Oxygen dissociation from ferrous oxygenated human hemoglobin:haptoglobin complexes confirms that in the R-state α and β chains are functionally heterogeneous

The adverse effects of extra-erythrocytic hemoglobin (Hb) are counterbalanced by several plasma proteins devoted to facilitate the clearance of free heme and Hb. In particular, haptoglobin (Hp) traps the αβ dimers of Hb, which are delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Since Hp:Hb complexes show heme-based reactivity, kinetics of O2 dissociation from the ferrous oxygenated human Hp1-1:Hb and Hp2-2:Hb complexes (Hp1-1:Hb(II)-O2 and Hp2-2:Hb(II)-O2, respectively) have been determined. O2 dissociation from Hp1-1:Hb(II)-O2 and Hp2-2:Hb(III)-O2 follows a biphasic process. The relative amplitude of the fast and slow phases ranges between 0.47 and 0.53 of the total amplitude, with values of koff1 (ranging between 25.6 ± 1.4 s−1 and 29.1 ± 1.3 s−1) being about twice faster than those of koff2 (ranging between 13.8 ± 1.6 s−1 and 16.1 ± 1.2 s−1). Values of koff1 and koff2 are essentially the same independently on whether O2 dissociation has been followed after addition of a dithionite solution or after O2 displacement by a CO solution in the presence of dithionite. They correspond to those reported for the dissociation of the first O2 molecule from tetrameric Hb(II)-O2, indicating that in the R-state α and β chains are functionally heterogeneous and the tetramer and the dimer behave identically. Accordingly, the structural conformation of the α and β chains of the Hb dimer bound to Hp corresponds to that of the subunits of the Hb tetramer in the R-state.

hemopexin ensure the complete clearance of the free heme, which is released into hepatic parenchymal cells by the CD91 receptor-mediated endocytosis of the hemopexin-heme complex.After conveying the heme intracellularly, hemopexin is released into the bloodstream and the heme is degraded 9,[14][15][16] .Moreover, haptoglobin (Hp) is devoted to trap the extra-erythrocytic αβ dimers of Hb; the formation of Hb dimers, and in turn of the Hp:Hb complexes, is favored by the low extra-erythrocytic Hb concentration and its oxygenated state.By CD163 receptor-mediated endocytosis, the Hp:Hb complexes are delivered to the reticulo-endothelial system where they are degraded to release the heme.Heme-oxygenase catalyzes the heme conversion to biliverdin that is further transformed to bilirubin.The bilirubin is then exported from the macrophage and carried from albumin to the liver for conjugation in the hepatocytes and subsequent biliary excretion 9,[15][16][17][18][19][20][21][22] .
In humans, two alleles of Hp (Hp1 and Hp2) are expressed as single polypeptide chains.In particular, Hp1 displays a single complement control protein (CCP) domain and a single serine protease-like (SP-like) domain, whereas Hp2 contains two CCP domains and one SP-like domain 23 .Both Hp1 and Hp2 are proteolytically cleaved into α and β chains, which are covalently linked by a disulfide bond(s).Hp1 and Hp2 alleles induce the formation of Hp1-1 dimers (covalently linked by Cys15 residues), Hp1-2 hetero-oligomers and Hp2-2 oligomers (covalently linked by Cys15 and Cys74 residues) 21 .The most abundant Hp2-2 species is the tetramer, but trimers and higher order oligomers have been reported [22][23][24][25] .
Each Hp β chain binds one αβ dimer of Hb, making extensive contacts with the Hb dimer-dimer interface 24,25 .Accordingly, values of (i) the dissociation equilibrium constants for the recognition of deoxygenated and oxygenated Hb (Hb(II) and Hb(II)-O 2 , respectively) dimers by Hp are 1 × l0 −7 M and 1.3 × l0 −6 M, respectively, and (ii) the second order rate constants for Hp:Hb complexation range between 5 × 10 5 M −1 s −1 and 9 × 10 5 M −1 s −1 26-28 .Since Hb(II)-O 2 dissociates into αβ dimers preferentially with respect to Hb(II), the reaction of Hp with Hb represents a probe of the R-T transition of Hb 27,29 .
As the Hp:Hb complexes show functional properties similar to those of the Hb R-state, e.g. they display a high ligand specificity and show neither "heme-heme interactions" nor the Bohr effect [30][31][32][33][34][35][36][37] , we decided to investigate the kinetics of O 2 dissociation from human Hb(II)-O 2 dimers bound to human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(II)-O 2 and Hp2-2:Hb(II)-O 2 , respectively).The relevance of this approach is related to the fact that in this way it is possible to characterize the O 2 dissociation from a pure population of α 1 β 1 (and α 2 β 2 ) dimers without any interference from tetrameric species.Therefore, it is possible to sort out the contribution of the α 1 β 1 (and α 2 β 2 ) inter-subunit contacts from the α 1 β 2 (and α 2 β 1 ) ones, which are destroyed upon dimerization 38 .O 2 dissociation from Hp1-1:Hb(II)-O 2 and Hp2-2:Hb(II)-O 2 follows a biphasic process, the fast process (i.e., k off1 values) being about 2-fold faster than the slow one (i.e., k off2 values).Values of k off1 and k off2 are similar to those for the deoxygenation of isolated α(II)-O 2 and β(II)-O 2 chains of Hb 39,40 and identical to those for the dissociation of the first O 2 molecule from tetrameric Hb(II)-O 2 41 .The close similarity for the observed heterogeneity between experiments of O 2 replacement by CO and those of O 2 dissociation by sodium dithionite allows to state unequivocally for the first time that the biphasicity cannot be referable to a negative cooperativity in the α 1 β 1 (and α 2 β 2 ) dimers.It clearly demonstrates that the α and β chains of the oxygenated R-state of Hb are functionally heterogeneous to the same extent both in the tetrameric and in the dimeric assembly.Accordingly, the conformation of the α and β chains of the Hb dimer bound to Hp corresponds to that of the α 1 β 1 (and α 2 β 2 ) dimers in the R-state tetramer 24,25,42,43 .

Materials
Human Hp1-1 and Hp2-2 were purchased from Athens Research & Technology, Inc. (Athens, GA, USA).Human oxygenated Hb was prepared as previously reported 44 .The oxygenated Hp:Hb complexes were prepared by mixing oxygenated Hb with Hp1-1 and Hp2-2 at pH 7.0 and 20.0 °C29 .The dimeric Hp:tetrameric Hb stoichiometry was 1:1.To avoid the occurrence of free Hb, a 20% excess of Hp1-1 and Hp2-2 was present in all samples.The absence of free Hb was checked by gel electrophoresis 31 .
CO was purchased from Linde AG (Höllriegelskreuth, Germany).The CO solution was prepared by keeping in a closed vessel the 5.0 × 10 −2 M phosphate buffer solution (pH = 7.0) under CO at P = 760.0mm Hg anaerobically (T = 20.0 °C).The solubility of CO in the aqueous buffered solution is 1.03 × 10 −3 M, at P = 760.0mm Hg and T = 20.0 °C44 .
All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).All chemicals were of analytical grade and were used without further purification.
Since with both methods, the O 2 dissociation time courses from Hp1-1:Hb(II)-O 2 and Hp2-2:Hb(II)-O 2 display two exponentials, they have been analyzed in the framework of either Fig. 1 or Fig. 2.
Values of the apparent first-order rate constants for O 2 dissociation from Hp1-1:Hb(II)-O 2 and Hp2-2:Hb(II)-O 2 (i.e., k off1 and k off2 ) were obtained according to Eqs 1 and 2 45 : The results (from at least four experiments) are given as mean values plus or minus the corresponding standard deviation.All data were analyzed using the GraphPad Prism program, version 5.03 (GraphPad Software, La Jolla, CA, USA).
Comparison of the three-dimensional structure of the αβ dimer of Hb bound to Hp (PDB code 5JDO; resolution 3.2 Å) 43 with the corresponding subunits of the Hb tetramer in the R-state (PDB code 2DN1; resolution 1.25 Å) 42 , including calculation of root mean square deviation (rmsd) values, has been carried out using SwissPDBViewer 46 .

Results and Discussion
Under all the experimental conditions, O 2 dissociation from Hp1-1:Hb(II)-O 2 and Hp2-2:Hb(III)-O 2 follows a biphasic process upon mixing the Hp1-1:Hb(II)-O 2 and Hp2-2:Hb(III)-O 2 solutions either with the dithionite solution or the CO solution in the presence of dithionite (Fig. 3).This is supported by statistical analysis (Table 1) and the residual distribution shown in Fig. 3 of the Supplementary Information section.
Interestingly, values of k off1 and k off2 are closely similar to both those for the deoxygenation of isolated α(II)-O 2 and β(II)-O 2 chains of Hb (28 ± 6 s −1 and 16 ± 5 s −1 , respectively 39 ; and 28 ± 3 s −1 and 18 ± 2 s −1 , respectively 40 ) and those for the dissociation of the first O 2 molecule from tetrameric Hb(II)-O 2 (21.2 ± 2.6 s −1 and 13.0 ± 1.4 s −1 , respectively) 41 (Table 1).Of note, while for isolated chains the O 2 dissociation rate constant is faster for the α-chains 39 , in the case of tetrameric Hb(II)-O 2 the dissociation of the first O 2 molecule from the tetra-ligated species seems to be faster for the β-subunit 41 .This assembly-linked subunit functional difference has been suggested to reflect a structural change(s) of the α-chain upon the tetrameric assembly leading to a slower O 2 dissociation rate constant.This finding has been also confirmed by subsequent observations on the kinetics of O 2 displacement from oxygenated Hb by CO 47,48 , where the subunit characterized by the faster phase displays a higher affinity for organic phosphate, which is known to bind at the β-dyad axis 49,50 .Therefore, it seems very reasonable to identify the β-subunit as the one characterized by the faster O 2 dissociation rate in the tetrameric R-state of Hb.
In the case of the Hp:Hb(II)-O 2 complexes, a different situation occurs since Hp binds the α 1 β 1 (and the α 2 β 2 ) dimers 24,25 ; therefore, parameters here reported (Table 1) reflect the functional features of this dimeric structure.In a previous paper on the O 2 binding properties of the Hp2-2:Hb(II) complex 33 the biphasic O 2 dissociation process was detected, but a two-fold higher rate was reported for the fast phase (i.e., 59.5 ± 5.5 s −1 ) whereas the slower rate is closely similar to what observed by us (Table 1).At the moment, we have no obvious explanation for this discrepancy between data here described and previous ones 33 , but indeed the close similarity for O 2 dissociation rates by both (i) mixing Hp:Hb(II)-O 2 complexes with dithionite (thus fully deoxygenating Hp:Hb(II)-O 2 to Hp:Hb(II), see Fig. 1) and (ii) displacing O 2 with CO (thus keeping always a fully liganded form, see Fig. 2), as observed by us (Table 1), rules out the possibility of a negative cooperativity within the α 1 β 1 dimer, definitely assigning the biphasicity to a different kinetic behavior of the two subunits.
Further, data shown in Table 1 confirm the view that: (i) the αβ dimers of Hb(II)-O 2 bound to Hp1-1 and Hp2-2 are in the R-state as reported for isolated α(II)-O 2 and β(II)-O 2 chains and (ii) Hp1-1 and Hp2-2 species affect to the same extent the O 2 dissociation from Hp1      Lastly, the similar reactivity of the αβ dimers of Hb(II)-O 2 bound to Hp and of the tetrameric R-state is in keeping with structural data.Indeed, both subunits of the αβ dimers of Hb(II)-O 2 bound to Hp 43 display a conformation superimposable to that of the α and β chains of tetrameric oxygenated Hb with the Fe(II) atom positioned in the plane of the heme group 42 (Fig. 5).This is confirmed by the very low backbone rmsd value (0.56 Å) calculated upon best fitting the three-dimensional structure of the αβ dimer of Hb bound to Hp (PDB code 5JDO) 43 to the corresponding subunits of the Hb tetramer in the R-state (PDB code 2DN1) 42 .
A final consideration is required with respect to the consequences of these data for the comprehension of the cooperative mechanism of O 2 binding to Hb.It has been shown that the ligand-linked quaternary structural change, responsible for the cooperativity, is connected to a shift of the α 1 β 2 (as well as of α 2 β 1 ) interfaces 2,51 , as also suggested by the fact that at very high ionic strength the dissociation into α 1 β 1 (and α 2 β 2 ) dimers brings about the disappearance of cooperativity 38 , indicating that dimers are in a R-like quaternary state.However, the evidence of a functional heterogeneity for the O 2 dissociation in the R state 41,48 raised the question on the role of the α 1 β 1 (and α 2 β 2 ) subunit interaction in the energetics of cooperativity.Although the stabilization of the dimeric assembly upon interaction of Hb with Hp has been established since long time 27 and a kinetic heterogeneity for the O 2 dissociation has been shown also for the Hp:Hb complex 33 , it is only the close similarity, reported in this paper for the first time, between the O 2 dissociation rates measured by full deoxygenation and O 2 displacement by CO (Fig. 3) that allows to state that no ligand-linked structural change occurs at the α 1 β 1 (and α 2 β 2 ) subunit interaction surface, as also supported by the substantial identity for the dimer structure in the Hp:Hb complex and in the tetrameric oxygenated Hb (Fig. 5).
-1:Hb(II)-O 2 and Hp2-2:Hb(III)-O 2 .In fact, the two CCP domains present in each Hp monomer are involved in the protein dimerization and do not participate to the recognition of the αβ dimers of Hb(II)-O 2 23-25 .

#k
off1 versus k off2 Student's t-test, p < 0.0001.Present study.b pH 7.0 and 20.0 °C33 .Since errors of k off1 and k off2 values are not available, errors have been calculated arbitrarily as the average ± the reported interval, for the homogeneous comparison.c Dissociation of the first O 2 molecule from Hb(II)-O 2 .pH 7.0 and 20.0 °C41 .d pH 7.0 and 20.0 °C39 .e pH 7.0 and 20.0 °C40 .

Figure 5 .
Figure 5. Structural superposition of the three-dimensional structure of the αβ dimer of Hb bound to Hp (cyan; PDB code 5JDO)43 to the corresponding subunits of the Hb tetramer in the R-state (light brown; PDB code 2DN1)42 .The heme-Fe moieties are shown in stick representation.Iron atoms are represented by orange spheres and O 2 molecules as red sticks.The figure has been drawn using the UCSF Chimera software52 .

Table 1 .
Values of k off for O 2 dissociation from Hp:Hb(II)-O 2 complexes.a pH 7.0 and 20.0 °C.Present study.