Glutathionylation Induced Structural Changes in Oxy Human

Nov 16, 2012 - Bindu Y. Srinivasu , Beena Bose , Gopa Mitra , Anura V. Kurpad , and Amit K. ... Amrita Mitra , Monita Muralidharan , Deepsikha Srivast...
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Glutathionylation Induced Structural Changes in Oxy Human Hemoglobin Analyzed by Backbone Amide Hydrogen/Deuterium Exchange and MALDI-Mass Spectrometry Gopa Mitra,# Monita Muralidharan,# Sreekala Narayanan, Jennifer Pinto, Krishnamachari Srinivasan, and Amit Kumar Mandal* Clinical Proteomics Unit, Division of Molecular Medicine, St. John’s Research Institute, St. John’s National Academy of Health Sciences, 100 ft Road, Koramangala, Bangalore -560034, India

ABSTRACT: Glutathionyl hemoglobin, a post-translationally modified form of hemoglobin, has been reported to serve as a marker of oxidative stress in several clinical conditions. This modification causes perturbations in the hemoglobin functionality by increasing oxygen affinity and reducing cooperativity. Moreover, glutathionylation of sickle hemoglobin was reported to lead to a significant reduction in the propensity of sickling of erythrocytes. The root cause of the above functional abnormality is not known in detail, as the crystal structure of the molecule is yet to be discovered. In this study, we investigated the effects of glutathionylation on quaternary structure of hemoglobin using hydrogen/deuterium exchange (H/DX) based mass spectrometry. H/DX kinetics of nine peptides from α and β globin chains of hemoglobin were analyzed to understand the conformational change in deoxy to oxy transition of normal hemoglobin and structural perturbations associated with glutathionylation of oxy hemoglobin. Significant structural changes brought about by the glutathionylation of oxy hemoglobin were observed in the following regions of globin chains: β86−102, β1−14, α34−46, β32−41, β130−146, β115−129, β73−81. Isotope exchange kinetics monitored through mass spectrometry is a useful technique to understand structural perturbation on post-translational modification of proteins in solution phase.



INTRODUCTION The redox status of erythrocytes is defined by the ratio of reduced (GSH) to oxidized (GSSG) glutathione (γ-L-glutamylL-cysteinyl-glycine), which is the most abundant thiol present as antioxidant in erythrocyte. In the event of cellular defense against reactive oxygen species (ROS), the GSH/GSSG ratio decreases and free thiols of cytosolic proteins are maintained in its reduced form. As a result of oxidative insult, the accumulation of GSSG within the cell may lead to posttranslational modification of proteins such as S-glutathionylation via thiol/disulfide exchange mechanism.1 Several studies have reported glutathionyl hemoglobin (GSHb) as a possible biomarker of oxidative stress in various medical conditions such as hyperlipidemia,2 diabetes mellitus,2 Friedreich’s ataxia,3 atherosclerosis,4 and chronic renal failure.5 Oxidative stress level can be better assessed from the analysis of glutathionylated proteins (GSSPs) compared to the measurement of GSH and/or GSSG, as GSSPs are more stable than GSSG and less susceptible to enzymatic reduction by glutathione reductase.6,7 © 2012 American Chemical Society

Hemoglobin (HbA) is a widely studied allosteric protein consisting of α and β globin chains in duplicate. Oxidative imbalance in erythrocytes causes glutathionylation of hemoglobin at βCys93 residue, which is the most accessible free thiol on the molecular surface.5 The above nonenzymatic posttranslational modification of hemoglobin was elevated on incubation of erythrocytes with tert-butylhydroperoxide.8 Compared to HbA, GSHb was found to have 6-fold tighter oxygen binding capacity.9 The Hill coefficient at half saturation (n50) is 1.5 for GSHb compared to 2.9 in HbA.9 The function of hemoglobin is mainly driven by the conformational transition between its deoxy and oxy forms. Therefore, it is likely that the reported functional abnormality in GSHb was induced by the structural perturbation associated with glutathionylation of hemoglobin in its deoxy and/or oxy states. Received: May 30, 2012 Revised: September 25, 2012 Published: November 16, 2012 2344

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reaction at 0 °C and pH 2.5, as discussed in our previous communication.10 Mass Spectrometry. All MS analyses were performed on a Waters Synapt HDMS mass spectrometer equipped with a MALDI source in the positive ion V mode, using 200 Hz solid state laser (λ = 355 nm). The mass spectrometer was calibrated using an external calibrant, PEG mix. Data Analysis. The experimental mass spectra were baseline-corrected and the isotope averaged centroid mass of each molecular ion (Mt) at a given time t was measured using the software HX Express version Beta (http://www.hxms.com/ HXExpress). The number of deuterium incorporated into a peptide D(t) was calculated11

Using NMR spectroscopy, it was shown that the tertiary structural changes in the oxy state of GSHb resulted in the movement of βVal67 residue toward the heme center and movement of helix F and FG corner in the β chain away from the heme center. The salt bridge between βAsp94 and βHis146 is destabilized.9 Quaternary structural changes included destabilization of the hydrogen bond between βAsp99αTyr42, βHis146-αLys40, and βHis97-αPro44 at the αβ interface.9 However, the crystal structure of the oxy state of GSHb has not been discovered to date. In our previous communication, we described the structural changes linked to glutathionylation of the deoxy state of HbA.10 In this study, we report glutathionylation-associated structural perturbation in the oxy state of HbA using hydrogen/ deuterium exchange (H/DX) based mass spectrometry.



D(t ) =

EXPERIMENTAL PROCEDURES

(M t − M 0 ) ×N (M∞ − M 0)

(1)

where M0 and M∞ are the observed masses at zero time control experiment and for a fully deuterated molecule, respectively,10 and N is the total number of backbone amide hydrogens in the peptide. The peptide backbone amide hydrogens can be categorized into fast (A), intermediate (B), and slow exchanging (C) groups and D(t) can be represented as follows:12

Materials. Pepsin crystalline, deuterium oxide (99.9%), 2,2′dithiodipyridine (2-PDS), Sephadex G-50, reduced glutathione (GSH), and sinapinic acid were purchased from Sigma (St. Louis, MO). Synthesis-grade trifluoroacetic acid (TFA) was obtained from Merck, Germany. Poly(ethylene glycol) (PEG) mix, the external calibrant for mass spectrometer, was purchased from Waters, Milford, MA. α-Cyano-4-hydroxycinnamic acid, MALDI matrix, was obtained from Fluka, Germany. All other chemicals used were of analytical grade. Isolation and Chemical Modification of Hemoglobin. Blood was collected from healthy volunteers with their prior consent. Isolation of hemoglobin was done from whole blood according to the procedure described previously.10 Glutathionyl hemoglobin was synthesized in vitro following protocol described earlier.10 GSHb with the abundance of more than 85% was used for structural characterization. Preparation of Oxyhemoglobin. Hemoglobin was maintained at 300 μM in 50 mM ammonium bicarbonate buffer (pH 7.4). Pure oxygen gas was continuously bubbled through the solution at a rate of 40 mL/min for an hour to make it completely saturated with oxygen. The solution was then diluted 15-fold with buffered D2O to start the isotope exchange kinetics with oxygen being continuously bubbled throughout the exchange experiment. The resulting solution was used for isotope exchange kinetics experiments. A similar procedure was followed for oxygenation of glutathionyl hemoglobin. Hydrogen−Deuterium Exchange Experiment. Hydrogen/deuterium exchange experiments were done according to the protocol described previously.10 In brief, the exchange reaction was initiated in hemoglobin by the addition of 15-fold excess of 50 mM ammonium bicarbonate/D2O buffer (pD 7.4) at 25 °C. At different time intervals, 10 μL of reaction mixture was added to 90 μL of ice-cold aqueous 0.1% TFA (pH 2.5) solution to stop the H/DX kinetics and 2 μL of aqueous pepsin solution was added immediately to the aforementioned acidified protein solution maintaining enzyme/substrate ratio 1:10 mol/mol, and digestion was continued for 5 min at 0 °C. 1 μL of digested sample was mixed with 1 μL of matrix solution (5 mg/mL) and 0.5 μL of the above mixture was spotted on the MALDI plate. The spot was dried rapidly using moderate vacuum in a desiccator and spectra were acquired immediately. Both 100% and 0% control experiments were done as reported previously.10 The issue of back-exchange of backbone amide hydrogens has been taken care of by quenching the H/DX

t

t

D = N − A e −k1 − B e −k 2 − C e −k 3

t

(2)

where k1, k2, and k3 are the average rate constants for fast, intermediate, and slow exchanging amide hydrogens, respectively. The exchange rate constants and the population of amide hydrogens in different groups were varied to minimize the sum of squared residuals (SSR). The SSR for each data set was calculated using the following equation: SSR =

∑ [(yiobs − yicalc )/yiobs ]2 i

(3)

yi obs and yi calc were calculated from eq 1 and eq 2, respectively.13



RESULTS AND DISCUSSION Glutathionylation, an oxidative stress induced post-translational modification of hemoglobin in erythrocytes, is an important event at the molecular as well as at the cellular level. It leads to modification in molecular functionality of hemoglobin through a change in the oxygen binding equilibrium.9 At the cellular level, glutathionylation of sickle hemoglobin leads to a significant reduction in the propensity of sickling of erythrocytes.14 The functional abnormality is most likely a result of glutathione-induced perturbation in the tertiary and quaternary structure of hemoglobin. To explore the probable perturbation in hemoglobin structure, particularly in its oxy state, we have used hydrogen/deuterium exchange based mass spectrometry (H/DX-MS). H/DX-MS has emerged as a powerful technique to investigate solution-phase conformational dynamics of proteins. Depending on the local environment, inductive and charge effect of the neighboring groups, different polar hydrogen exchanges with deuterium from the surrounding solvents at different rates. Except for peptide amide backbone hydrogens, which exchange on a convenient and measurable time scale, all other solvent-accessible polar hydrogens in protein molecule exchange very rapidly. Mass spectrometric analysis of proteolytic peptides obtained from deuterated protein provides information that eventually helps in understanding the conformational dynamics of the region of 2345

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Figure 1. MALDI mass spectra for the peptide with m/z 1308.6 obtained on hydrogen/deuterium exchange kinetics. Panels A and B represent the isotope exchange kinetics for the peptide fragment in HbA and GSHb, respectively.

nine peptic peptides are listed in Table 1. The parameters have been provided for both oxy HbA and oxy GSHb as well as for the previously reported deoxy HbA. The analysis of tandem mass spectra followed by sequence assignment confirmed that the peptic peptide of HbA with m/z 1921.9 was a β globin fragment consisting of residues 86−102 (ATLSELHCDKLHVDPEN). The same fragment in GSHb digest appeared at 2226.1 m/z, showing a shift in mass consistent with the presence of glutathione moiety attached to βCys93 through disulfide linkage. The number of exchangeable peptide amide hydrogens was 15 and 17 for fragments 1921.9 m/z and 2226.1 m/z, respectively. Glutathione moiety contributed two additional amide hydrogens in 2226.1 m/z. Figure 2A represents the best fit curve obtained from deuterium incorporation kinetics data for peptide 1921.9 m/z in the oxy state of HbA (HbAoxy) (trace 1) and 2226.1 m/z in glutathionyl HbAoxy (GSHboxy) (trace 2) fitted to eq 3. A close review of our data obtained in the present study for HbAoxy and that reported previously for deoxy HbA (HbAdeoxy) showed that, upon transition from deoxy to oxy state of normal hemoglobin, the rate constants increased significantly for the fast and intermediate exchanging group of amide hydrogens (Table 1). The population of the fast exchanging amide hydrogen increased from 8.20% to 29.73%, which was compensated by a decrease in the population of the intermediate and the slow

origin of experimental peptide in the intact protein molecule. The novelty of the H/DX-MS approach is linked to its high precision in measurement, requirement of picomoles of protein, and short time duration for the analysis of the entire molecule. In the hemoglobin molecule, the cysteine residue in the α globin subunit is completely inaccessible. Between the two cysteine residues in the β globin subunit of HbA, βCys93 is the preferred site for glutathione binding.5 All H/DX-MS experiments were carried out using glutathionyl hemoglobin synthesized in vitro where the molecular abundance was greater than 85%. Figure 1 represents deuterium exchange kinetics of peptic peptide with m/z 1308.6 obtained from both HbA and GSHb molecules in their oxy state. On replacement of one hydrogen atom by deuterium, the mass increases by one unit, and as more hydrogens are replaced by deuterium with time, the envelope corresponding to the peptide molecular ion shows mass shift progressively toward higher mass. While panel A represents the envelope for the peptide in HbA, panel B represents the same in GSHb. The peptide backbone amide hydrogens were assigned into three groups, fast (A), intermediate (B), and slow (C) exchanging in each individual peptide fragment, and the corresponding average rate constants for exchange kinetics were k1, k2, and k3, respectively. The number of differentially grouped peptide amide hydrogens and rate constants for all 2346

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Table 1. Kinetic Parameters for Isotope Exchange of Different Peptic Peptidesa peptide mass (m/z)

residues

1921.9

β86−102 ATLSELHCDKLHVDPEN

1868.9

β130−146 YQKVVAGVANALAHKYH

1494.8

β1−14 VHLTPEEKSAVTAL

1585.8

α34−46 LSFPTTKTYFPHF

1308.6

β32−41 LVVYPWTQRF

1635.8

β115−129 AHHFGKEFTPPVQAA

2910.4

α1−29 VLSPADKTNVKAAW GKVGAHAGEYGAEAL

1798.9

β15−31 WGKVNVDEVGGEALGRL

b

967.5

β73−81 DGLAHLDNL

molecule

N

k1(min−1)

k2(min−1)

HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy HbAdeoxy HbAoxy GSHboxy

15 15 17 16 16 16 12 12 12 10 10 10 8 8 8 12 12 12 27 27 27 16 16 16 8 8 8

1.5419 7.8536 2.7340 0.0427 0.0220 0.1491 0.6677 6.8633 1.4629 0.1553 7.9650 4.9998 0.0240 0.0802 1.0647 0.0199 0.0924 0.9887 1.3112 9.3418 2.8777 0.7153 7.6332 1.6751 0.2964 7.6826 7.9639

0.0893 0.1000 1.6988 0.0018 0.0058 0.0088 0.0552 0.1526 1.4220 0.0456 0.0933 0.1581 0.0080 0.0045 0.0527 0.0013 0.0002 0.3933 0.0495 0.1239 0.7210 0.0723 0.1191 0.0111 0.0011 0.0378 0.0169

k3(min−1) 1.60 1.25 1.18 2.00 6.27 3.89 1.00 3.20 3.75 2.00 2.38 5.99 1.40 2.27 3.30 4.15 6.58 4.05 1.90 2.74 5.28 1.00 3.66 4.97 2.00 3.88 5.67

× × × × × × × × × × × × × × × × × × × × × × × × × × ×

10−05 10−05 10−03 10−03 10−04 10−03 10−04 10−04 10−03 10−05 10−04 10−07 10−05 10−03 10−03 10−06 10−05 10−04 10−03 10−03 10−02 10−04 10−03 10−03 10−04 10−04 10−04

A (%)

B (%)

C (%)

8.20 29.73 69.94 55.31 43.75 48.31 48.83 48.37 84.42 11.10 1.40 19.54 37.38 39.07 26.25 35.42 28.79 31.71 34.96 49.68 58.00 24.80 30.80 68.50 32.00 4.80 18.75

41.93 28.80 10.24 34.00 43.75 13.75 35.33 37.97 8.42 28.70 40.03 10.36 25.13 28.97 24.75 11.92 25.77 26.46 53.81 41.19 23.89 39.40 28.30 6.50 15.25 32.30 47.25

49.87 41.47 19.82 10.75 12.50 37.94 15.92 13.66 7.17 60.20 58.58 70.10 37.50 31.96 49.00 52.67 45.44 41.83 11.19 9.13 18.11 35.80 40.90 25.00 52.75 62.90 34.00

a

Total numbers of exchangeable amide hydrogens for each peptide are given by N, while A, B, and C represent the number of fast, intermediate, and slow exchangeable hydrogens, respectively. The corresponding rate constants are given by k1, k2, and k3, respectively. bFor the peptide 1921.9, after glutathionylation, the number of exchangeable hydrogens is 17, taking into account two hydrogens from glutathione moiety.

important role in the allosterism of hemoglobin.20 Therefore, the movement of the aforementioned residues on transition from deoxy to oxy state might have increased the solvent accessibility of the peptide 1921.9, thus resulting in an increase in the isotope exchange rates. The fitted parameters for H/DX kinetics of the peptide 2226.1 m/z showed that, upon glutathionylation of HbAoxy, there was an increase in the fast exchanging hydrogen population from 29.73% to 69.94% (Table 1). This increase in the fast exchanging population was compensated by a decrease in the intermediate and slow exchanging amide hydrogens. In GSHboxy, the rate constants for the fast exchanging hydrogens decreased by 3-fold while that of the intermediate and slow exchanging hydrogens increased by 17-fold and 99-fold, respectively (Table 1). Hence, on glutathionylation, there is an overall increase in the conformational flexibility in this part of the molecule. On the basis of NMR data, Craescu et al. showed that the characteristic intrasubunit salt bridge βAsp94-βHis146 and inter-subunit hydrogen bond at the αβ interface, βAsp99-αTyr42 in HbAdeoxy, were perturbed significantly on glutathionylation.9 It was reported that, in Hb Creteil, a hemoglobin variant with high oxygen affinity, βCys93 is located in the pocket between the FG corner and F and H helices.21 By comparing the NMR spectra of Hb Creteil and GSHboxy, Craescu et al. reported that, for both the molecules, the conformational perturbation of the β globin chain near the oxy heme pocket was similar.9 According to the experimental data and our previously reported H/DX kinetics data of GSHbdeoxy, we observed that the increases in the fast

exchanging hydrogens (Table 1). This suggests that the peptide 1921.9 became more solvent-exposed upon oxygenation. Perutz MF reported that the salt bridge between the carboxyl group of βAsp94 and the imidazole group of βHis146 in HbAdeoxy is broken on oxygenation.15 This in turn might have resulted in loosening of the region, leading to more solvent exposure and hence an increase in the isotope exchange rate. In the deoxy state, the side chain of βTyr145 is hydrogen-bonded to the carboxyl group of βVal98, while in the liganded state, the bond is present only for a short period.16 Oxygen binding results in a decrease in the distance between proximal histidine (βHis92) and porphyrin ring, which triggers a series of changes in the tertiary structure in the molecule. The side chains of βLeu88, βLeu91, and βHis92 undergo slight conformational changes to stay in contact with the heme unit.16 Upon oxygenation, βCys93 shifts its orientation from solvent-exposed state to the pocket between helix F and H.16 αβ inter-subunit interactions play an important role in the cooperativity of hemoglobin, and significant structural change takes place during deoxy to oxy transition in this region of the molecule.17 The residue βAsp99 forms inter-subunit hydrogen bonds with αTyr42 in the αβ interface, which stabilizes the deoxy state (T).18 The residue βAsn102 stabilizes the oxy state (R) by forming a hydrogen bond with αAsp94.19 Shih et al. reported that, on deoxy to oxy transition, the polarity of the βGlu101 residue, which is positioned between βAsp99 and βAsn102, influences the switchover of the two neighboring interchain hydrogen bonds on its either side. This switchover in hydrogen bond plays an 2347

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with the previously reported data for HbAdeoxy are shown in Table 1. The kinetics data showed that, on oxygenation of HbAdeoxy, the rate constant and the population of fastexchanging hydrogens decreased by 1.94-fold and 11.56%, respectively. For intermediate exchanging hydrogens, the rate constant increased by 3.22-fold with an increase in population by 9.75%. The change in population and rate constants for all three groups of hydrogens indicated that oxygenation of hemoglobin might result in a decrease in the solvent exposure and rigidity in the conformation of this region of molecule. It was reported that a new set of salt bridges involving βAsn139βArg104, βLys132-βGlu7, and a hydrogen bond involving βHis146-βLys144 are formed in the oxy state of hemoglobin.22 The penultimate Tyr145 and C-terminal His146 of β globin chain are part of this peptide. In HbAdeoxy, the C-terminal residue is anchored by two salt bridges. The α-carboxyl group of βHis146 is linked to ε-amino group of αLys40 and its imidazole to βAsp94.15 Tyr145 residue is also firmly anchored inside the pocket between helices F and H through van der Waals force and by hydrogen bonds between its hydroxyl group and carbonyl group of Val98.15 On oxygen binding to hemoglobin, the helix F moves toward the center of the molecule, narrowing the pocket occupied by Tyr145 and ultimately expelling it. The expelled Tyr145 pulls His146 along with it and breaks its salt bridges.15 Perutz had proposed a central role for the salt bridge involving C-terminal residues of hemoglobin in its cooperativity.15 Disruption in these contacts causes the shift of R-T equilibrium toward the R state, thus resulting in an increased oxygen affinity and loss of allosteric function. βHis146 was reported to contribute 40% to the alkaline Bohr effect of hemoglobin.23 Louie et al. reported that several allosterically sensitive hydrogens which are present at the β C-terminus account for about 70% of allosterically significant structural changes.24 Therefore, a change in the aforementioned interactions might have imparted rigidity to the oxy conformer resulting in solvent shielding. The fitted parameters of H/DX kinetics showed that, on glutathionylation of HbAoxy, there was more than a 6-fold increase in the rates for both the fast and the slow exchanging hydrogens with 1.5-fold increase in the rate for the intermediate one. The fast and slow exchanging population increased by 4.56% and 25.44%, respectively, whereas the intermediate group decreased by 30%. The observed data indicated that glutathionylation of HbAoxy led to an increase in flexibility in the conformation for this peptide. Compared to our earlier report of a dramatic increase in the population of the fast exchanging amide hydrogens on glutathionylation of deoxy hemoglobin, the magnitude of the increase in the fast exchanging population on glutathionylation of oxy hemoglobin was modest. On the basis of NMR data, Craescu et al. showed that the salt bridge between βHis146 and βAsp94 was absent in GSHbdeoxy. The displacement of βHis146 on glutathionylation might lead to breakage of βHis146-αLys40 salt bridge.9 A significant increase in the fast exchange kinetics in GSHbdeoxy was indicative of removal of the above-mentioned structural constraints. The existence of additional salt bridges and hydrogen bonds specific to oxy state might be the reason for relatively lower flexibility in the conformation of GSHboxy compared to GSHbdeoxy. The peptic peptide with m/z 1494.8 is an N-terminal fragment of the β globin chain (β1−14) (VHLTPEEKSAVTAL) consisting of 12 exchangeable amide hydrogens. Figure 2C illustrates the best fit curve of H/DX kinetics for both HbAoxy (trace 1) and GSHboxy (trace 2) molecules. Oxygen-

Figure 2. Representation of hydrogen/deuterium exchange kinetics of peptic peptides. The Y-axis is labeled with number of deuterium incorporated and X-axis with the corresponding exchange time. Trace 1 in each panel (filled diamond) represents the best fit curve of the exchange kinetics for the corresponding peptide obtained from native hemoglobin, and trace 2 in each panel (hollow diamond) represents the same for the peptide obtained from glutathionyl hemoglobin. Panel A represents peptide fragment 1921.9 m/z (β86−102). Panel B represents peptide fragment 1868.9 m/z (β130−146). Panel C represents peptide fragment 1494.8 m/z (β1−14).

exchanging population on GSHbdeoxy to GSHboxy transition and on Hbdeoxy to Hboxy transition were 22.88% and 21.53%, respectively.10 The oxygenation of deoxy glutathionyl hemoglobin resulted in an increase in the fast exchange rate by 1.35fold, while oxygenation of deoxy normal hemoglobin led to an increase in the same by 5.1-fold.10 Therefore, in the case of fast exchange kinetics in the above two transitions, the population increase was very similar but the increase in the rate was much larger for normal hemoglobin compared to glutathionyl hemoglobin. Thus, covalent binding of glutathione might introduce conformational constraints in the molecule causing βCys93 to adopt oxy-like conformation, which might be the probable cause of the higher oxygen affinity of glutathionyl hemoglobin. Sequence assignment of the peptide fragment with m/z 1868.9 confirmed that it is the C-terminal β chain fragment spanning residues 130−146 (YQKVVAGVANALAHKYH) with 16 exchangeable amide hydrogens. Figure 2B represents the best fit curve of H/DX kinetics data of the peptide 1868.9 for both HbAoxy (trace 1) and GSHboxy (trace 2) molecules. The fitted parameters of HbAoxy and GSHboxy molecules along 2348

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ation of HbAdeoxy resulted in an increase in rates by 10.28-fold, 2.76-fold, and 3.20-fold for the fast, intermediate, and slow exchanging amide hydrogens, respectively (Table 1). The population of all exchange groups remained almost unaltered. Hence, oxygenation might increase the solvent accessibility of this peptide. The residues βVal1 and βHis2 contribute amino groups to the binding site of 2,3-diphosphoglycerate (DPG) and is defined as an allosteric effector of hemoglobin.25 DPG binds between two β chains. Its presence reduces oxygen affinity, which ultimately facilitates unloading of oxygen in tissues.26,27 Hence, DPG stabilizes the deoxy state of hemoglobin. In the oxy state of hemoglobin, the salt bridges between DPG and β subunits are ruptured resulting in the liberation of DPG.15 Therefore, on deoxy to oxy transition, the solvent accessibility of this region in β globin chain of HbAoxy might increase, as observed in our experiment. On glutathionylation of HbAoxy, the population of the fast exchanging hydrogens increased by 36.05%, whereas the intermediate and the slow exchanging hydrogens decreased by 29.55% and 6.49%, respectively (Table 1). On glutathionylation, the rate constants for the intermediate and slow exchanging hydrogens increased by 9.32- and 11.72-fold, respectively, whereas that of fast exchanging hydrogens decreased by 4.2-fold. Therefore, glutathionylation of HbAoxy might cause an overall increase in the conformational flexibility of the residues in peptide 1494.8 with a shift in the population of amide hydrogens from intermediate to fast exchanging groups resulting in a decrease in the average rate of the latter. Glutathionylation had been reported to increase the oxygen affinity of hemoglobin by 6-fold.9 A comparison of kinetic data of GSHb in its deoxy and oxy states clearly depicts that there is a higher degree of conformational freedom in the oxy state of the molecule. Similar comparison between deoxy and oxy states of HbA showed that all rate constants increased for the oxy state, but the number of exchangeable amide hydrogens remained unaltered. In the case of GSHb, the number of fast exchanging hydrogens increased significantly upon oxygenation. Hence, oxygenation of GSHbdeoxy was accompanied by a relatively larger increase in conformational freedom in the region of peptide 1494.8 compared to that in the HbAdeoxy molecule. Therefore, the GSHbdeoxy to GSHboxy transition is entropically more favored compared to HbAdeoxy to HbAoxy transition. This might be the probable reason behind the higher degree of oxygen affinity of GSHb. The tandem mass spectra for the proteolytic fragment ion 1585.8 m/z showed that it is a part of the α globin chain of hemoglobin consisting of residues 34 to 46 (LSFPTTKTYFPHF) with ten exchangeable backbone amide hydrogens. Figure 3A shows the fitted curve of H/DX kinetics data of the peptide 1585.8 in HbAoxy (trace 1) and GSHboxy (trace 2) molecules. Upon oxygenation of HbAdeoxy, the population for the fast exchanging hydrogens decreased from 11.10% to 1.40% and that of the intermediate exchanging hydrogens increased from 28.70% to 40.03% with a very small decrease in the population for slowest exchanging hydrogens (Table 1). The above change in population is indicative of a reduction in the conformational flexibility of the residues in this region of the molecule on HbAdeoxy to HbAoxy transition. As the reported rate constants represent an average of all amide hydrogen exchange rates in a particular group, a shift in the population from fast to intermediate exchanging groups might have resulted in an effective increase in the average value of corresponding rate constants in both groups. The constituent

Figure 3. Representation of hydrogen/deuterium exchange kinetics of peptic peptides. The Y-axis is labeled with the number of deuterium incorporated and X-axis with the corresponding exchange time. Trace 1 in each panel (filled diamond) represents the best fit curve of the exchange kinetics for the corresponding peptide obtained from native hemoglobin and trace 2 in each panel (hollow diamond) represents the same for the peptide obtained from glutathionyl hemoglobin. Panel A represents peptide fragment 1585.8 m/z (α34−46). Panel B represents peptide fragment 1308.6 m/z (β32−41). Panel C represents peptide fragment 1635.8 m/z (β115−129).

residues of peptide 1585.8 belong to the αβ interface of the tetramer, a portion where inter-subunit interactions play an important role in the deoxy to oxy transition of the hemoglobin molecule. According to Hashimoto et al., αThr38 was found to undergo considerable displacement on deoxy to oxy (T to R) transition and reported to be an important residue in the oxygen binding of hemoglobin.28 In the deoxy state of hemoglobin, βHis97 is positioned between residues αThr41 and αPro44. T to R transition results in the migration of the βHis97 residue between αThr38 and αThr41 and the formation of a hydrogen bond with αThr38.16,29 Perutz MF reported that there is an increase in the number of van der Waals interactions on the T to R transition at the αβ interface of tetramer.15 Hence, the observed decrease in the conformational flexibility associated with oxygenation of hemoglobin might be due to the appearance of the aforementioned noncovalent interactions at the αβ interface, which are important for the cooperativity of hemoglobin. Comparison of GSHboxy and HbAoxy data shows that, on glutathionylation of 2349

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sufficient information regarding structural changes in this region of hemoglobin associated with glutathionylation is lacking, the observed H/DX data indicated that glutathionylation of the oxy state of hemoglobin resulted in an increase in the conformational flexibility in this region of the molecule. The peptic peptide 1635.8 m/z was found to be a β globin chain fragment containing residues 115−129 (AHHFGKEFTPPVQAA) with 12 exchangeable peptide amide hydrogens. Traces 1 and 2 in Figure 3C represent fitted curves of H/ DX kinetics data for peptide 1635.8 in HbAoxy and GSHboxy, respectively. Comparison of the observed H/DX data for HbAoxy and previously reported data for HbAdeoxy showed that oxygenation of hemoglobin led to an increase in the rate constants for the fast and the slow exchange steps and a decrease in the intermediate exchange step (Table 1). The population of the fast and slow exchanging protons decreased by 6.63% and 7.23%, respectively, whereas the population of the intermediate exchanging protons increased by 13.85% (Table 1). The above changes in the kinetic parameters on oxygenation of hemoglobin indicated that there was a change in the solvent exposure for the constituent residues in peptide 1635.8. Oxygen binding of hemoglobin was found to introduce a new salt bridge between βGlu121 and βLys17.23 In another hemoglobin variant Hb Roma, mutation βA115V resulted in an increase in oxygen affinity.33 Therefore, above constituent residues of the peptide 1635.8 are involved in the oxygen binding of hemoglobin. Hence, this region might be susceptible to an alteration in solvent exposure due to oxygenation. Glutathionylation of HbAoxy showed a significant increase in the rate constants for all the exchange steps in the peptide 1635.8. A small increase in the fast and intermediate exchanging populations was balanced by the decrease in the same for the slow exchanging population. Previously, we reported that glutathionylation of HbAdeoxy resulted in a substantial increase in the fast and intermediate exchange rate constants with a significant increase in the respective population. There is not enough information available concerning the structural changes in this region of molecule associated with glutathionylation, but it is clear from the observed H/DX data, that glutathionylation had a more profound effect on the structural perturbation of the deoxy state than the oxy state of hemoglobin. Introduction of the new salt bridge (βGlu121-βLys17) on oxygenation might be the cause behind the relatively greater rigidity in the conformation in this region of GSHboxy compared to GSHbdeoxy. Pepsin digestion of hemoglobin generated a peptide fragment with m/z 2910.4, which corresponds to residues 1 to 29 in the α globin chain (VLSPADKTNVKAAWGKVGAHAGEYGAEAL) with 27 exchangeable amide hydrogens in it. The fitted curves of H/DX kinetics data for fragments 2910.4 in HbAoxy (trace 1) and GSHboxy (trace 2) molecules are shown in Figure 4A. On oxygenation of HbAdeoxy, there was an increase in the rate constants by 7.12-fold, 2.50-fold, and 1.44-fold for the fast, intermediate, and slow exchanging hydrogens, respectively. The fast exchanging population increased by 14.72%, which was compensated by the decrease in population for the intermediate and slow exchanging groups of hydrogens (Table 1). This indicated that on oxygenation there was an enhanced solvent exposure in this region of the molecule. It was reported that in deoxy hemoglobin, the α amino group of Nterminal αVal1 is involved in a salt bridge with the α carboxyl of arginine αArg141.16 αVal1 contributes 20−30% in the alkaline Bohr effect and is also involved in the binding of allosteric

HbAoxy, the rate constant for the fast exchange step decreased by 1.6-fold whereas that of the intermediate exchange step increased by 1.7-fold with a large decrease in the rate for the slow exchange step. The fast and slow exchanging hydrogen populations increased by 18.14% and 11.52%, respectively. The above increase in population was compensated by a decrease in the same for intermediate exchanging hydrogens. The observed change in kinetic parameters indicated that the conformational flexibility in GSHboxy was comparatively higher than that of HbAoxy. Previously, we reported that glutathionylation of HbAdeoxy resulted in an increase in the fast exchanging population by 31.6% with a decrease in population for the intermediate and slow exchanging hydrogens.10 The slow and the intermediate H/DX exchange rates increased and decreased, respectively, while the fast exchanging rate remained unaltered.10 On the basis of NMR data, Craescu et al. showed that three inter-subunit (αβ) interactions were perturbed on glutathionylation of deoxy hemoglobin, viz., αLys40-βHis146, αTyr42-βAsp99, and αPro44-βHis97.9 Garel et al. reported a change in the conformation of four α globin residues, αLys40, αThr41, αTyr42, and αPro44, on glutathione binding to βCys93 of HbAdeoxy.14 Comparing the kinetic parameters obtained in present study and previously reported data, we observed that the conformational perturbation associated with glutathionylation was relatively higher in GSHbdeoxy than in GSHboxy.10 As inter-subunit interactions are crucial for cooperativity of hemoglobin, the decrease in conformational rigidity caused by the disappearance of a number of noncovalent interactions might have contributed significantly to the reduction in the cooperativity of glutathionyl hemoglobin. Sequence analysis of peptic fragments showed that peptide with m/z 1308.6 originated from the β globin subunit of hemoglobin. The fragment 1308.6 consists of eight exchangeable peptide amide hydrogens spanning residues β32 to β41 (LVVYPWTQRF). Figure 3B represents the best fit curve of H/DX data for the fragment in HbAoxy (trace 1) and GSHboxy (trace 2). Oxygenation of HbAdeoxy resulted in an increase in the rate constants for the fast and slow exchange steps and a decrease in the rate for intermediate exchange step. The changes in population of exchangeable amide hydrogens across all the groups were small. Thus, differentially populated hydrogens rearranged among the groups (Table 1). In HbAdeoxy, βTrp37 remained buried in the hinge region of the αβ interface.30 A hydrogen bond is formed between the indole NH group of βTrp37 and the carboxylate side chain of αAsp94.30 Ligand binding induces expulsion of the C-terminus αTyr140 resulting in disruption of the quaternary structural constraints associated with βTrp37.31 Hemoglobin with the following mutation, βW37S, resulted in an increased oxygen affinity and reduced cooperativity, thus indicating involvement of βTrp37 residue in oxygen binding.32 In HbAdeoxy, the carbonyl group of βVal34 is bonded to the side chain of αArg141, whereas in HbAoxy, αArg141 appears to be free.16 It was also reported that in HbAoxy βArg40 forms a hydrogen bond with αThr41.16 Therefore, the differential behavior of the rate constants and populations in deoxy to oxy transition of hemoglobin might be due to the proximity of this region to the αβ interface along with disappearance and appearance of several quaternary interactions. On glutathionylation of HbAoxy, rate constants for all exchange steps increased with a decrease in the population of the fast and intermediate exchange group and an increase in the slow exchanging group. Though 2350

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chloride.34 αAsp6 interacts with other residues in the internal cavity between α chains and was found to contribute to the Bohr effect.16 Disappearance of the above interactions on oxygenation might lead to an increase in the flexibility in conformation of the peptide 2910.4. Glutathionylation of HbAoxy resulted in a decrease in the fast exchanging rate constants and an increase in the intermediate and slow exchanging rate constants. The fast and the slow exchanging hydrogen population increased on glutathionylation, whereas that of the intermediate one decreased (Table 1). Thus, glutathionylation caused significant conformational rearrangements among backbone amide hydrogens in this part of oxy hemoglobin. The peptic peptide with m/z 1798.9 is a β globin chain fragment containing residues 15 to 31 with 16 exchangeable amide hydrogens (WGKVNVDEVGGEALGRL). Figure 4B illustrates the best fit curve of H/DX kinetics for both HbAoxy (trace 1) and GSHboxy (trace 2) molecules. Comparison of currently observed and previously reported H/DX kinetics data for HbAoxy and HbAdeoxy showed that on oxygenation the rate constants for all exchange steps increased with a simultaneous increase in the number of the fast and slow exchanging population by 6% and 5.1%, respectively, and a decrease in the intermediate exchanging population by 11.1% (Table 1). The change in kinetic parameters suggested that the conformational flexibility in the region of this peptide in hemoglobin molecule increased on oxygenation. It was reported that recombinant hemoglobin with mutation βL28W and βL28F had very low oxygen affinity.35 In the deoxy state of hemoglobin, βLeu28 is positioned in the B10 helix. On oxygenation of hemoglobin, proximity of the B10 helix to the ligand binding site makes it an important part of the molecule.35 NMR data of the recombinant hemoglobin showed that the mutation affected the tertiary structure near the heme pocket, while there was no significant perturbation in the αβ interface.35 On glutathionylation of the oxy state of hemoglobin, the rate constants for the fast and the intermediate exchanging step decreased. There was a small increase in the rate constant for the slow exchanging step. The increase in fast exchanging hydrogen population by 37.7% was balanced by the decrease in the same for the intermediate and slow exchanging hydrogens (Table 1). Though the changes in rate constant and in population of different groups of hydrogens were not unidirectional, but it is clear from H/DX kinetics data that, on glutathionylation,

Figure 4. Representation of hydrogen/deuterium exchange kinetics of peptic peptides. The Y-axis is labeled with the number of deuterium incorporated and the X-axis with the corresponding exchange time. Trace 1 in each panel (filled diamond) represents the best fit curve of the exchange kinetics for the corresponding peptide obtained from native hemoglobin, and trace 2 in each panel (hollow diamond) represents the same for the peptide obtained from glutathionyl hemoglobin. Panel A represents peptide fragment 2910.4 m/z (α1− 29). Panel B represents peptide fragment 1798.9 m/z (β15−31). Panel C represents peptide fragment 967.5 m/z (β73−81).

Scheme 1

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Isotope exchange kinetics showed significant conformational perturbations of the residues in the peptides α34−46 and β32− 41, which are present at the αβ interface of the protein and play an important role in the deoxy to oxy transition. The increased flexibility in GSHb might be caused by the disappearance of a number of noncovalent interactions at the αβ interface which eventually manifests as reduced cooperativity. Significant structural perturbation was also observed in the peptides β130−146, β115−129, and β73−81. Among the peptides studied, the conformational rearrangement of residues occurred in the peptides α1−29 and β15−31. Glutathionylation of proteins is emerging as an important post-translational modification because of its direct correlation with oxidative stress. The novelty in our approach lies in establishing the correlation between functional abnormality and structural perturbation of hemoglobin on glutathionylation using isotope exchange based mass spectrometry where the crystal structure of the molecule is not yet known. The H/DXMS method has been found to be a simple technique to investigate structural perturbation and consequently structure function correlation of post-translationally modified proteins in solution phase. In addition, the requirement of picomoles of proteins and short time duration for the entire analysis makes the technique novel compared to other spectroscopic tools.

different groups of exchangeable hydrogen rearranged among themselves. Sequence assignment from MS/MS spectra of the peptide fragment 967.5 m/z confirmed that it was a β globin chain fragment consisting of residues 73 to 81 (DGLAHLDNL) with eight exchangeable peptide amide hydrogens. Figure 4C illustrates the best fit curve of H/DX kinetics for both HbAoxy (trace 1) and GSHboxy (trace 2) molecules. On oxygenation, the fast exchanging population decreased by 27.2%, whereas the intermediate and the slow exchanging groups increased by 17.05% and 10.15%, respectively. The rate constants for the fast and intermediate exchange steps increased dramatically with a 2-fold increase for the slow exchange rate (Table 1). Therefore, on oxygenation of hemoglobin the fast exchanging amide hydrogens moved toward the intermediate group with a concomitant shift in the intermediate exchanging population to the slow exchanging group. It clearly indicated that oxygenation led to a decrease in the solvent accessibility in this region of peptide in the hemoglobin molecule. Glutathionylation of the oxy state resulted in an increase in the population of the fast and the intermediate exchanging hydrogen to a similar extent with a decrease in the population of the slow exchanging group. Rate constants for the intermediate group decreased, whereas that of the slow exchanging group increased while the fast exchange rate remained unchanged. Therefore, glutathionylation of HbAoxy state induced an increase in the conformational flexibility for this peptide in hemoglobin to a small extent. Both α and β globin subunits of hemoglobin have α-helical conformation. α chain comprises seven helices (lacks D helix), while the β chain consists of eight helices designated as A−H.36 The amino acid sequences of those helices are marked in Scheme 1. The shaded area in the scheme denotes the nine experimental peptides where H/D exchange kinetics was monitored. The site of glutathionylation (βCys93) has been marked with an asterisk. On the basis of these nine peptides, sequence coverage for α and β were 29.7% and 67.8%, respectively.



AUTHOR INFORMATION

Corresponding Author

*Phone: +91-80-25532037. Fax: +91-80-25501088. E-mail: [email protected]. Author Contributions #

The authors share equal contribution.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We acknowledge Nano Science Mission DST, Govt. of India, for providing mass spectrometry facility in St John’s Research Institute. We acknowledge all volunteers who provided sample in the study. We also acknowledge Dr. Anura V. Kurpad, Professor, St John’s Research Institute, for helping us in data analysis.



CONCLUSION The present study aimed to analyze the structural perturbation associated with glutathionylation of oxy hemoglobin using hydrogen/deuterium exchange based mass spectrometry. Using experimental H/DX-MS data of deoxy and oxy hemoglobin, we tried to understand the reported structure function correlation of normal hemoglobin. In the next step, we attempted to correlate the observed H/DX kinetics with structural perturbation of hemoglobin on glutathionylation and finally to its function. The isotope exchange parameters indicated that the glutathionylation of βCys93 induced conformational constraints in the region β86−102 causing glutathionyl hemoglobin to adopt oxy hemoglobin-like conformation. Analysis of H/DX kinetics of the peptide β1−14 showed that the enhanced solvent accessibility on oxygenation of hemoglobin might be due to the release of DPG, which was bonded through salt bridges prior to its release. Comparison of exchange kinetics indicated that the formation of GSHboxy state is entropically more favored than the HbAoxy state, which might be the probable rationale behind the higher oxygen affinity of GSHb compared to HbA. In general, glutathionylation caused an overall increase in the conformational flexibility of the molecule. The inter-subunit interactions that were reported to be crucial for the cooperativity of the molecule were perturbed.



REFERENCES

(1) Gilbert, H. F. (1995) Thiol/disulfide exchange equilibria and disulfide bond stability. Methods Enzymol. 251, 8−28. (2) Niwa, T., Naito, C., Mawjood, A. H. M., and Imai, K. (2000) Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry. Clin. Chem. 46, 82−88. (3) Piemonte, F., Pastore, A., Tozzi, G., Tagliacozzi, D., Santorelli, F. M., Carrozzo, R., Casali, C., Damiano, M., Federici, G., and Bertini, E. (2001) Glutathione in blood of patients with Friedreich’s ataxia. Eur. J. Clin. Invest. 31, 1007−1011. (4) Nonaka, K., Kume, N., Urata, Y., Seto, S., Kohno, T., Honda, S., Ikeda, S., Muroya, T., Ikeda, Y., Ihara, Y., Kita, T., and Kondo, T. (2007) Serum levels of S-glutathionylated proteins as a risk-marker for arteriosclerosis obliterans. Circ. J. 71, 100−105. (5) Mandal, A. K., Woodi, M., Sood, V., Krishnaswamy, P. R., Rao, A., Ballal, S., and Balaram, P. (2007) Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry. Clin. Biochem. 40, 986−994. (6) Lou, M. F. (2003) Redox regulation in the lens. Prog. Retin. Eye Res. 22, 657−682.

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(7) Giustarini, D., Dalle-Donne, I., Colombo, R., Petralia, S., Giampaoletti, S., Milzani, A., and Rossi, R. (2003) Protein glutathionylation in erythrocytes. Clin. Chem. 49, 327−330. (8) Murakami, K., and Mawatari, S. (2003) Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of alphahemoglobin to membrane. Arch. Biochem. Biophys. 417, 244−250. (9) Craescu, C. T., Poyart, C., Schaeffer, C., Garel, M. C., Kister, J., and Beuzard, Y. (1986) Covalent binding of glutathione to hemoglobin. II. Functional consequences and structural changes reflected in NMR spectra. J. Biol. Chem. 261, 14710−14716. (10) Mitra, G., Muralidharan, M., Pinto, J., Srinivasan, K., and Mandal, A. K. (2011) Structural perturbation of human hemoglobin on glutathionylation probed by hydrogen-deuterium exchange and MALDI mass spectrometry. Bioconjugate Chem. 22, 785−793. (11) Zhang, Z., and Smith, D. L. (1993) Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci. 2, 522−531. (12) Hoofnagle, A. N., Resing, K. A., and Ahn, N. G. (2003) Protein analysis by hydrogen exchange mass spectrometry. Annu. Rev. Biophys. Biomol. Struct. 32, 1−25. (13) Kemmer, G., and Keller, S. (2010) Nonlinear least-squares data fitting in Excel spreadsheets. Nat. Protoc. 5, 267−281. (14) Garel, M. C., Domenget, C., Caburi-Martin, J., Prehu, C., Galacteros, F., and Beuzard, Y. (1986) Covalent binding of glutathione to hemoglobin. I. Inhibition of hemoglobin S polymerization. J. Biol. Chem. 261, 14704−14709. (15) Perutz, M. F. (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228, 726−739. (16) Baldwin, J., and Chothia, C. (1979) Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism. J. Mol. Biol. 129, 175−220. (17) Vallone, B., Bellelli, A., Miele, A. E., Brunori, M., and Fermi, G. (1996) Probing the alpha 1 beta 2 interface of human hemoglobin by mutagenesis. Role of the FG-C contact regions. J. Biol. Chem. 27, 12472−12480. (18) Kavanaugh, J. S., Rogers, P. H., Arnone, A., Hui, H. L., Wierzba, A., DeYoung, A., Kwiatkowski, L. D., Noble, R. W., Juszczak, L. J., Peterson, E. S., and Friedman, J. M. (2005) Intersubunit interactions associated with Tyr42 alpha stabilize the quaternary-T tetramer but are not major quaternary constraints in deoxyhemoglobin. Biochemistry 44, 3806−3820. (19) Kister, J., Kiger, L., Francina, A., Hanny, P., Szymanowicz, A., Blouquit, Y., Promé, D., Galactéros, F., Delaunay, J., and Wajcman, H. (1995) Hemoglobin Roanne [alpha 94(G1) Asp–>Glu]: a variant of the alpha 1 beta 2 interface with an unexpected high oxygen affinity. Biochim. Biophys. Acta 1246, 34−38. (20) Shih, D. T., Jones, R. T., Imai, K., and Tyuma, I. (1985) Involvement of Glu G3 (101) beta in the function of hemoglobin. Comparative O2 equilibrium studies of human mutant hemoglobins. J. Biol. Chem. 260, 5919−5924. (21) Arnone, A., Thillet, J., and Rosa, J. (1981) The structure of hemoglobin Creteil (beta 89 Ser replaced by Asn) is similar to that of abnormal human hemoglobins having sequence changes at Tyr 145 beta. J. Biol. Chem. 256, 8545−8552. (22) Shaanan, B. (1983) Structure of human oxyhaemoglobin at 2.1 A resolution. J. Mol. Biol. 171, 31−59. (23) Kilmartin, J. V., Breen, J. J., Roberts, G. C., and Ho, C. (1973) Direct measurement of the pK values of an alkaline Bohr group in human hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 70, 1246−1249. (24) Louie, G., Tran, T., Englander, J. J., and Englander, S. W. (1988) Allosteric energy at the hemoglobin beta chain C terminus studied by hydrogen exchange. J. Mol. Biol. 201, 755−764. (25) Arnone, A. (1974) Mechanism of action of hemoglobin. Annu. Rev. Med. 25, 123−130. (26) Benesch, R., and Benesch, R. E. (1967) The effect of organic phosphates from the human erythrocyte on the allosteric properties of hemoglobin. Biochem. Biophys. Res. Commun. 26, 162−167.

(27) Chanutin, A., and Curnish, R. R. (1967) Effect of organic and inorganic phosphates on the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 121, 96−102. (28) Hashimoto, M., Ishimori, K., Imai, K., Miyazaki, G., Morimoto, H., Wada, Y., and Morishima, I. (1993) Site-directed mutagenesis in hemoglobin: functional and structural study of the intersubunit hydrogen bond of threonine-38(C3) alpha at the alpha 1-beta 2 interface in human hemoglobin. Biochemistry 32, 13688−13695. (29) Safo, M. K., Burnett, J. C., Musayev, F. N., Nokuri, S., and Abraham, D. J. (2002) Structure of human carbonmonoxyhemoglobin at 2.16 Å: a snapshot of the allosteric transition. Acta Crystallogr., Sect. D: Biol. Crystallogr. D58, 2031−2037. (30) Kavanaugh, J. S., Weydert, J. A., Rogers, P. H., and Arnone, A. (1998) High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity Tstate. Biochemistry 37, 4358−4373. (31) Li, R., Nagai, Y., and Nagai, M. (2000) Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A. Chirality 12, 216−220. (32) Sasaki, J., Imamura, T., Yanase, T., Atha, D. H., Riggs, A., Bonaventura, J., and Bonaventura, C. (1978) Hemoglobin Hirose, a human hemoglobin variant with a substitution at the alpha1beta2 interface. Subunit dissociation and the equilibria and kinetics of ligand binding. J. Biol. Chem. 253, 87−94. (33) Manconi, B., De Rosa, M. C., Cappabianca, M. P., Olianas, A., Carelli, A. C., Mastropietro, F., Ponzini, D., Amato, A., and Pellegrini, M. (2010) A new beta-chain haemoglobin variant with increased oxygen affinity: Hb Roma [beta115(g17)Ala–>Val]. Biochim. Biophys. Acta 1800, 327−335. (34) Kilmartin, J. V. (1977) The Bohr effect of human hemoglobin. Trends Biochem. Sci. 2, 247−250. (35) Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) A biophysical investigation of recombinant hemoglobins with aromatic B10 mutations in the distal heme pockets. Biochemistry 44, 7207−7217. (36) Nichols, W. L., Zimm, B. H., and Ten Eyck, L. F. (1997) Conformation-invariant structures of the alpha1beta1 human hemoglobin dimer. J. Mol. Biol. 270, 598−615.

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