these T. bernacchii Hb structures (Fig. 8). The similarity of the matrix relative to the structures deoxy-HbTb and HbTbCO (Fig. 8A) and the matrix computed from the structures of aeHbTb and HbTbCO (Fig. 8C) provides convincing evidence that the structural alterations that occur on hemichrome formation coincide with the mod- ifications associated with the structural transition from the R to the T functional states [10,11]. Discussion In this study, the oxidation process of five Hbs isolated from three Antarctic fish species was investigated by combining spectroscopic and crystallographic techniques. In particular, Hbs extracted from T. newnesi (Hb1Tn, Hb2Tn and HbC), T. bernacchii (HbTb) and G. acuticeps (HbGa) were con- sidered. These three notothenioid species occupy well-separated places in the phylogenetic tree [26]. In fact, two of these species (T. newnesi and T. bernacchii) belong to the family Nototheniidae (subfamily Trematominae), whereas G. acu- ticeps belongs to the family Bathydraconidae. A compar- ative analysis of the sequences of these AFHbs reveals the occurrence of substitutions in important regions of the protein, e.g. the heme pocket and the a 1 b 2 interface. The five Hbs analyzed in this study also show different functional properties. Indeed, whereas the oxygen affinity of Hb1Tn [6], Hb2Tn [6], and HbGa [12] is only slightly affected by pH, the other two Hbs [6,7] exhibit a strong Root effect. Despite these differences, here we demonstrate that these Hbs share a common oxidation pathway, which is remark- ably different from that exhibited by other tetrameric Hbs, including those extracted from the investigated species living in temperate climates [23,24]. In addition to the commonly observed aquomet and hydroxymet forms, oxidation of AFHbs leads to the formation of a significant amount of a reversible hemichrome form. This finding, which is in line with a preliminary analysis of the oxidation process of Hb1Tn [8], is particularly surprising as hemichrome forma- tion is often associated with denatured states of tetrameric Hbs [27]. The strong tendency of AFHbs to form hemichromes is strenghthened by the observation that bishistidyl complexes were invariably detected despite changing the oxidizing agent (air or ferricyanide), the ionic strength of the medium, and the temperature (4 and 20 °C). By analyzing the oxidation process of a Root-effect Hb (HbTb) at pH 6.0, we have demonstrated that hemichrome formation also occurs when the protein is constrained to the T state. Fig. 8. Difference distance matrices. (A) deoxy-HbTb vs. HbTbCO; (B) deoxy-HbTb vs. aeHbTbO; (C) aeHbTbOx vs. HbTbCO. In each map, blue regions represent residues that move closer in the second structure, whereas the converse happens in the red regions. The picture was generated using the program ESCET [20]. Ó FEBS 2004 Oxidation of Antarctic fish hemoglobins (Eur. J. Biochem. 271) 1657 The crystal structures of HbTb oxidized either by air or ferricyanide reveal that a and b chains follow different oxidation processes. In fact, the formation of a bishistidyl complex occurs only in the heme iron of the b subunits. On the other hand, the electron density indicates a water molecule bound to the heme iron of the a chains. This finding suggests that the a and b chains possess a significantly different degree of freedom in the tetrameric structure of AFHbs. In the absence of information on the oxidation products of isolated Hb chains, it is difficult to decide whether the two chains are intrinsically endowed with a different flexibility or their mobility is differently constrained in the tetramer. The study of isolated chains of human Hb has demonstrated that a chains are more ready to form hemichrome than b chains [28], indirectly support- ing the latter possibility. The different ligation state of the a and b chains may account for the anomalous behaviour of this partial hemichrome form when it is treated with reducing agents. Unlike hemichromes of other hemoproteins, which can be reduced to hemochrome, the oxidized form of HbTb is reduced by dithionite to deoxy HbTb. This may be explained by considering that the a chains are necessarily reduced to the deoxy state. This process drives the allosterically regulated protein toward the deoxy state. As reported for Hb1Tn(hemi) [9], the quaternary struc- tures of the fully oxidized forms of HbTb, aeHbTbOx and fcHbTbOx, are intermediate between the R and T states. Comparative analyses on these three structures, derived from three different crystalline forms, provide information on the invariant features as well as on the overall flexibility of this intermediate R/T state. In all three structures, the scissoring- like motion of the b-heme pocket produces a rearrangement of the b FG corner. His b97 takes a position that is intermediate between those taken by this residue in R and T structures. These alterations are transferred through the a 1 b 2 interface to helix F of the a chain. The position of the helix is locked by displacement of the Tyr a141 side chain, which takes a conformation similar to that reported in the T state. Despite these conserved structural elements, the three structures display significant differences with regard to the overall structure. This can be inferred from the overall root mean square deviations between the structures that lie in the range 0.40–0.70 A ˚ . This finding suggests that this R/T state is endowed with a certain degree of flexibility despite the structural constraint of the bishistidyl complex at the b heme. A molecular-graphics analysis carried out to identify the specific structural basis responsible for the unusual oxida- tion of AFHbs did not provide a conclusive answer. However, some amino-acid substitutions occurring in the CD region (residues 42–52) and the heme pocket (residues 60–95) of the b subunit have been identified as potential candidates that may facilitate hemichrome formation in AFHbs. Although rather flexible in all mammalian Hb structures, the CD region of the b subunit is completely disordered in AFHbs structures in both the R [7,15] and R/T hemichrome [9] states. As reported for the nonsymbi- otic rice Hb [29], a high flexibility of the CD region may be essential for the direct coordination of distal His to the heme iron in AFHbs. The greater mobility of this region may be ascribed to the presence of an extra glycine residue (Gly43 in HbCTn and Gly44 in Hb1Tn, Hb2Tn, HbTb, HbGa) in the AFHbs sequences compared with human Hb and to replacement of Pro51 (human sequence) with Ala. In this context, it is noteworthy that the formation of bishistidyl complexes in the a chain of crystalline horse hemoglobin [30] and in neuroglobin [31] is associated with large displacements of the CD corner. The formation of the bishistidyl complex may also be facilitated by replacement of Ala70 of the human Hb sequence with Gly residue in AFHbs. Indeed, in human Hb [32], the methyl group of Ala70 is placed between two substituents of the heme group, and probably prevents the shift of the heme required for the formation of the bishistidyl complex. It can be also surmised that flexibility of the CD region may be essential for the direct coordination of distal His to the heme iron as well as for heme dissociation. It cannot be excluded, however, that hemichrome forma- tion may be favored by a greater overall flexibility of AFHbs. Indeed, by analogy with proteins from psychrophilic organ- isms [33], AFHbs may have acquired an enhanced plasticity, which allows the distortions required for hemichrome formation to be fully active at very low temperatures. Finally, in the last few years it has been shown that bishistidyl complexes are functional states of several important monomeric and dimeric globins, such as neuroglobins [34], truncated Hbs [35,36] and nonsymbiotic Hbs [29,37]. Although, on the basis of the available data, similar roles cannot be postulated for tetrameric Hbs, the present data show that bishistidyl complexes are, however, accessible states of a subclass of tetrameric Hbs. Further- more, it has been shown that a significant amount of oxidized Hb is present in mammalian [38] as well as in fish [39] erythrocytes. 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In the early stages of the oxidation process (2–9 h, Fig. 3) the exposure of HbTbCO. matrix. Results Spectroscopic studies of the oxidation process of AFHbs Oxidation of AFHbs exposed to air. The oxidation process of five AFHbs (Hb1Tn, Hb2Tn, HbCTn,