Recombinant Hemoglobin rHb0.1 with Cross-Linked Alpha Subunits Preferentially Crystallizes in the β₄ Oligomeric State, Potentially Driven by a βG18(H116I) Mutation

Abstract

Hemoglobin rHb0.1 is a recombinant version of the red blood cell transport protein carrying cross-linked α-subunits (V1M and G15A) and β-subunit mutations (V1M, G16A, and H116I), which is used as a construct to develop recombinant hemoglobin-based oxygen carriers (rHBOCs). Spectroscopic studies revealed the altered stability of rHb0.1 compared to heterotetrameric (α₂β₂) HbA. Heme dissociation kinetic analysis revealed nearly 2-fold faster rate constants (k _slow and k _fast) for rHb0.1 compared to HbA, with such differences necessitating structural studies for a greater insight into the variabilities. Here, we report the first crystal structure of the oxygen-bound β₄-rHb0.1 at 2.0 Å resolution, obtained from crystallization conditions designed for α₂β₂ rHb0.1, reflecting preferential crystallization of a preexisting β₄ population in solution. The overall quaternary architecture in the oxy-bound state closely resembles ferric, deoxygenated, and carbomonoxy β₄ HbA and liganded α₂β₂ R-state HbA. Unlike ferric β₄-HbA, the oxy-bound β₄-rHb0.1 structure prevents the formation of a disulfide bond between Cys112­(G14) residues of β1/β4 and β2/β3 subunits. A detailed comparison of the quaternary structures of oxy-bound (O₂-β₄) and ferric β₄ (PDB: 6FQF) showed minimal conformational changes upon ligand binding to the β₄ tetramer (RMSD across all 146 pairs: 0.714), demonstrating the remarkable structural conservation of β₄ tetramers in different redox states. Lower expression of the di-α subunit compared to the β-subunit in solution seems to have triggered β₄ homotetramer formation, as revealed by the crystal structure, with the H116I mutation in the β-subunit promoting this homotetramerization through extensive hydrophobic β-β interactions, offering insights into hemoglobin assembly, stability, and functional divergence from HbA.

1. Introduction

Vertebrate hemoglobin is a heterotetramer consisting of two α-globin and two β-globin polypeptide chains, each containing a heme group tightly bound within its globin pocket. The dynamic interactions and conformational changes between the α- and β-subunits enable cooperative oxygen binding, enabling hemoglobin to efficiently load oxygen in the lungs and release it in peripheral tissues. Structurally, the distal histidine (named E7) in both α- (His 58) and β-subunits (His 63) of HbA forms a hydrogen bond with the O₂ molecule bound to the heme. The heme-bound O₂ is further protected from solvent exposure by the distal His E7, which acts as a selective gate for the ligand. Balanced synthesis of α- and β-subunits is crucial for cooperative oxygen binding, structural stability, and normal physiological function. Disruption of the α- to β-subunit ratio (1:1) leads to abnormal oligomeric states, which are associated with hematological disorders such as thalassemias and unstable hemoglobinopathies. − In α-thalassemia, the formation of hemoglobin H (β₄ homotetramer) results from decreased production of α-globin chains. , The two globin chains differ markedly in their propensity to undergo self-association. β-chains tend to spontaneously form tetrameric units even at moderate concentrations (>1 mg/mL), whereas α-chains exhibit minimal or no aggregation. These differences together with imbalances in α- and β-globin synthesis contribute to hemoglobinopathies and underscore the need for recombinant technologies, which not only provide stable and well-defined hemoglobin variants for structural and functional studies but also enable the engineering of molecules to better understand and treat these disorders.

Recombinant hemoglobin-based oxygen carriers (rHBOCs) are developed from highly purified, cell-free human hemoglobin that has been expressed in heterologous host organisms such as transgenic bacteria, yeast, mice, or swine. These engineered Hbs provide a promising alternative to conventional blood transfusion therapies. − rHBOCs provide two major advantages over conventional blood-derived sources. First, the risk of viral or prion contamination is significantly reduced, as the production process excludes human or animal blood. Second, recombinant technology enables site-directed mutagenesis, allowing targeted modifications of hemoglobin properties, making HBOCs as promising oxygen carriers. Pioneering efforts by Somatogen and Nagai groups led to the development of a genetically fused di-α gene, placed within an operon alongside a β-globin gene, generating a tetrameric hemoglobin that remains intact and resists dissociation into α₁β₁ dimers under physiological conditions, even at low protein concentrations. This genetically cross-linked tetramer was termed rHb0.1, with “0.1” denoting a single glycine linker between the two α-subunits. ,, rHb0.1 served as the foundation for early rHBOC prototypes. The substitution of Val with Met at the N-termini of the α- and β-subunits in rHb0.0 reduced oxygen affinity, but the introduction of a peptide linkage between the two α-chains in rHb0.1 enhanced oxygen affinity while reducing cooperativity. Graves et al. compared the α- and β-subunits of sperm whale Hb and human adult Hb (HbA) to identify amino acids contributing to enhanced stability. Two notable differences were identified at positions αGly15 and βGly16, where sperm whale Hb contains alanine residues instead of glycine. Based on this finding, they engineered human recombinant Hb (rHb) variants bearing αGly15Ala and βGly16Ala mutations, both singly and in combination, and evaluated their effects on these substitutions on the structural stability and expression efficiency of the resulting holoproteins.

A complementary comparative strategy involved examining differences between the adult β-subunit and the γ-subunit found in fetal hemoglobin (HbF). HbF, which consists of two α- and two γ-chains, exhibits a conformation similar to HbA but demonstrates significantly greater resistance to acid, alkaline, and alcohol-induced denaturation. , Furthermore, the α₁γ₁ dimer dissociates three times more slowly than the α₁β₁ dimer, with an increased rate of holo-assembly. − Sequence alignment of the γ- and β-chains initially suggested that replacements such as γCys112Val and γTyr130Trp were responsible for the enhanced stability of HbF. However, functional studies revealed that these substitutions had limited effect on HbA stability. , The amino acid residues at the α1β1 interface of adult hemoglobin (HbA) differ from those at the α1γ1 interface of fetal hemoglobin (HbF) at four key positions: specifically, β51 Pro, β112 Cys, β116 His, and β125 Pro in HbA are replaced by γ51 Ala, γ112 Thr, γ116 Ile, and γ125 Glu in HbF. These substitutions likely enhance the structural stability of HbF and reduce its tendency to dissociate into monomers compared to HbA. Notably, their findings indicate that the presence of isoleucine at γ116, instead of histidine at the corresponding β116 position (G18), is crucial for the formation of a stable γ-chain dimer. Adachi et al. demonstrated that replacing His116 in the β-subunit with Ile in the γ-subunit increases the hydrophobicity at the α1γ1 interface, resulting in greater α1β1 interface stability. , As a result, the β His116-to-Ile mutation was incorporated into recombinant hemoglobin both in the presence and absence of Gly-to-Ala substitutions in the A helix.

Under physiological conditions, the formation of α₂β₂ tetramers is strongly favored, but β homotetramers (β₄) may form in α-deficient states or under certain engineered conditions. Notably, mutations within the β-chain can alter subunit interfaces and oligomerization behavior. In particular, the H116I mutation in the β-chain has been reported to promote β₄ tetramer formation.

In the present study, rHb0.1 was crystallized under high-salt conditions (1.2 M trisodium citrate, pH 8.8) with the expectation of obtaining an α₂β₂-like heterotetrameric structure. Surprisingly, the X-ray structure revealed a β₄ homotetramer, despite SDS-PAGE and other analyses confirming the presence of both α- and β-chains in solution. This novel finding provides a unique opportunity to investigate the structural and stability features of β₄ hemoglobin and to compare them with those of native HbA, offering insights into hemoglobin assembly mechanisms and the pathophysiology of β-rich states. The crystal structure of rHb0.1 was resolved at 2.0 Å resolution and compared with the HbA structure (PDB ID: 2DN1) to examine the role of the G-18 residue in stabilizing the α1β1 and β1β2 interfaces in α₂β₂ and β₄ tetramers, respectively, as well as its contribution to the assembly of α and non-α chains. This also enables a complete crystallographic analysis of oxy-bound β₄ Hb, which holds considerable medical importance due to its occurrence in the RBCs of millions of individuals affected by α-thalassemia worldwide. The formation of this protein reflects complex dissociation and reassociation events, offering valuable insights into subunit interactions, thereby explaining the molecular basis of β-rich states observed in α-thalassemia.

2. Experimental Procedures

2.1. Cloning, Expression and Purification

The gene encoding recombinant human hemoglobin wild type (rHb0.1), subcloned into pET28a vector using two restriction enzymes namely, BamHI and HindIII, was kindly provided by Prof. John S. Olson (Rice University, USA). The construct includes di-α gene (V1M, G15A) covalently linked through a glycine linker and a single β gene (V1M, G16A, H116I) carrying an N-terminal 6X His tag. The pET28a plasmid with the rHb0.1 gene was confirmed through DNA Sanger sequencing. The recombinant rHb0.1 plasmid was used to express the rHb0.1 protein using BL21 DE3 cells.

For protein expression, the cells were grown in Terrific Broth (TB) (HiMedia, Mumbai, India) media at 37 °C with shaking at 200 rpm until the optical density at 600 nm reached around 0.6–0.8. , The culture was then induced at 30 °C without IPTG for induction and allowed to grow for 16–18 h at 150 rpm in a shaker incubator (Eppendorf, New Brunswick Innova 44, Swedesboro, New Jersey, USA). , Cells were pelleted by centrifugation at 7000g for 10 min at 4 °C and resuspended in lysis buffer containing 0.1 M potassium phosphate (pH 7.4), 0.1 M NaCl, and 1 mM PMSF (BioBasic Inc., Konrad Cr, Unionville, Canada). , DNase (30 μg per g of cell pellet) and lysozyme (300 μg per g of cell pellet) from BioBasic Inc.(Konrad Crescent, Unionville, Canada) were then added to the lysed pellet followed by incubation at room temperature for 30 min at 150 rpm. ,

The resuspended cells were then lysed using sonicator W-385 Heat Systems (Ultrasonics Inc., St. Louis, MO, USA), and cellular debris was removed by centrifugation at 12000 x g for 45 min at 4 °C. ^26,27 The Nickel-Sepharose (GE Healthcare, Little Chaltfont, United Kingdom) beads were first equilibrated with a binding buffer containing 0.1 M potassium phosphate (pH 7.4), 0.1 M NaCl, and 10 mM imidazole. The clarified lysate was then applied to the beads to allow protein binding. − The flow-through was collected, and beads were subsequently washed with the same binding buffer. The recombinant rHb0.1 protein was then eluted using 50–100 mM imidazole (Hi-Media, Mumbai, India) prepared in the binding buffer. The eluted fractions were concentrated and further purified by size exclusion chromatography using a Superdex 75 16/600 GL (GE Healthcare Life Sciences, Buckinghamshire, UK) column equilibrated with buffer containing 0.1 M potassium phosphate and 0.1 M NaCl (pH 7.4). Protein purity was assessed by measuring the Abs_Soret/Abs_280 nm absorbance ratio, with values of 3.5 or higher indicating high purity. , This was further confirmed by 15% SDS-PAGE followed by staining with Coomassie brilliant blue R250. , Amicon ultrafiltertion units with a 10 kDa molecular weight cutoff was used to concentrate the pooled pure fractions of rHb0.1 for subsequent spectroscopic and biochemical analyses and for protein crystallization.

2.2. Reverse-Phase HPLC

Reverse-phase chromatography was performed with 300 × 4.6 mm Kinetex C-18 (Phenomenex Inc., Torrance, USA) column on a Waters HPLC system (Waters, Milford, MA). Samples were analyzed by injecting 50 μg of protein onto the column. The method involved two buffer solvents; solvent A containing 0.1% trifluoroacetic acid (Thomas Baker Chemicals Pvt. Ltd., Mumbai, India) in water and solvent B containing 0.1% TFA in acetonitrile (Thermo Fisher Scientific India Pvt. Ltd., Maharashtra, India). The mobile phase was run at a flow rate of 0.3 mL/min. Solvent A was used to equilibrate the column for 5 min. The protein was subsequently eluted using a linear gradient of solvent B, increasing from 40% to 55% acetonitrile consisting of 0.1% trifluoroacetic acid for 40 min. , For cleaning the column, the proportion of solvent B was increased to 90% linearly for 3 min and then decreased to 10% over a 1 min interval. The spectra of Hb were observed at 280 nm. The peaks were analyzed utilizing Empower 2.0 software from Waters (Milford, MA, USA). This was carried out to compare alpha and beta subunits of both HbA and rHb0.1.

2.3. Spectroscopic Characterization of rHb0.1

• a) UV–visible absorbance spectrophotometry. UV–vis spectra were recorded across a wavelength range of 260–700 nm using a Cary Varian Bio100 UV–vis spectrophotometer (Varian Inc., Palo Alto, California, USA). Approximately 0.1 mg/mL of protein was analyzed in 0.1 M phosphate buffer (pH 7.0) at room temperature. The native, folded, and ligand-bound states of hemoglobin were assessed on the basis of their spectral profiles. Before crystallization trials were set up, rHb0.1 was assessed by UV–vis absorbance spectroscopy to verify the quality, oxidation state, and ligand-binding status. Oxidized (Fe³⁺) forms were generated separately, while oxygenated (Fe²⁺-O₂) forms were obtained by dithionite reduction of the ferric protein followed by desalting through a G-25 column, which allows reoxygenation to the Fe²⁺-O₂ state.
• b) Fluorescence spectroscopy. Fluorescence spectra were recorded over a wavelength range of 300–440 nm to determine the intrinsic fluorescence due to tryptophan residues present in rHb0.1 and were compared with those of HbA using a Cary Eclipse spectrofluorometer (Varian Inc., Palo Alto, California, USA) attached to a Peltier temperature controller. ∼0.1 mg/mL of protein was prepared in 0.1 M of phosphate buffer (pH 7.4). Buffer was taken as a blank. Both excitation and emission slit widths were set to 5 nm. The protein was excited at 292 nm, and the data represent the mean of three independent measurements.
• c)
  Circular dichroism spectroscopy. CD spectroscopy was used to investigate the secondary structure of rHb0.1 in the far-UV region from 190 to 260 nm and compared with HbA using JASCO 815 spectropolarimeter (Jasco Corporation, Tokyo, Japan) equipped with the Peltier thermostat system. For almost all experiments, ∼4 μM of protein was used (in 100 mM phosphate buffer pH 7.4) with a 1 mm path length quartz cell. A scan rate of 100 nm/min was used for all CD measurements. The data were reported as the mean residue ellipticity (MRE in °cm²/dmol), which is defined as ,

  $$\mathrm{M}\mathrm{R}\mathrm{E}={\theta }_{\mathrm{o}\mathrm{b}\mathrm{s}}(\mathrm{m}\mathrm{d}\mathrm{e}\mathrm{g})/10\times \mathrm{n}\times \mathrm{C}\mathrm{p}\times \mathrm{l}$$ 1

where θ_obs is the observed ellipticity in degrees (°), C_p is the molar concentration, n is the number of residues, and “l” is the path length of the cuvette in cm. The α-helical content of the protein was determined from the MRE value at 222 nm. , The Percentage of different secondary structures including α-helix and β-sheet) were estimated using K2D3 software (https://cbdm-01.zdv.unimainz.de/~andrade/k2d3/mainz.de/~andrade/k2d3/). ,

2.4. Heme Dissociation Kinetics of rHb0.1 and HbA

Heme dissociation kinetics of rHb0.1 protein was carried out following the protocol reported earlier. ,, Briefly, ferric rHb0.1 holoprotein (3 μM) was incubated with 30 μM H64Y/V68F apo-myoglobin in 0.1 M potassium phosphate buffer, pH 7.4 containing 0.45 M sucrose. , Heme transfer was monitored spectrophotometrically by measuring the change in absorbance at Soret peak (410 nm) as “green” holo-metmyoglobin (H64Y/V68F), which exhibits a characteristic absorbance at 600 nm, was formed. , The assay was performed at 37 °C for 5 h. Normalized absorbance values were calculated using the ratio of A_{Soret peak}/A₆₀₀ and plotted as a function of time (min). , The obtained data were analyzed by fitting to a two-phase exponential model using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA) and are presented as the mean values from three independent experiments.

2.5. Hydrogen Peroxide-Mediated Oxidation

The oxidation of ferrous Hbs (40 μM) with a molar excess H₂O₂ (400 μM) was monitored using a Cary Varian Bio100 UV–vis spectrophotometer (Varian Inc., Palo Alto, California, USA). , The Hb samples were incubated with H₂O₂ for 5, 10, 15, and 20 min, after which catalase (200 units/ml) was added for 1 min to ensure the complete decomposition of residual H₂O₂. , Formation of ferryl Hb was further assessed using a previously described method. Briefly, 2 mM sodium sulfide (Na₂S) was added to the reaction mixture to convert ferryl Hb into sulfhemoglobin (sulfHb). ,− Fresh Na₂S stock solutions were prepared for each experiment. , The amount of ferryl Hb formed was quantified by measuring the absorbance of sulfHb at 620 nm using its corresponding extinction coefficient (ε_620 nm = 20.8 mM^–1). ,,,

2.6. Kinetics of Ferryl Hemoglobin Formation and Decay

In a separate set of experiments, ferryl Hb was generated by adding 10 eq of H₂O₂ to metHb (40 μM) in 50 mM phosphate buffer (pH 7.4) to ensure maximal formation of the ferryl heme species. ,, After a reaction time of 2 min, excess catalase (200 units/ml) was introduced to terminate the reaction and eliminate residual H₂O₂, thereby preventing heme bleaching. The decay of the ferryl species back to metHb was monitored at room temperature (25 °C). Absorbance spectra were recorded between 500 and 700 nm for 1 h at 30 s intervals. , Changes in absorbance were followed by measuring the decrease at 542 nm, and the resulting time courses for ferryl Hb reduction were fitted to a single exponential function using GraphPad Prism 8.4.3. In contrast, the kinetics of metHb formation were analyzed by fitting the increase in absorbance at 630 nm to a rectangular hyperbolic function.

2.7. Crystallization, Data Collection, and Structure Determination

The oxy-bound form of rHb0.1 was concentrated to nearly 15 mg/mL and prepared for crystallization trials. Crystals of rHb0.1 were obtained using the sitting drop vapor diffusion method at 293 K. Diffraction-quality crystals were obtained in a solution of 1.2 M trisodium citrate, pH 8.8, and 15% PEG 8000. Unexpectedly, during attempts to crystallize the recombinant human hemoglobin rHb0.1 α₂ (glycine-linked) β₂ protein, the crystals of β₄ homotetramer Hb, reported in the present study, were obtained. Crystals were obtained across a broad pH range (4.5–8.8), with the block-shaped crystals formed at higher pH identified as the Hb β₄ homotetramer. Data collection was performed at 100 K, and the crystals were cryoprotected in a 20% ethylene glycol solution. X-ray diffraction data for the oxy-bound rHb0.1 were collected at the Molecular Crystallographic Unit (MCU) at the Institute Instrumentation Centre (IIC), IIT Roorkee. Diffraction data were collected to a resolution of 2.0 Å. The crystals belonged to the space group P 1 2₁ 1. Initial phases for rHb0.1 were obtained by molecular replacement using MOLREP within the CCP4i suite, with the homologous structure of the deoxy rHb1.1 (PDB ID: 1ABW) serving as the template. Refinement was performed iteratively using REFMAC, while model building and adjustments were carried out in COOT. In the final stages of refinement (including TLS refinement), 25 water molecules were incorporated into the model based on features observed in the Fourier electron density maps. The refined coordinates were deposited in the Protein Data Bank under the accession code 9WW1. Pymol, Chimera, and Ligplot were used for structural analysis and preparation of images.

2.8. Ellman’s Assay to Estimate Free Thiols in rHb0.1

A 4 mg/mL stock solution of DTNB (5,5′-dithio-bis­(2-nitrobenzoic acid) (Sigma-Aldrich, St. Louis, MO, USA) was prepared by dissolving DTNB in 0.1 M sodium phosphate buffer (pH 8.0) and stored in foil-wrapped tubes to protect it from light exposure. A stock solution of l-cysteine hydrochloride monohydrate was prepared at 3.0 mM concentration by dissolving in 0.1 M sodium phosphate buffer (pH 8.0) to prepare the standard curve with different concentrations of cysteine (0, 15, 30, 50, and 100 μM). To check whether the formation of disulfide bonds is dependent on the oxidation state of iron, both oxy-bound and ferric states of rHb0.1 at 15 μM concentration were incubated in 0.1 M sodium phosphate buffer (pH 8.0). Subsequently, 6.5 μL of DTNB was added to obtain a final reaction volume of 368 μL, and the mixture was incubated at room temperature for 15 min. The absorbance of the DTNB- Cys complex was measured at 450 nm in 96-well plate using SpectraMax iD3 (Molecular Devices, LLC., 3860 N First Street San Jose, USA). The intrinsic absorbance of hemoglobin at 450 nm was used as the blank and subtracted from the total absorbance to obtain the net absorbance of the DTNB-thiol (TNB) complex. Monitoring the reaction at 412 nm was not feasible due to the intense heme absorbance in the Soret region that interferes with the signal. ,

3. Results

3.1. Recombinant rHb0.1 Hemoglobin Expresses α- and β-Subunit in Disproportionate Stoichiometry

The 6×-His tagged rHb0.1 protein was expressed under the control of T7 promoter and purified in soluble form. The expressed protein consists of one glycine-linked di-α subunit and two β-subunits each carrying a V1M mutation. In addition, rHb0.1 contains stabilizing mutations di-α­(G15A) and β­(G15A/H116I), which enhance apoglobin stability and facilitate its expression inE. coli. On SDS-PAGE, the purified rHb0.1 protein migrated with apparent molecular masses of ∼16 kDa for β-chain and ∼32 kDa for di-α-chain, consistent with their predicted molecular weights. The presence of both di-α and β chains in rHb0.1 was detected using MS/MS analysis as peptide mass fingerprinting (Supplementary Section S3). Moreover, the expression of β-subunit was 4-fold higher than that of the α-subunit, as evident by the SDS-PAGE gel (Figure A). The SEC profile shows that rHb0.1 elutes at around 70 mL similar to native HbA whose molecular weight is 64 kDa (Figure B).

1.

(A) 15% SDS-PAGE profile of rHb0.1 following size-exclusion chromatography, where F denotes the elution fraction numbers. (B) Size-exclusion chromatography using Superdex 75 10/300 column shows that rHb0.1 adopts a tetrameric quaternary structure, comparable to native HbA, which has a molecular weight of approximately 64 kDa.

The reversed-phase HPLC analysis of HbA and rHb0.1 reveals distinct differences in the expression levels of both α- and β-subunits. Chromatograms were recorded by monitoring absorbance at 280 nm for protein and 360 nm for free heme species. In HbA, well-resolved peaks corresponding to α- and β-chains were observed with an equal ratio of α- and β-chains. In contrast, the rHb0.1 profile shows a more intense β peak compared to di-α chains, consistent with the overexpression of β-chains (Figure ). These findings corroborate the SDS-PAGE gel profile and confirm the differential expression of di-α and β-subunits.

2.

Reversed-phase HPLC analysis of purified HbA and rHb0.1 using Kinetex C18 column. (A) and (B) The elution profiles of the HbA and rHb0.1 proteins, respectively, were monitored at 280 nm. The release of free heme moiety was tracked at 360 nm, while protein elution was simultaneously followed at 280 nm.

3.2. Absorbance Spectra of rHb0.1 Revealed Structural and Functional Integrity of the Heme Environment

The UV–visible absorbance spectra of Hbs are highly informative regarding their oxidation state, ligand binding, iron coordination, and overall structural integrity of the proteins. Spectra were recorded over the wavelength range of 260–700 nm. , All spectra exhibited features characteristic of Hb, confirming that the purified protein was a globin containing a heme prosthetic group. At pH 7.0, rHb0.1 in ferric (Fe III⁺) or oxidized state displayed a characteristic Soret peak at 406 nm with a Q-band at 539 and 630 nm, whereas HbA showed similar Q-bands with a Soret maximum at 405 nm. The reduced form of rHb0.1 exhibited a Soret peak at 428 nm and a Q-band at 558 nm, along with a low-intensity broad peak near 630 nm, indicating the presence of a minor high-spin Fe³⁺ population (Figure A); whereas HbA showed a Soret peak at 429 nm and a Q-band at 556 nm. A characteristic red shift of the Soret wavelength (414 nm) and splitting in Q-bands (540 and 575 nm) upon binding with oxygen indicated that rHb0.1 was functionally active. HbA showed similar spectral features with a Soret peak at 413 nm and Q-band splitting (540 and 575 nm) (Figure B). Together, these spectral properties confirm that rHb0.1 maintains the structural integrity of the heme environment and retains an oxygen-binding ability comparable to that of native HbA.

3.

Spectroscopic characterization of rHb0.1 and HbA at pH 7.0. (A) and (B) Full spectrum showing characteristic Soret and Q-bands corresponding to different redox and ligand-bound forms. Inset figure shows a magnified view from 500 to 700 nm, highlighting shifts in the Q-bands. The presence of a single Q-band at 558 nm in the reduced, ligand-free form suggests a pentacoordinate heme iron environment within the globin, similar to HbA. (C) The intrinsic tryptophan fluorescence spectrum of rHb0.1 exhibited an emission maximum at 338 nm upon excitation at 292 nm, indicating that the Trp residues are located in a hydrophobic, buried environment, consistent with a properly folded protein. (D) rHb0.1 displayed a spectrum characteristic of a predominantly α-helical conformation. The far-UV CD spectra of rHb0.1 and HbA showed distinct negative minima at 208 and 222 nm, confirming the presence of significant α-helical secondary structure.

3.3. Trp Fluorescence and CD Spectroscopy Revealed Apolar Environment Consistent with Folded Protein Structure and α-Helical Structure Similar to Native HbA

Intrinsic tryptophan fluorescence spectroscopy is a reliable method for monitoring alterations in the immediate surroundings of aromatic amino acids like Tyr, Trp, and Phe. It offers insights into the naturally folded tertiary structure of the polypeptide. When we employed intrinsic fluorescence with specific excitation at 292 nm for both rHb0.1 and HbA, we observed emission peaks at 333 and 338 nm, respectively (Figure C). These findings suggest that the six tryptophan (Trp) residues are largely buried within the hydrophobic core of the tightly packed globin structure.

Purified rHb0.1 exhibited characteristic CD minima at 208 and 222 nm, confirming its α-helical conformation. Similarly, it exhibited a CD spectrum comparable to native HbA, which is predominantly α-helical (Figure D). The secondary structure content was estimated from the far-UV CD spectrum using K2D3 software. At 25 °C, the CD spectrum of rHb0.1 indicated 67.35% α-helix and 9.97% β-sheet, which was comparable to HbA, showing 67.79% α-helix and 9.97% β-sheet (Table S1). Three-dimensional structures of HbA show that rHb0.1 preserves an α-helical secondary structure with negligible β-sheets, consistent with native hemoglobins.

3.4. rHb0.1 Exhibited Faster Heme Dissociation Rates Compared to HbA

Monitoring the kinetics of heme release provides valuable insights into the Hb stability in vitro, which in turn reflects their functional properties. For rHb0.1 and HbA, time-dependent hemin dissociation from the met form was recorded at 37 °C over a 5-h period, as longer incubations often led to distorted absorbance profiles due to precipitation of apoHb. Biphasic hemin dissociation kinetics were observed in both proteins, indicating the contributions of the two distinct subunits (α and β). The fast phase corresponds to hemin release from ferric β-subunits, whereas the slower phase is associated with hemin release from the α-subunits. , Interestingly, rHb0.1 exhibited faster dissociation kinetics, with rate constants of 0.96 h^–1 for the α-chain and 16.2 h^–1 for the β-chain, as reported in our recent study. On the other hand, HbA exhibited lower rate constants of 0.54 h^–1 for the α-chain and 8.54 h^–1 for the β-chain (Figure A and B, Table ). This nearly 2-fold increase in the heme dissociation rate of both α- and β-chains in rHb0.1 compared to HbA is unexpected since this construct carries a di-alpha glycine linker, which prevents αβ dimer dissociation and hence is expected to lower the heme dissociation kinetics. These findings prompted further structural investigation to determine whether the observed kinetics arise from altered subunit assembly, which we addressed through crystallographic analysis. Accordingly, we now interpret the heme dissociation data as reflecting ensemble-averaged behavior arising from the coexistence of α₂β₂ and β₄ species in solution. Importantly, the comparative conclusion that rHb0.1 exhibits faster heme dissociation than HbA remains valid, as HbA exists exclusively as an α₂β₂ heterotetramer under the same experimental conditions, whereas rHb0.1 populates additional β-rich assemblies that are known to exhibit enhanced heme dissociation.

4.

Heme dissociation kinetics of HbA (black) and rHb0.1 (blue) in 0.1 M phosphate buffer at pH 7.4, 37 °C. (A) MetHb (“brown”) at 3 μM was incubated with 30 μM H64Y/V68F apoMb, which exhibits a “green” spectral shift upon hemin binding. A 10-fold molar excess of the scavenger protein (H64Y/V68F apoMb) was used to ensure that the measured kinetics primarily reflected heme release from the ferric hemoglobin. Importantly, hemin release from both the α- and β-subunits of rHb0.1 was found to be approximately two times faster than that from the corresponding subunits of native HbA. To monitor this process, the absorbance ratio (A_Soret peak/A_600 nm) was normalized and plotted as a function of time. Each trace represents the average of three independent experiments, with standard deviations shown as error bars. The curves were fitted using a biphasic exponential model, where the fast phase corresponds to hemin dissociation from β-subunits and the slower phase reflects dissociation from α-subunits. (B) Bar graphs representing the average of heme transfer rate (h^–1) from both the α- and β-subunits of both rHb0.1 and HbA.

1. Heme Dissociation Rates of HbA and rHb0.1 (N = 3).

Protein	kfast(h–1)	kslow (h–1)
HbA	8.5 ± 0.51	0.54 ± 0.06
rHb0.1	16.8 ± 0.013	0.96 ± 0.0005

3.5. rHb0.1 and HbA Show Similar Susceptibility to H₂O₂-Mediated Ferryl and SulfHb Formation

Reactions of rHb0.1 and HbA with hydrogen peroxide (H₂O₂) were examined to evaluate their susceptibility to oxidative stress and associated spectral alterations. Both proteins were treated with 10 mol equiv of H₂O₂ (heme:H₂O₂ = 1:10), and the spectral changes were recorded over time (Figure A,B). The initial spectra represent oxyHb, characterized by well-defined absorption maxima at 541 and 576 nm. Upon incubation with H₂O₂ for 5, 10, 15, and 20 min, spectral features corresponding to ferrylHb were observed. FerrylHb could subsequently be derivatized to sulfHb (characterized by a peak near 620 nm) by the addition of Na₂S, before autoreducing back to the ferric state. Importantly, as shown in both figures, HbA and rHb0.1 displayed comparable spectral transitions, and the magnitude of sulfHb formation under the same experimental conditions was nearly identical, indicating that both proteins are equally prone to H₂O₂-induced ferryl formation and subsequent sulfHb conversion (Figure C).

5.

H₂O₂-induced oxidation of Hbs. (A) and (B) Absorbance spectra of HbA and rHb0.1, respectively, recorded following treatment of the ferrous Hb with 10 equiv of H₂O₂. The black solid traces represent the spectra of the untreated ferrous HbA and rHb0.1, respectively. Formation of SulfHb was assessed immediately after the addition of 2 mM sodium sulfide (Na₂S), which produces a characteristic absorbance peak at 620 nm in oxidized samples. (C) The amount of sulfHb generated upon exposure to a 1:10 heme:H₂O₂ ratio was quantified calculated using an extinction coefficient, ε_620 nm = 20.8 mM^–1. Data are presented as mean ± SD from three independent experiments. Statistical comparisons between HbA and rHb0.1 were performed using an unpaired t-test, indicating no significant difference (p > 0.05, not significant, ns).

3.6. Comparative Kinetics of Ferryl Decay Reveal Slower Autoreduction in rHb0.1

To assess potential differences in autoreduction rates, we monitored the decay of ferryl species for both HbA and rHb0.1 to their ferric states by following absorbance changes at 542 nm. As shown in Figure A and B, a typical transition from ferric to ferryl and back to ferric for HbA and rHb0.1 is depicted. Under these conditions, the ferryl species in rHb0.1 decayed more slowly than in HbA, consistent with a lower autoreduction rate constant for the recombinant protein. , The decay kinetics of ferryl rHb0.1 were fitted to a single exponential expression with a rate constant equal to 1.08 h^–1, compared with 2.16 h^–1 for ferryl HbA, indicating that ferryl rHb0.1 decays almost 2 times more slowly than ferryl HbA (Table and Figure C). As shown in Figure D, the kinetics of metHb formation from the ferryl intermediate were measured by following the time-dependent increase in absorbance at 630 nm for both rHb0.1 and HbA. The time courses for both proteins were well described by a rectangular hyperbolic function, consistent with a saturating exponential rise. The derived rate constants were 1.44 h^–1 for rHb0.1 and 3.96 h^–1 for HbA (Table ), indicating that rHb0.1 form ferric Hb at a significantly slower rate (2.7-fold) compared to HbA. These results demonstrate that the autoreduction of ferryl species to metHb is kinetically less favorable in rHb0.1, in agreement with the slower decay profile. The results show that rHb0.1 exhibits slower ferryl-to-metHb autoreduction, prolonging ferryl persistence and oxidative stress potential.

6.

Autoreduction kinetics of ferryl HbA and rHb0.1 to the ferric forms. (A) and (B) Time-resolved absorbance spectra acquired during the autoreduction of HbA and rHb0.1, respectively. Ferryl species were generated by reacting 40 μM (heme) hemoglobin with 400 μM H₂O₂ in 50 mM phosphate buffer (pH 7.4) at room temperature. Spectral changes were monitored using a Cary Varian Bio100 UV–vis spectrophotometer with spectra collected every 30 s over 1 h in the 500–700 nm range. The spectra measured at time 0 () and after 1 h of incubation (), and the ferric state (-·-) are shown for comparison. (C) The decay kinetics of ferryl HbA (black) and ferryl rHb0.1 (blue) to their corresponding ferric forms were followed at 542 nm and fitted to a single exponential curve. (D) The formation of ferric species for HbA (black) and ferric rHb0.1 (blue) was monitored by tracking the increase in absorbance at 630 nm.

2. Rate Constants for Ferryl Hb Decay and Ferric Formation Measured in Phosphate Buffer (N = 3).

Sample	Ferryl decay rate constant(h–1)	Ferric formation rate constant(h–1)
HbA	2.16 ± 0.83	3.96 ± 0.36
rHb0.1	1.08 ± 0.54	1.44 ± 0.72

3.7. X-ray Structure Revealed That rHb0.1 Crystallized as β₄-Homotetramer

The rHb0.1 crystallized as red, brick-shaped crystals that diffracted to a resolution of 2.0 Å (Figure A and B). It turns out that the rHb0.1 protein crystallized as β₄-homotetramer instead of α₂β₂ heterotetramer. The crystal structure of β₄-rHb0.1 was refined to an R-factor of 20.7% (R_free= 23.7%) using molecular replacement with the 2.0 Å structure of rHb1.1 (PDB: 1ABW) as the search model. The oxy-bound heme was modeled and refined at full occupancy within the heme pocket surrounded by His 63 and His 92, consistent with the observed electron density (Figure C). Ramachandran analysis of main-chain torsion angles (ψ, φ) shows that 98.96% of residues in O₂-β₄ occupy the most favored regions, with only 0.35% of residues located in disallowed regions (Table ). The heme moieties and ligand densities are distinctly resolved for all β-subunits. The electron density is clearly resolved for both the backbone and side chains of most residues within the β-subunits. The solved crystal structure revealed a correctly folded β₄ homotetramer with well-ordered heme incorporation (Figure D). Surface analysis of the tetramer highlighted the significance of residue Ile116, positioned at the dimer–dimer interfaces (β1-β2 and β3-β4), where it contributes to intermolecular contacts critical for structural stabilization (Figure E-F). These findings suggest that Ile116 plays a key role in maintaining tetrameric integrity, thereby supporting the structural and functional assembly of β₄ hemoglobin.

7.

Crystallization and structural features of the β₄ hemoglobin tetramer. (A) Representative crystal of the β₄ tetramer grown under optimized crystallization conditions (1.2 M trisodium citrate, pH 8.8, 15% PEG 8000). (B) X-ray diffraction pattern obtained from β₄ crystals, demonstrating well-ordered diffraction suitable for structural analysis at a resolution of 2.0 Å. (C) The Fo-Fc omit map (contoured at 3σ) at 2.0 Å resolution, highlighting the oxygen molecule within the heme pocket. (D) Solved crystal structure of oxy-bound β₄ homotetramer. (E) and (F) Surface representations of the hemoglobin tetramer highlighting the β1-β2 (left) and β3-β4 (right) subunit interfaces, respectively. The critical residue Ile116, located at the dimer–dimer interface, is shown in yellow. Dashed lines indicate the plane of subunit interaction.

3. Summary of Data Collection and Refinement Statistics for Oxy-Bound β₄ rHb0.1.

Crystal	β4 oxy bound rHb0.1
Data collection
Resolution range	22.49–2.0 (2.071–2.0)
Unit cell dimensions a, b, c (Å)	53.80, 80.94, 62.68
α, β, γ	90, 90.39, 90
Space group	P 1 21 1
Completeness (%)	99.84 (99.56)
Rmerge	0.06 (0.60)
Rmeas	0.07 (0.74)
R pim	0.03 (0.42)
CC 1/2	1.00 (0.61)
Mean I/sigma I	16.45 (1.65)
Wilson B-factor	23.08
Refinement
Reflections used in refinement	36345 (3621)
Reflections used for Rfree	1817 (166)
Rwork	0.20 (0.29)
Rfree	0.24 (0.33)
Number of non-hydrogen atoms	4765
Macromolecules	4540
Ligands	200
Solvent	25
Protein residues	584
RMS (bonds) (Å)	0.012
RMS (angles) (°)	1.95
Favored (%)	98.96
Allowed (%)	0.69
Outliers (%)	0.35
Average B factor (Å2)	20.32
Macromolecules (Å2)	20.26
Ligands (Å2)	20.97
Solvents (Å2)	26.19

^aValues given in parentheses correspond to the highest resolution shell.

^bRwork was calculated as 100 × Σhkl ||F_o| – |F_c| |/Σhkl |F_o|, where F_o and F_c represent the observed and calculated structure factors, respectively.

^cThe R_free is determined using a test set comprising 5% of reflections that are excluded from the refinement process.

^dRMS stands root-mean-square.

Molecular interaction maps highlighted the differences in subunit interfaces between the rHb0.1 β₄ tetramer and native α₂β₂ HbA (Figure A and B). In the β₄ tetramer, residue Ile116 (β-subunit) is central to stabilizing the β-β interface through multiple hydrophobic contacts and hydrogen-bonding networks, involving neighboring residues such as Cys112, Ala115, Gly119, and Pro125 (Figure A). In contrast, the α2β2 interface of HbA features His116 (β-subunit), which participates in hydrogen bonding and polar interactions with α-subunit residues, including Gln131, Gln 127, Phe122, and Arg30, alongside hydrophobic contacts that maintain quaternary stability (Figure B). These distinct interfacial residues (Ile116 vs His116) illustrate how sequence differences reshape subunit packing and interfacial stabilization, potentially influencing cooperative function and structural assembly.

8.

Interaction analysis maps of β-subunit interfaces in hemoglobin. (A) and (B) Schematic diagrams of interfacial interactions for β1-β2 (β₄ rHb0.1) and α1-β1 (PDB: 2DN1) contacts. Hydrogen bonds are depicted as green dashed lines, whereas hydrophobic interactions are shown as red arcs. The diagrams illustrate the distinct sets of hydrogen bonding and van der Waals interactions that stabilize the two interfaces, emphasizing the central role of Ile116 and surrounding residues in subunit packing and interfacial stability.

It is demonstrated that the tertiary and quaternary structures of oxy-β₄ rHb0.1 are very similar to those of the liganded α₂β₂ R-state tetramer (PDB: 2DN1) and ferric-β₄ (6FQF). All four crystallographically independent chains possess ferrous heme iron­(II) exhibiting exogenous six-coordinate, low-spin geometry, without any evidence of a bis-histidyl coordination (Figure ). Consistently, the structural arrangement of the heme pockets does not support endogenous coordination, which would require the Cα-Cα distance between His63 (E7) and His92 (F8) to be less than 13 Å (Table . ,, The oxy-bound form of our recombinant hemoglobin mutant exhibits a Cα–Cα distance of ∼14 Å between the distal histidine (HisE7) and the proximal histidine (HisF8), which is consistent with the exogenous coordination typically observed in native oxy-hemoglobin (Figure ). This distance supports a coordination mode in which the Fe­(II) is ligated by HisF8 and molecular oxygen, with HisE7 positioned to stabilize the bound O₂ through hydrogen bonding rather than direct coordination. As summarized in Table , the Fe–O(1) bond distance between the bound oxygen and the heme iron across all four subunits is consistent with that reported for the oxy form of human hemoglobin (∼1.87 Å). , Furthermore, analysis of the heme iron–iron distances in oxy-β₄-HbA (Figure and Table ) reveals values comparable to those reported in the CO-bound β₄ and deoxy-β₄ crystal structures. ,, The relative orientation of the bound ligand and the distal histidine varies slightly among the four subunits. The distance between the O₂ molecule and the Nε atom of the distal histidine is 2.67 Å in β1, 3.08 Å in β2, 2.88 Å in β3, and 2.74 Å in β4. In contrast to the α₂β₂ tetramer, the β₄ tetramer exhibits high oxygen affinity and lacks cooperative oxygen binding. , As anticipated, the overall structure of oxy-β₄ rHb0.1 closely resembles that of the oxy-HbA (PDB: 2DN1). Indeed, when comparing oxy-β₄ with the β-subunit of HbA (PDB ID: 2DN1), the root-mean-square deviation (RMSD) for the backbone atoms is 0.494 Å. Distinct differences are observed in the side-chain orientations of residues F41, F42, K66, and L141, together with altered positioning of the heme propionate groups relative to HbA. Although the distance between K66 and the bound O₂ differs slightly between rHb0.1 (8.8 Å) and HbA (9.6 Å), this separation is too large for K66 to exert a direct electrostatic influence on ligand binding. Instead, the more relevant structural feature is the hydrogen-bonding interaction between the bound O₂ molecule and the distal His63. In rHb0.1, the O₂–His63 distance in β1 subunit is shortened to 2.67 Å compared with 3.02 Å in HbA, indicating a stronger hydrogen bond, which may contribute to the observed difference in oxygen affinity (Figure ). The structural comparison of rHb0.1 oxy-bound β₄ homotetramer (yellow) with native HbA or ferric β₄ hemoglobin (pink, PDB: 6FQF) shows that the overall fold is well conserved, with only subtle conformational differences. The two structures exhibit a high level of similarity, as evidenced by a backbone root-mean-square deviation (RMSD) of 0.714 Å obtained upon superposition. However, closer inspection of the heme pocket highlights minor positional shifts in key residues such as Lys66 and Leu141 along with propionate group of heme (Figure A and B). These small changes may influence heme stabilization, ligand binding, and oxygen affinity. Importantly, the proximal histidine (His92) remains coordinated to the heme iron, while the distal histidine (His63) is positioned to regulate O₂ binding, consistent with functional hemoglobin.

9.

Coordination environment of the heme iron in the four β subunits of the oxy-β₄-rHb0.1 hemoglobin. Structural representations of the heme (indigo) and its proximal and distal interactions in each β subunit (β1-β4). Key coordinating residues are shown in pink, with hydrogen-bonding and Fe-ligand interactions indicated as dashed lines along with bond distances (Å). The distal His 63 (E7) and proximal His 92 (F8) interactions are conserved across all subunits, with minor variations in bond lengths that may contribute to subtle differences in heme stabilization and ligand-binding properties.

4. Structural Parameters and Heme Coordination Distances in the β Subunits of Oxy-β₄-rHb0.1.

Chains	O2---Fe(II)	O2---Nε2(HisE7)	Fe(II)--- Nε2(HisF8)	Cα(HisF8)---Cα(HisE7)
β1	1.86	2.67	2.05	14.25
β2	1.87	3.08	2.04	14.32
β3	1.83	2.88	2.01	14.36
β4	1.98	2.74	2.08	13.95

10.

Spatial arrangement of heme groups in the β₄ hemoglobin tetramer. The four heme prosthetic groups are shown in indigo-colored stick representation with iron atoms depicted as orange spheres. Distances (Å) between the central iron atoms are indicated with dashed lines, highlighting the geometric relationship and packing within the tetrameric assembly. The relatively close Fe–Fe separations (25–37 Å) suggest a compact quaternary arrangement that may influence cooperative or allosteric properties of the β₄ hemoglobin structure.

5. Heme Iron–Iron Distances (Å) of Oxy-β₄-rHb0.1.

Chains	β2	β3	β4
β1	36.82	35.97	25.53
β2		25.36	35.97
β3			37.07

11.

Structural comparison of the oxy-bound β subunit heme pocket in HbA (blue, 2DN1) and rHb0.1 (yellow) reveals a main-chain RMSD of 0.494. Distinct differences are observed in the side-chain orientations of residues F41, F42, K66, and L141, along with altered positioning of the heme propionate groups relative to HbA. Right inset: magnified view of rHb0.1 shows that His 63 lies 2.67 Å from the bound O₂ molecule (red sticks) compared to 3.02 Å in HbA, which may contribute to differences in oxygen affinity.

12.

Structural comparison of rHb0.1 oxy-bound beta homotetramer with ferric β₄ HbA (6FQF). (A) The oxy-bound β₄ rHb0.1 structure is superimposed with its nearest structural homologue for comparative analysis. A ribbon representation of oxy-bound β₄ rHb0.1 (yellow) and β₄ ferric HbA (pink) is shown. (B) A comparison of the amino acid arrangement within the heme pocket of both structures reveals a backbone RMSD of 0.714 Å. (C) and (D) illustrate the alternative conformations of Cys112­(G14) observed in ferric-β₄-HbA and oxy-β₄-rHb0.1, respectively. In the ferric-β₄-HbA structure, Cys112­(G14) from β1 and β4 subunits are positioned within bonding distance, enabling the formation of a disulfide covalent linkage. In contrast, the oxy-β₄-rHb0.1 structure lacks this disulfide connectivity, as the β1-β2 Cys112­(G14) residues are separated by ∼6.5 Å, exceeding the permissible sulfur–sulfur distance for disulfide bond formation.

Analysis of the electron density maps at the interchain interfaces particularly the β1β4 and β2β3 regions, reveals no evidence of disulfide bond formation between the Cys112 (G14) residues (Figure C and D) unlike ferric-β₄-HbA (PDB: 6FQF). Moreover, disulfide bond formation is absent in previously reported crystal structures of CO-β₄ , and deoxy-β₄ hemoglobin. , In these structures, the Cys112 (G14) residues adopt dual conformations with their sulfhydryl groups positioned opposite each other across the β1β4 (β2β3) interface. These residues are proposed to undergo coordinated conformational shifts in a concerted manner, analogous to the synchronized movement of windshield wipers. ,, Furthermore, Cys112 (G14) in the β-chain has been identified as a key residue contributing to the stablility of β₄ homotetramers and αβ dimers within the α₂β₂ heterotetramer. , Consistent with this, X-ray crystallographic analysis of β₄ hemoglobin at 2.0 Å resolution revealed that Cys112 (G14) is positioned at the β-β subunit interface, with the side chains from the β1 and β2 oriented toward the molecular dyad axis. In the ferric-β₄-HbA structure, the Cys112 residues from β1 and β4 subunits are positioned within bonding distance, enabling the formation of a disulfide linkage across the interface. In contrast, in the oxy-β₄-rHb0.1 structure, the β1-β2 Cys112 residues are separated by ∼6.5 Å, which exceeds the permissible sulfur–sulfur distance (∼2.05 Å) for disulfide bond formation, thereby precluding covalent linkage (Figure C and D). These residues are surface-exposed and may participate in weak interactions with neighboring residues.

3.8. Oxidation State of Iron Does Not Drive Disulfide Formation in rHb0.1

Using l-cysteine hydrochloride monohydrate as a standard, we quantified the concentration of DTNB-accessible thiols in solution. Oxy-rHb0.1 exhibited approximately 1.84 ± 0.24 μM free thiols, while ferric-rHb0.1 showed ∼2.45 ± 0.49 μM free thiols per μM protein (Figure A, B, and C). Notably, oxidation to the ferric state did not result in a decrease in accessible thiols, as would be expected if intersubunit Cys112–Cys112 disulfide bonds were forming. Instead, the comparable thiol content observed in the ferric state indicates that Cys112 residues largely remain in the reduced form in solution. These biochemical data, together with the crystallographic observation that Cys112 sulfur atoms are separated by ∼6.5 Å in the oxy-β₄ rHb0.1 structure, strongly suggest that the absence of a disulfide bond arises from unfavorable geometry rather than oxidation state alone.

13.

Ellman’s assay to quantify free thiols in oxy- and ferric rHb0.1. (A) Representative Ellman’s assay demonstrates color development of the DTNB–thiol adduct for oxy-rHb0.1 and ferric-rHb0.1 (15 μM each) along with an l-cysteine HCl monohydrate standard (15–100 μM). Absorbance of rHb0.1 at 450 nm was used as a blank and subtracted from all readings. (B) Standard calibration curve generated using l-cysteine HCl monohydrate, monitored at 450 nm (R² = 0.998). (C) Quantification of free thiols in oxy- and ferric rHb0.1, expressed as μM cysteine equivalents. Data are shown as mean ± SD; differences were not statistically significant (ns).

4. Discussion

The present study provides a biochemical and structural characterization of recombinant hemoglobin rHb0.1, a rationally engineered construct containing stabilizing mutations di-α­(G15A)­β­(G15A/H116I) alongside a V1M substitution in the β-chain. Our results reveal that rHb0.1 displays novel structural and functional properties that diverge from those of native HbA. SDS-PAGE and reverse-phase HPLC analyses demonstrated that rHb0.1 overexpresses β-subunits relative to di-α subunits, in agreement with prior observations that β-globins are intrinsically more stable and soluble than α-globins. The preferential expression of β- over α-chains likely drives the formation of β-homotetramers in solution and captured in crystal, as confirmed by our structural analysis. Isolated α- and β-chains show very different propensity for solubility, stability, and aggregation. In the ferrous state, the β₄ homotetramer exhibits high solubility and stability, while free α-subunits are unstable, tend to precipitate, and are typically stabilized in vivo by binding to α-hemoglobin stabilizing protein (AHSP). ,

Spectral studies confirmed that rHb0.1 retains the global spectral signatures of native HbA, including characteristic Soret and Q-band transitions across redox and liganded states. Intrinsic Trp fluorescence indicated a well-folded hydrophobic core, while CD spectra showed a high α-helical content (∼67%), comparable to HbA. These findings verify that the engineered construct folds into a canonical globin tertiary structure. However, thermal and pH stability assays revealed that rHb0.1 is less stable than HbA, with lower melting temperature (52 °C vs 54 °C) and increased susceptibility to pH-induced unfolding. Despite the stabilizing mutations, these results suggest that altered subunit assembly and quaternary structure impose limitations on global stability.

Our data demonstrate that rHb0.1 exhibits faster heme dissociation rates from both α- and β-subunits compared to HbA, with nearly 2-fold increase in rate constants. This observation was unexpected, given that the di-α glycine linker was designed to prevent αβ dimer dissociation and thereby enhance heme retention. The enhanced dissociation rates may instead reflect destabilization of the α-subunit heme pocket or altered quaternary contacts in the β₄ assembly. Reactions with hydrogen peroxide revealed that both HbA and rHb0.1 form ferryl species and subsequently convert to sulfHb with similar yields, indicating comparable susceptibility to oxidative stress. However, kinetic analyses revealed that rHb0.1 displayed significantly slower ferryl decay (1.08 h^–1 vs 2.16 h^–1 for HbA) and slower conversion to ferric hemoglobin (1.44 h^–1 vs 3.96 h^–1). These data suggest that rHb0.1 autoreduction pathways are intrinsically slower, possibly due to altered electron transfer dynamics within the β₄ tetramer. The persistence of ferryl intermediates is particularly relevant since prolonged ferryl states can promote oxidative damage and cytotoxicity, posing challenges for therapeutic use.

Importantly, our biochemical analyses indicate that rHb0.1 exists in solution as a mixture of α₂β₂ and β₄ species, with crystallization preferentially capturing the preexisting β₄ assembly rather than inducing de novo β₄ formation, as evidenced by mass spectrometry analysis (Supplementary Section S4). Given the higher expression level of the β chain relative to the glycine-linked di-α chain, β-β encounters in solution are statistically favored, making β₂ and β₄ assemblies plausible. The lower abundance of the di-α subunit thus shifts this equilibrium toward β₄ formation, which is subsequently stabilized by the H116I mutation through enhanced hydrophobic β-β interfacial interactions. Comparison with the rHb1.1 crystal structure (PDB: 1ABW), which lacks the H116I mutation and assembles as di-α/β subunits rather than αβ homotetramer, together with the failure of native HbA to crystallize under identical conditions, supports a contributory role of the H116I substitution in stabilizing β-homotetramer formation. Collectively, these findings suggest mechanistic insight into how subunit stoichiometry and sequence variation can influence hemoglobin quaternary assembly, stability, and functional divergence from HbA. The structural analysis demonstrated well-ordered heme groups, appropriate Fe–O₂ coordination, and overall quaternary geometry resembling the R-state of liganded HbA, albeit without cooperativity. Consistent with prior reports, β₄ tetramers exhibited high oxygen affinity and lacked cooperative binding behavior, supporting the functional divergence from HbA.

The previous findings show that the γ116Ile→His substitution in γ-chains led to an in vitro assembly rate with α-chains comparable to β-chains but also caused destabilization of γ₄ tetramers. , These results highlight the importance of amino acid hydrophobicity at position G18 in both β- and γ-chains in modulating the equilibrium between monomer–dimer and tetramer states, which is a critical determinant of α-chain assembly. , Their findings suggested that the hydrophobic nature of β116 Ile facilitates stabilizing interactions with nearby β-chain residues (such as 115 Ala and 122 Phe), enhancing β₁-β₂ interactions and promoting the formation of stable β₂ dimers, ultimately slowing α-chain incorporation in vitro. In contrast, when β116 is substituted with His, such hydrophobic interactions may be disrupted during αβ assembly. Instead, the formation of αβ heterodimers may rely more on hydrogen bonding involving residues on the α-chain G-helix (e.g., 110 Ala, 114 Pro, and 117 Phe), as supported by X-ray crystallographic data. ,

Together, these findings underscore the dual role of stabilizing mutations; while they enable soluble expression of recombinant hemoglobins, they can also stabilize β₄ quaternary assembly and heme dynamics in unanticipated ways. The coexistence of β₄ tetramer, increased heme dissociation rates, reduced thermal/pH stability, and slower ferryl decay all point to intrinsic structural differences that compromise functional resilience compared to HbA. These properties must be carefully considered in the context of HBOC design, where both heme stability and resistance to oxidative stress are paramount. These findings emphasize the need for next-generation HBOC designs that achieve a balance between globin chain stability, stoichiometry, and native functionality.

Interestingly, several of the biochemical and structural features observed in rHb0.1 parallel those seen in naturally occurring hemoglobinopathies. For instance, the predominance of unstable subunit assemblies and the propensity for homotetramer formation mimic the pathological mechanisms of β-thalassemia and related disorders, where imbalances in globin chain expression lead to precipitation, oxidative stress, and cytotoxicity. Likewise, the increased heme loss and persistence of ferryl intermediates resemble features of unstable hemoglobin variants associated with hemolytic anemia. For instance, the βY35F variant (Philly) has an altered α1β1 interface, which leads to decreased cooperativity and increased oxygen affinity, causing hemolytic anemia and reticulocytosis. In contrast, the βV111F (Peterborough) and βV111A (Stanmore) variants exhibit decreased oxygen affinity. The altered interfacial residue at β116 also emphasizes how single-point substitutions at key structural “hotspots” can dramatically influence assembly pathways, a phenomenon well documented in both α- and β-chain variants. Thus, rHb0.1 not only provides insight into the engineering strategies of recombinant hemoglobins but also serves as a valuable structural and functional model to understand how subtle sequence perturbations in globin genes can translate into hemoglobinopathies.

5. Conclusion

In summary, our findings highlight the dual impact of stabilizing mutations in recombinant hemoglobins. While such modifications facilitate soluble expression, they can also introduce unanticipated alterations in quaternary assembly, heme dynamics, and oxidative stability. rHb0.1 emerges as a structurally well-folded yet functionally divergent hemoglobin variant. In solution, rHb0.1 exists as a heterogeneous ensemble of α₂β₂ and β₄ species, with crystallographic analysis revealing a preferentially stabilized β₄ homotetramer. Altered interfacial contact residues and distinctive redox and heme-dissociation kinetics collectively distinguish rHb0.1 from native HbA. These findings not only provide mechanistic insights into how quaternary structure influences hemoglobin stability and reactivity but also highlight the inherent challenges of engineering hemoglobin variants for therapeutic applications, while offering broader insight into hemoglobin assembly and hemoglobinopathies.

Glossary

Abbreviations

rHb

Recombinant hemoglobin

rHBOC

Recombinant hemoglobin-based oxygen carrier

RBCs

Red blood cells

Tm

Transition thermal midpoint

GuHCl

Guanidium hydrochloride

HPLC

High-performance liquid chromatography

His

Histidine

HbA

Native human hemoglobin

Cys

Cysteine

Ile

Isoleucine

β4

β homotetramer

HbF

Fetal hemoglobin

All relevant data are provided within the main text and the Supporting Information, including the supplementary tables. The crystal structure has been deposited in the Protein Data Bank (PDB) under the accession number 9WW1.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11376.

• Structure-based sequence alignment of recombinant hemoglobin β-subunit variants; percentage of α-helix and β-sheet of rHb0.1 and HbA; thermal and pH stability measurements; identification of α- and β-globin chains in rHb0.1 by LC-MS/MS; intact mass analysis using a SCIEX QTRAP 6500 mass spectrometer (PDF)

K.Y., S.K., P.K., and A.N.: writing, review and editing; K.Y. and S.K.: conceptualization; S.K., P.K., and A.N.: supervision; S.K. and P.K.: resources; S.K., P.K., and K.Y.: investigation; S.K. and A.N.: funding acquisition; S.K., K.Y., and S.V.: formal analysis; K.Y., P.K., and S.V.: validation; K.Y. and S.V.: methodology; K.Y.: writing original draft; K.Y.: visualization; K.Y. and S.V.: software; K.Y.: data curation.

We gratefully acknowledge financial support to S.K. from the Life Science Research Board, Defense Research and Development Organisation (LSRB-DRDO, O/o DG­(TM)/81/48222/LSRB-317/SH&DD/2017), Delhi, India. S.K. also acknowledges financial support from the University of Delhi (Institution of Eminence grant IOE/FRP/LS/2020/27). A.N. gratefully acknowledges research support from the Institution of Eminence, University of Delhi (IoE/2024–25/12/FRP, IoE/2023–24/12/FRP, IoE/2021/12/FRP), the Department of Science and Technology (DST-SERB, Grant No. CRG/2020/003380), and the Council of Scientific and Industrial Research (CSIR, Scheme No. 27/0388/23/EMRII).

The authors declare no competing financial interest.