Structural and Functional Significance of the N- and C‑TerminalAppendages in Arabidopsis Truncated HemoglobinNitika Mukhi,† Sonali Dhindwal,‡ Sheetal Uppal,§ Abhijeet Kapoor,§ Richa Arya,§ Pravindra Kumar,‡

Jagreet Kaur,*,† and Suman Kundu*,§

†Department of Genetics, University of Delhi South Campus, New Delhi 110021, India‡Department of Biotechnology, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India§Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India

*S Supporting Information

ABSTRACT: Plant hemoglobins constitute three distinct groups: symbiotic,nonsymbiotic, and truncated hemoglobins. Structural investigation of symbioticand nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similarto bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. Whilemultiple structures have been reported for plant hemoglobins of the first twocategories, for plant truncated globins only one structure has been reported of late.Here, we report yet another crystal structure of the truncated hemoglobin fromArabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of whichone is distinctly coordinated to the heme iron, unlike the only available crystalstructure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in itscrystallographic asymmetric unit; however, dimer was evident in the crystallographicsymmetry, and the globin indeed existed as a stable dimer in solution. The tertiarystructure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension anddisordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletionmutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had aninsignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide bindingkinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role inintramolecular or intermolecular interactions probably regulating protein−ligand and protein−protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influencedcoordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicatedthat the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements,dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix.

Globins are pervasive in all kingdoms of life, ranging fromunicellular bacteria to higher eukaryotes.1−3 Apart from

the well-characterized symbiotic hemoglobins, plant genomesare endowed with multiple nonsymbiotic hemoglobins(nsHbs), the significance of which can be anticipated by theirubiquitous occurrence in almost all land plants.4,5 The nsHbsare further categorized into three separate classes: class I(nsHb1), class II (nsHb2), and class III or truncatedhemoglobins (trHbs).6 Members of each class bear uniquekinetic and structural fingerprints, suggestive of their distinctphysiological role.7 Structurally, land plant hemoglobins (Hbs)are divided into two distinct lineages: those that hold a classical3/3 α-helical globin fold (like symbiotic and class I and class IInsHbs) and the rest with 2/2 α-helical fold globins, like theclass III Hbs.6−8 TrHbs, marked by the presence of a typical 2/2 globin fold, are found to be ubiquitously present in plants,bacteria, and unicellular eukaryotes, with a typical size of 110−140 amino acid residues.9,10 Plant truncated hemoglobins(ptrHbs) (class III) share 40−45% sequence similarity with

bacterial trHbs. In contrast to bacterial trHbs, which typicallyhave shorter sequences, ptrHbs have longer polypeptide chains,the size sometimes being larger than those of even class I and IInsHbs but still retaining the putative truncated 2/2 α-helicalglobin fold.9−11

Arabidopsis thaliana encodes a single copy for each of thethree classes of nonsymbiotic globin genes: AHb1 (class I),AHb2 (class II), and AHb3 (truncated hemoglobin).11,12 AHb1and AHb2 share 60% sequence homology between them, andboth display a classical 3/3 α-helical globin fold.8−12 Withdifferences in expression pattern and tissue localization, theseglobins have been proposed to play diverse physiologicalroles.13−15 AHb1 displays a relatively high oxygen bindingaffinity16 and is primarily believed to be engaged in NO binding

Received: September 14, 2015Revised: February 25, 2016Published: February 25, 2016

Article

pubs.acs.org/biochemistry

© 2016 American Chemical Society 1724 DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

because of its NO dioxygenase activity.16−18 AHb2, incomparison, has an oxygen affinity lower than that of AHb1and has been proposed to facilitate the supply of oxygen todeveloping tissues.19 In Arabidopsis, class II nsHb have beenproposed to participate in fatty acid metabolism and shootorgan development.20,21 AHb3 is expressed in both Arabidopsisroots and shoots and was found to be downregulated byhypoxia.11,22 In contrast to class I and class II nsHbs, much lessis known about plant trHbs. Structures of class I and class IInsHbs were obtained for several plant Hbs.8,23−25 However,only recently was a solitary ptrHb structure reported.26

Characterizing the three-dimensional structure is fundamentalto gaining insights into the function of a protein, with structuresof Hbs having played a significant role in understanding theirfunctions and evolutionary relationship. Every three-dimen-sional structure of novel Hbs deciphered in the past twodecades has ushered in new insights about this ubiquitousprotein family. Due to a lack of multiple crystal structures forptrHbs, their physiological role in land plants is still undercontest; their structural characteristics and functional potentialare not fully recognized or explored.Attempts were thus made to crystallize trHb from the model

plant A. thaliana, and we could successfully determine thethree-dimensional structure of AHb3 [Protein Data Bank(PDB) entry 4C44]. Simultaneously and independently,however, Reeder et al. also determined the structure of AHb3at 1.7 Å (PDB entry 4C0N).26 PDB records are testimony tothe fact that our coordinates were submitted almost at the sametime (PDB entry 4C0N, August 6, 2013; PDB entry 4C44,August 30, 2013), but for technical reasons, we were late inreporting the crystal structure. To overcome this deficiency, weextended our investigation beyond the three-dimensionalstructure for additional insight into the structure−functionrelationship of the novel globin, especially in solution.As in the previous crystal structure,26 the tertiary architecture

of the protein exhibited a bacterial-like 2/2 α-helical globin foldin its core with an added N-terminal α-helical extension anddisordered C-termini. However, subtle differences were alsoseen in the two structures, like the presence of different ligandsin the heme pocket. For insight into the functional andstructural significance of the N- and C-terminal appendages inptrHbs, N- and C-terminal deletion mutants, AHb3-Δ25N andAHb3-Δ25C, respectively, as well as the double mutant AHb3-Δ25NΔ25C were investigated. It was observed that theremoval of 25 residues from the N- or C-terminus did notcause any significant perturbation of the overall core structureor globin fold of the protein. Molecular dynamics (MD)simulations of the crystal structure of AHb3 or models ofAHb3-Δ25C and AHb3-Δ25N displayed stable trajectoriesthroughout the simulation, further precluding their role in themaintenance of the structural architecture or integrity of theprotein. AHb3-Δ25C was also similar to wild-type AHb3 inmost of its biochemical properties, suggesting the functionalirrelevance of the C-terminus in general characteristics of theglobin. However, the C-terminally truncated mutant displayed asignificant difference in its azide binding kinetics in the ferricstate. The C-terminus, thus, might be vital for recognition ofinteracting or binding ligands and proteins, which isreminiscent of intrinsically disordered domains or proteins.27

However, it was evident that the N-terminus played asignificant role in the dimerization of the globin, thecoordination of heme iron, the hydration state of the hemepocket, and the tunnel topology in the protein matrix as judged

from its spectroscopic, structural, and other biochemicalproperties.

■ MATERIALS AND METHODS

Protein Expression and Purification. cDNA (Unigeneclone U83861) for Arabidopsis truncated hemoglobin (AHb3)was amplified via polymerase chain reaction (PCR) and clonedin bacterial expression vector pET21c (Novagen, Darmstadt,Germany) between BamHI and XhoI restriction sites. Forexpression of wild-type AHb3, transformed BL21(DE3)bacterial cells were initially grown in Terrific broth at 37 °Cto an optical density of 0.6 (at 600 nm) followed by inductionat 25 °C (without IPTG) for 16 h at 200 rpm. Cells were thenharvested and lysed by both chemical and mechanical treatmentas described previously for other globins.8,28 Purification of six-His-tagged recombinant protein was performed by Ni-Sepharose (GE Healthcare, Little Chalfont, United Kingdom)affinity chromatography. Protein was further purified byemploying DEAE Sephadex anion exchange (GE Healthcare)and S-200 Sephacryl (GE Healthcare) size exclusionchromatography. An ASoret/A280 absorbance ratio of ≥3.5indicated the purity of the protein, which was also confirmedby sodium dodecyl sulfate−polyacrylamide gel electrophoresis(SDS−PAGE). The purified recombinant protein was thensubjected to reduction and oxidation of heme iron by sodiumdithionite and potassium ferricyanide, respectively, followingdesalting on a G-25 Sephadex (GE Healthcare) column toobtain a homogeneous population of ferric globin. Pure proteinwas stored at −80 °C for further use.

Crystallization and Data Collection. Before crystalliza-tion trials, the quality and oxidation state (ferric) of AHb3 wereassessed by examining the absorbance spectrum, and ifrequired, it was converted completely into the ferric stateusing potassium ferricyanide at pH 7.0. Crystallization screens(Hampton) were used to identify suitable crystallizationconditions. Crystals of ferric AHb3 were grown using thesitting drop vapor diffusion method at 20 °C by mixing equalvolumes of a 2 mM protein solution and a precipitant solutioncontaining 1.2 M sodium potassium tartarate tetrahydrate in 0.1M Tris (pH 8.5). Well-defined crystals were observed inapproximately a few weeks. Before data collection, the proteincrystals were cryoprotected by being directly transferred to themother liquor drop containing 10% (v/v) ethylene glycol as acryoprotectant. The diffraction data were collected at 100 Kusing Cu Kα X-ray radiation generated by a rotating-anodegenerator (Bruker-Nonius Microstar H, Billerica, MA)equipped with a MAR345 imaging plate detector on an in-house facility at the Indian Institute of Technology Roorkee(India).

Structure Determination and Refinement. Diffractiondata, obtained as described above, were reduced and scaledusing the HKL2000 program suite.29 Initial phases for thestructure were obtained by the molecular replacement methodwith the crystal structure of Geobacillus stearothermophilustruncated hemoglobin (PDB entry 2BKM) as a search model(35% sequence homology) using MOLREP from the CCP4.6.3software suite.30,31 Crystallographic refinement was performedusing REFMAC 5.32,33 COOT was used for electron densitymap and model building.34 The PyMol visualization tool wasutilized for structural analysis and production of figures.35 Theatomic coordinates and structure have been submitted to thePDB36 as entry 4C44.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1725

Cloning, Expression, and Purification of TruncatedRecombinant AHb3 Proteins. AHb3-Δ25N, AHb3-Δ25C,and AHb3-Δ25NΔ25C deletion mutants (from which 25amino acids were deleted from either the N-terminus, the C-terminus, or both) were generated using a PCR-basedapproach. The primer pairs used for the amplification of thedeleted variants were 5′-CACGGATCCAGGAGTCCAATCT-GTTCG-3′ and 5′-TGCCTCGAGTTCTGCTGGTTTATTG-3′, 5′-CACGGATCCAGATGCAATCGCTGCAAG-3′ and 5′-TGCCTCGAGCAGCTCGTTTCCAGCCAC-3′, and 5′-CA-CGGATCCAGGAGTCCAATCTGTTCG-3′ and 5′-TGCCT-CGAGCAGCTCGTTTCCAGCCAC-3′. The PCR-amplifiedgenes were then independently cloned into the pET21cbacterial expression vector between BamHI and XhoI sitesand the clones verified by DNA sequencing. Subsequently,individual variants were expressed and purified in the same wayas for wild-type AHb3 protein. The purified proteins were thenreduced, oxidized, and stored at −80 °C for further use asdescribed above.Quaternary Structure Determination of AHb3 and Its

Truncated Derivatives. HPLC Method. For protein quater-nary structure determination, size exclusion chromatographywas performed using the HPLC system from Waters (model2489, Milford, MA) fitted with a UV−vis detector. Prior to theanalysis, the column (Biosuite 125, 4 μm UHR SEC, 4.6 mm ×300 nm) was pre-equilibrated using 0.1 M phosphate buffer(pH 8.0). Protein concentrations ranging from 200 μM to 1mM were investigated at a flow rate of 0.5 mL/min. Theretention time was recorded while the absorbance at 280 nmwas monitored to obtain the chromatogram. Proteins of knownmolecular masses were used as markers for reference(ribonuclease A, ∼14 kDa; carbonic anhydrase, ∼29 kDa;ovalbumin, ∼45 kDa; conalbumin, ∼76 kDa).Cross-Linking Assay. To investigate the oligomerization

potential of AHb3 and AHb3-Δ25C, the glutaraldehyde-dependent cross-linking assay was used as describedpreviously.8 In brief, 100 μg of appropriate protein wasincubated with varying concentrations of a freshly preparedsolution of glutaraldehyde ranging from 0.01 to 1% for 5 min at37 °C. The reaction was terminated by adding 10 μL of 1 MTris-HCl (pH 8.0). Cross-linked protein was analyzed usingSDS−PAGE.Spectroscopic Characterization of AHb3-Δ25N,

AHb3-Δ25C, and AHb3-Δ25NΔ25C. All the absorbancemeasurements were taken using a Cary Varian 100 Bio UV−visspectrophotometer (Varian Inc.) in the range from 260 to 700nm with protein samples at a concentration of ∼0.5 mg/mL, asdetermined by Bradford’s assay. For spectral measurement offerrous globin and ligand-bound proteins, deoxy samples weregenerated by reducing ferric protein with sodium dithionite.Following reduction, CO-bound protein was prepared bydirectly purging CO gas in the deoxy sample. Oxygen-boundsamples were prepared by desalting the reduced hemoglobinover a G-25 column. CD spectra were recorded on a JASCO J-815 spectropolarimeter (JASCO Corp., Tokyo, Japan) using acylindrical quartz cell with a path length of 1 mm for the far-UVregion between 190 and 260 nm.Ligand Binding Kinetic Studies. Stopped-flow and laser

flash photolysis-based spectroscopic methods were employed todetermine the effect of deletions on the gaseous ligand bindingkinetics of AHb3 as described elsewhere for pentacoordinatedHbs.37−40 O2 dissociation rates and CO association rates forAHb3 and its mutants were measured at 25 °C in a stopped-

flow spectrometer using an SFM400 module equipped withfour syringes in association with an MPS70 syringe controllerand an MOS50 spectrophotometer from BioLogic ScienceInstruments (Bio-Logic SAS, Claix, France). The kinetic traceswere monitored using absorbance measurements at 414 nm forO2 dissociation and 431 nm for CO association. Laser flashphotolysis was conducted to determine the O2 association rateconstant for the proteins. Oxygenated protein samples wereprepared as described above, and the oxy samples werecollected directly into gastight syringes from the desaltingcolumn. Subsequently, they were transferred to a 3 mL quartzcuvette with a path length of 1 cm sealed with a rubber septum.The O2-bound protein samples were then subjected to flashphotolysis using an LKS.60 flash system (Applied PhotophysicsLtd., Leatherhead, Surrey, United Kingdom) containing aNd:YAG pulsed laser at 1064 nm, frequency doubled to 532nm with an energy of 10 or 20 Hz. White light from a Xe lampwas used to probe the samples. The kinetic tracescorresponding to changes in the Soret peak wavelength foreither formation of oxy-bound globin or depletion ofdeoxyglobin were recorded and rates extracted as describedelsewhere.41,42

Azide Binding. Azide binding in the ferric proteins wasperformed with a stopped-flow spectrometer using an SFM400module equipped with four syringes in association with anMPS70 syringe controller and an MOS50 spectrophotometerfrom BioLogic Science Instruments (Bio-Logic SAS). Thekinetic traces were monitored using absorbance measurementsat 409 nm for ligand association. The azide binding in therespective ferric proteins was spectroscopically confirmed byrecording the shift in the Soret peak and Q-bands upon ligandbinding.

Biochemical Characterization. Autoxidation Measure-ments. Autoxidation rates were measured by monitoring theabsorbance changes at 581 nm for the proteins investigated, asdescribed previously.28 Briefly, 0.3 mM oxyprotein was dilutedinto ∼100 mM potassium phosphate (pH 8.0) supplementedwith 1 mM EDTA and 3 mmol/mol of heme catalase andsuperoxide dismutase. Changes in the entire visible spectrawere recorded using a Cary Varian 100 Bio UV−visspectrophotometer (Varian Inc.) in scanning kinetics mode.Igor Pro (Wavemetrics Inc.) was used for data plotting andanalysis.

Stability Investigations and pH Titration Profiles. Thestability of the proteins against changes in pH was probed usingbuffers in the pH range of 2.0−11.0.28 Prior to spectroscopicmeasurements, appropriate protein at 3 μM was diluted in abuffer at the desired pH and incubated for 3−4 h at 25 °C. Togenerate pH titration profiles, ferric proteins (wild-type AHb3and its mutants) were subjected to the pH range mentionedabove while recording their optical spectrum between 260 and700 nm. The apparent pKa was calculated by plotting thedifference in absorbance at 424 and 406 nm against the changein pH as reported by Reeder et al.26 The globins tend toaggregate and precipitate at pH 5.5 and hence the titrationprofiles included the pH range of 5.5−11. Thermal stability wasprobed at pH 7.0 in the range of 25−80 °C with a 5 minincubation at each temperature followed by absorbancemeasurement.

Heme Dissociation Assay. To assess the effect of N- and C-terminal deletions on the heme retention capability of AHb3, ifany, heme was extracted from the globins using Teale’smethod.43 Briefly, the pH of the sample was adjusted to 2.0

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1726

using ice-chilled 0.1 M HCl. An equal volume of cold ethylmethyl ketone was added to the sample immediately, followedby brief shaking. The samples were then incubated for 5 min onice to separate the two phases. Usually, heme partitions in thetop organic phase and the resulting apoglobin in the bottomaqueous phase. The rate constants for heme dissociation weremeasured as described by Hargrove et al.44 The transfer ofheme from 3 μM holoprotein of interest to 30 μM H64Y/V68Fapomyoglobin was measured at pH 7.0 by monitoring changesin absorbance at 410 and 600 nm. Curve fitting to a single-exponential equation was accomplished using Igor Pro(Wavemetrics Inc.), and rate constants for heme dissociationwere calculated accordingly.28

Modeling of AHb3 To Include or Exclude TerminalAmino Acid Side Chains. The crystal structure of wild-typeAHb3 was used as the template to model the derivatives. Themodel to include the C-terminus, because its electron densitywas missing in the crystal structure, was built using I-TASSER.45 The N-terminally truncated model was obtainedsimply by deleting the coordinates for the corresponding sidechains from the PDB file of the crystal structure of AHb3.Molecular Dynamics (MD) Simulation. MD simulation

studies were conducted for the X-ray crystal structure of AHb3(with missing C-terminal density) and AHb3 with a deleted N-terminus (pre-A helix). All the simulations were executed usingNAMD46 and the CHARMM2247 force field with CMAPcorrection. MD simulations were performed using periodicboundary conditions and TIP3P water, and the system wasneutralized by adding counterions. A 2 fs time step was usedwith a 12 Å cutoff for VDW interactions and full particle-meshEwald electrostatics. All simulations were initiated by firstminimizing the structure followed by constant volume heating(to 310 K) for 10 ps. This was followed by constant-temperature and constant-pressure (1 atm) dynamics for 50 ns,including a 1 ns equilibration run. Trajectory analysis was doneusing Visual Molecular Dynamics (VMD).48 The average root-mean-square deviation (rmsd) on all protein atoms wascalculated compared to the X-ray structure for the wild-typeand mutated protein structures over their entire trajectorysimulations using the VMD rmsd trajectory application. Allprotein structures were visualized, displayed, and analyzed usingPyMol.35

Analysis of Protein Matrix Tunnels. Tunnel analyses ofthe crystal structure of AHb3 and bacterial truncatedhemoglobins were conducted using online molecular channelanalysis tool MOLE 2.0.49 MD pocket calculations wereperformed on MD simulated trajectories of AHb3 and AHb3-ΔN25.50 The tunnels and pockets were analyzed and visualizedusing PyMol.35 Some paradigm 3/3 globins were also analyzedsimilarly and compared with the truncated hemoglobins.

■ RESULTSCrystal Structure of Arabidopsis Truncated Hemoglo-

bin Displayed a 2/2 Globin Fold, an ExtendedPolypeptide Terminus, and a Heme Pocket ArchitectureAs Reported Previously, with Distinctive Observationsof an Open Tunnel, a C-Terminal Orientation, and a “ϕ”Helix. The three-dimensional structure of AHb3 wasdetermined to 2.65 Å resolution by the molecular replacementmethod. The crystallographic data collection and refinementstatistics are summarized in Table 1. The protein was found tobe a monomer in the crystallographic asymmetric unit. Theoverall structure of AHb3 was found to be similar to that

reported recently by Reeder et al.26 with a disordered C-terminus, an extended N-terminus, and a 2/2 α-helicalsandwich fold similar to those of other bacterial truncatedhemoglobins (Figure 1A). As reported earlier, the electrondensity map at the active site displayed additional density in thedistal pocket, best fitted with two water or hydroxide moleculesstabilized by H-bonding and electrostatic interactions viaGlnE11 in assistance with TrpG8 and TyrCD1. The hemepocket, described in detail by Reeder et al.,26 is endowed withan array of polar and hydrophobic residues, with mainlyTyrB10, PheCD1, GlnE11, and TrpG8 as putative ligandstabilizing partners, similar to bacterial truncated hemoglo-bins.51−55 The primary structure of plant truncated hemoglo-bins displayed long terminal extensions on either side of theprotein core, as reflected in the crystal structures, as well, whichaligned with the sequence of bacterial truncated Hbs.11 Incontrast to the C-terminal region, the sequence of the N-terminal region is highly conserved across all plant truncatedhemoglobins (Supplementary Figure 1). In AHb3, the 25residues at the N-terminal extension formed a pre-A helixcomposed of two short helical regions, which lay almostperpendicular to each other. The N-terminal helical extension(pre-A helix) was anchored to the protein core via electrostaticcontacts between residues in the pre-A helix and the H-helix, asdescribed before26 (Figure 1B).What was not emphasized in the earlier report of the AHb3

crystal structure26 was the presence of an almost continuoustunnel through the protein matrix connecting the heme distalpocket to the external surface, thereby providing direct accessof ligand from solvent to the central heme cavity, and viceversa, as depicted by the flow of water molecules from the

Table 1. Crystallographic Data for Arabidopsis Hemoglobin3

Crystallographic Dataspace group P4332wavelength (Å) 1.54resolution 50.0−2.65cell dimensions

a = b = c (Å) 122.08α = β = γ (deg) 90

no. of unique reflections 9881completeness (%) (last shell) 100.0 (100.0)Rsym (%) (last shell) 0.09 (0.64)I/σ (last shell) 14.56 (2.0)multiplicity (last shell) 5.68 (5.7)

Refinementno. of reflections (working/test) 9020/504no. of residues 150no. of water molecules 37resolution range (Å) 43.20−2.65Rcryst (%) 20.27Rfree (%) 25.21average B factor (Å2) 40.12water atoms 40.20all atoms 40.0rmsd for bond lengths (Å) 0.009rmsd for bond angles (Å) 1.44Ramachandran plot

favored (%) 98.0allowed (%) 99.8outliers (no. of residues) 3

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1727

solvent to the heme (Figure 1C). Such an open tunnel mayinfluence the binding of the ligand to the truncatedhemoglobins. Interestingly, unlike its homologues from group

II truncated hemoglobins, AHb3 retains Phe at both positionsE14 and E15 (Table 2), similar to Mycobacterium trHbN,suggestive of a similar regulatory role. While the density of 25

Figure 1. Structural features of Arabidopsis hemoglobin 3 (AHb3). (A) The X-ray crystallographic structure of AHb3 displays a 2/2 globin fold withpentacoordinated heme and an extended N-terminus. (B) Electrostatic interactions anchoring the N-terminal helical extension of AHb3 to the globincore. (C) The surface representation of AHb3 displays an open cavity occupied by an array of water molecules from the external surface to the hemepocket. (D) Overlay of the crystal structure of AHb3 (blue) with the I-TASSER-modeled structure (green) display extended and exposed C-terminiprimarily constituted of loop regions. (E) Superposition of the I-TASSER-modeled structure of AHb3 (blue) with Hell’s gate HbIV (red) showingsimilar C-terminal tails. (F) Cartoon representation of the characteristic “ϕ” helix, present between helices E and F hosting Tyr85 and Lys89 forinteraction with heme propionates and certain other distal/proximal pocket residues.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1728

residues at the C-termini was found missing in the diffractiondata, the I-TASSER (ab initio)-modeled structure of C-terminipredicted predominantly loop regions, whose flexibility mighthave precluded proper X-ray diffraction (Figure 1D). Acomparison of the crystal structure of AHb3 with the C-terminal inclusive model demonstrated the N- and C-terminalappendages to be external to the core and available forintramolecular or intermolecular interactions (Figure 1A,B,D).The conformation of the predicted C-terminus was found to besimilar to that of the I-helix of Hell’s gate hemoglobin (PDBentry 4NK1) (Figure 1E), suggesting a similar yet unknownrole.56 Another distinctive feature observed in the presentcrystal structure (Figure 1F) was the presence of an extra “ϕ”helix between residues 84 and 89 (between helices E and F),with the helix positioning Tyr85 and Lys89 for interaction withheme propionate (Figure 2D). This feature is characteristic ofgroup II truncated hemoglobins.The AHb3 Crystal Structure Reveals Coordinated

Heme Pocket Water Molecules. The crystal structure ofAHb3 reported here was compared to the one reportedrecently,26 and both structures displayed common signatureswith a similar fold, and characteristics as mentioned above, withsimilar active site architecture (Figure 2A). The majordifference lay in the presence of different putative ligands inthe active sites of the two (Figure 2A, red circles), which werebelieved to be water molecules in the former but hydroxyl ionin the latter (Figure 2B). Considering the resolution of thecrystal structure and the electron densities in the distal hemepocket, both hydroxide and water molecules could probably fit,and ambiguity cannot be ruled out. However, our structure bestfitted with two water molecules. Two hydroxyls in the distalpocket did not seem likely in the absence of any literatureevidence of the same. On the contrary, the presence of orderedwater molecules in the heme pocket has been reportedpreviously for other heme proteins, including myoglobin andheme oxygenase. One of the water molecules was 2.06 Å fromthe iron atom and displayed unambiguous electron density,indicating coordination at the heme iron (Figure 2C). Watermolecules found in the heme pockets of hemoglobins areusually more than 6 Å from iron; AHb3 thus presents a noveltyin its use of water molecules in the heme pocket. The hydroxylgroup in the previously reported crystal structure seems also tobe coordinated to the heme iron at a similar distance, acting asa ligand. The water/hydroxide molecules seemed to favor anetwork of H-bonding interactions (Figure 2B,C), which might

stabilize bound gaseous ligands like O2 or CO, as well,influencing kinetics.Reeder et al.26 considered hydroxyl ligand in their crystal

structure based on the optical spectrum of AHb3 and thenature of the acid−alkali transition of the ferric globinmonitored by probing the difference spectra at 424 and 406nm. For comparative purposes, similar investigations wereperformed here, and the absorbance spectra of AHb3 in thebuffer that crystallized the protein in the report presented hereand in the buffer that crystallized the globin in ref 26, were seento be distinctly different in both the Soret wavelength and theQ-bands (Figure 2D). The blue shift in the Soret peak (413nm) in the crystallization condition of Reeder et al. comparedto ours (409 nm) clearly indicated the possibility of hydroxyl asthe ligand in the former. The presence of a shoulder at ∼620nm (closer to 630 nm) in our globin spectrum also indicates ahigher probability of aquo-met Hb. The pKa between the Fe3+-H2O and Fe3+-OH transition of wild-type ferric AHb3 underour experimental conditions was found to be ∼7.8 (Figure 2E),unlike the value of 7.17 determined by Reeder et al.,26

supporting the inclusion of hydroxyl ligand in the earlierstructure and water in the present structure.

Structural Conservation among Plant and BacterialTruncated Hemoglobins. Despite significant variation in theamino acid sequence, comparative structural analysis of AHb3with the bacterial truncated hemoglobins exhibited conserva-tion across the family. Figure 3 displays the structural overlay ofAHb3 with representative crystal structures of bacterialtruncated hemoglobins from each class of the family. Theaverage rmsd value with respect to AHb3 varies from 3.3 Å forgroup I to 0.97 Å for group II to 3.8 Å for group III. Clearly, thetertiary structure of AHb3 was more similar with that ofmembers of group II truncated hemoglobins with a rmsdranging from 0.7 to 1.2. The only major difference was thepresence of the extra terminal extensions in AHb3, of which theC-terminus significantly protruded from the core domain(Figure 3). A similar kind of N- and C-terminal extensions haspreviously been reported for some bacterial truncatedhemoglobins (MtrHbN and HGbIV) and protoglobins,suggesting their evolutionary significance.56−58 AHb3 sharesseven conserved residues with the bacterial truncated Hbs inthe heme pocket at topological positions: B9, B10, CD1, E7,E11, E14, and F8 (Figure 3). Except HGbIV, residues lining thedistal pocket of AHb3 were conserved across members of thetrHbO group (Table 2), with minor differences in their spatialorientation (Figure 3), which may lead to variation in ligandbinding regulation mechanisms.Comparative analyses of tunnel systems (well-documented

for truncated hemoglobins in the literature by Bolognesi etal.59,60) in the crystal structure of AHb3 and the bacterialtruncated hemoglobins as well as some established 3/3 globinsshowed significant differences in their topology (Supplemen-tary Figure 2) across the groups, suggestive of diverse ligandmigration pathways for entry and/or exit, which probablyevolved to play the physiological role of a particular globin inthe context in which it functions. The classical 3-on-3 plantglobins like soybean leghemoglobin (Figure 2A) display a well-conserved ligand migration route via the E7 His gate andassociate on the distal side of the heme pocket, required fortheir role in oxygen transport. However, alternate tunnelsystems exist in dimeric plant nonsymbiotic hemoglobins. Forexample, we had shown in an earlier study8 that the 3-on-3globin AHb1 has a novel tunnel connecting the distal heme

Table 2. Residues Lining the Active Site of Plant andBacterial Truncated Hemoglobins

PDB entrya B10 CD1 E7 E11 E14 E15 G8

4C44 Tyr Phe Ala Gln Phe Phe Trp1IDR Tyr Phe Leu Gln Phe Phe Val1DLY Tyr Phe Gln Gln Ala Phe Val1NGK Phe Phe Ala Leu Phe Leu Trp2BKM Tyr Phe Thr Gln Phe Leu Trp4NK1 His Phe Ser Arg Asp Phe Trp1UX8 Tyr Phe Gln Gln Phe Leu Ala21G3 Tyr Phe His Ile Phe Trp Trp

a4C44, A. thaliana AHb3; 1IDR, Mycobacterium tuberculosis trHbN;1DLY, Chlamydomonas eugamentos trHb; 1NGK, M. tuberculosistrHbO; 2BKM, Geobacillus stearothermophilus trHb; 4NK1, Hell’sgate globin IV; 1UX8, Bacillus subtilis trHb; 2IG3, Campylobacter jejunitrHb.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1729

pocket of both the monomers. Such tunnels in nsHbs mayfunction as an intermediate docking site for the incoming ligandthat may also support NO dioxygenase activity. The tunnelsystem in bacterial truncated hemoglobins is however complexand is composed of two orthogonal branches centered at thedistal face of the heme (Bolognesi et al. and SupplementaryFigure 2). The tunnel opens up at two distinct accessible sites

on the protein surface.58,59 The long tunnel (∼20 Å) connectsthe region between the AB and GH corners to the distal site,whereas the short tunnel branch (∼8 Å) holds the regionsbetween the G- and H-helices to the heme. The tunnel iscomposed of mainly hydrophobic residues with a tunnelvolume of 330−360 Å3.58,59 Similar to other bacterial truncatedhemoglobins, AHb3 hosts a two-branch protein matrix tunnel

Figure 2. Water molecules are coordinated in the distal pocket of the AHb3 crystal structure. (A) Overlay of the two crystal structures that displaysno significant differences in the backbone conformation or overall fold: blue for PDB entry 4C44 and green for PDB entry 4C0N. (B) Magnifiedview of the heme pockets in the two structures, with PDB entry 4C44 displaying two water molecules forming a H-bonding network. In PDB entry4C0N, a hydroxyl ligand was observed. (C) Fo − Fc electron density map in the distal site of AHb3 (4C44) best fitted with a water molecule tightlycoordinated to the heme iron in the active site. (D). Comparative spectral imprints of ferric AHb3 wild-type protein under the crystallizationconditions of 4C44 (black) and 4CON (red). (E) pH titration of ferric AHb3 displaying the acid−alkaline transition of met AHb3. The observedpKa between aqua and hydroxyl derivatives was found to be ∼7.8.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1730

system. However, the branches seem to exist on only one sideof the heme center. The residues lining the tunnel are mainlyhydrophobic with a tunnel volume of ∼400−700 Å3. Suchdifferences suggest that AHb3 may not have a role in O2transport or NO dioxygenase function via these tunnels.Deletion of the N- and C-Terminal Extensions in

Arabidopsis Truncated Hemoglobin. To understand thefunctional and structural relevance of the N- and C-terminalextensions of AHb3, we generated truncated versions of AHb3lacking 25 residues from the N-terminus (AHb3-Δ25N), the C-terminus (AHb3-Δ25C), or both (AHb3-Δ25NΔ25C). All thedeleted variants expressed as soluble red protein, suggestingthat the mutants were capable of holding the heme. However,the expression yields of AHb3-Δ25N and AHb3-Δ25NΔ25Cwere very low and were frequently observed to undergo facileaggregation and precipitation during downstream processing.Quaternary State of AHb3 and Its Mutants in

Solution. HPLC Analysis Indicated AHb3 Is a DimericGlobin. It was necessary to inspect the quaternary state ofthe protein in solution, because such states affect the functionof globins. HPLC analysis revealed AHb3 to exhibit an elutiontime equivalent to that of a protein with a molecular mass of∼40 kDa, indicating that the globin is a dimer (Figure 4). The

deletion of 25 residues from the C-terminus resulted in aHPLC profile that indicated the mutant protein to have amolecular mass of ∼29 kDa, indicating an equilibrium mixtureof monomeric and dimeric populations (Figure 4A). However,AHb3-Δ25N and AHb3-Δ25NΔ25C derivatives displayedprotein peaks corresponding to ∼90 kDa, suggesting thepresence of the proteins in the higher oligomeric state (Figure4A). The absence of the N-terminus makes the globin stickyand aggregation-prone, as also suggested by the broad HPLCelution peaks and the associated shoulders (Figure 4A).

A Chemical Cross-Linking Assay Validated the DimericNature of AHb3. To further confirm the effect of thepolypeptide terminal deletions on the quaternary structure ofAHb3, we performed a chemical cross-linking assay. Aglutaraldehyde cross-linking assay is one of the methods widelyused for obtaining preliminary information about thequaternary association of proteins.62 Purified proteins wereincubated with varying concentrations of glutaraldehyde withsubsequent separation by SDS−PAGE (12%). It was observedthat AHb3 predominantly existed in the dimeric state (Figure4B), while AHb3-Δ25C existed as an equilibrium mixture ofmonomer and dimer in solution (Figure 4B), with a higherpopulation of the monomeric state. AHb3-Δ25N and AHb3-

Figure 3. Structural comparison of AHb3 with bacterial truncated hemoglobins. Overlay of the crystal structure of AHb3 (blue) with group I(Mycobacterium trhbN; yellow), group II (Mycobacterium trhbO; green), and group III (C. jejuni trHbP; orange) truncated hemoglobins that displayconserved globin fold architecture. The extended N-termini of Mtb trHbN and AHb3 were observed distinctly. The magnified view of the hemepockets of the globin displays conserved side chains that differ only in their spatial orientation.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1731

Δ25NΔ25C, however, precipitated heavily in the presence ofthe cross-linker and hence did not migrate successfully onSDS−PAGE. Nonetheless, this observation indicated the innateability of these mutants to form higher-order oligomers. Due tothe high aggregation potential observed for the doublytruncated mutant, AHb3-Δ25NΔ25C, it was not investigated

further in detail. The N-terminus thus played a distinct role inmaintaining AHb3 in a dimeric state.

Crystallographic Evidence of Dimerization. The crystalstructure of AHb3 is a monomer in the crystallographicasymmetric unit, but a stable dimeric assembly was observed inthe crystallographic symmetry (Figure 4C). This was also

Figure 4. Analysis of the oligomeric state of AHb3 and its truncated derivatives. (A) Chromatographic elution profiles of AHb3 (green), AHb3-Δ25C (blue), AHb3-Δ25N (magenta), and AHb3-Δ25NΔ25C (cyan) from the HPLC gel filtration column. The graph displays the absorbance at280 nm as a function of retention time in minutes. In the absence of the N-terminus, the globin appears to have attained an oligomeric state. Proteinsof known molecular masses were used as markers for reference (I, ribonuclease A, ∼14 kDa; II, carbonic anhydrase, ∼29 kDa; III, ovalbumin, ∼45kDa; IV, conalbumin, ∼76 kDa). (B and C) The oligomerization potential of AHb3 and AHb3-Δ25C was also assessed using glutaraldehyde as across-linking agent followed by electrophoresis on a 12% SDS−polyacrylamide gel. A concentration-dependent cross-linking assay was performed for(B) wild-type AHb3 and (C) AHb3-Δ25C using varied concentrations of glutaryldehyde ranging from 0.1 to 1%. While AHb3 is predominantly inits dimeric state, AHb3-Δ25C represented a mixed population of monomer and dimer with a higher population of the monomeric state. (D) Surfacerepresentation of dimeric AHb3 in crystallographic symmetry. (E) Close-up of the crystallographic contacts between the two monomers in thedimeric interface that includes H-bonding and electrostatic interactions between residues in the H-helix, F-helix, and G-helix.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1732

predicted by PISA analysis (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html)61 encompassing the N-terminal α-helicalextension in the dimeric interface. The total buried surfacearea in dimeric AHb3 was predicted to be 4841.8 Å2, which ison the order of that seen in other dimeric globins (data notshown). The dimerization interface involved interaction

between the N-terminus and H-helix (Figure 4C) as reportedin detail by Reeder et al.26 and hence not described here.

The N- and C-Terminal Extensions Do Not Influencethe General Biochemical Properties of AHb3, but the N-Terminal Extension Induces Hexacoordination at theHeme Iron. CD Spectroscopy. In an attempt to address the

Table 3. Spectral (Soret peak) and Ligand Binding Kinetic Parameters for AHb3 and Its Mutants

O2 and CO (gaseous ligand) Binding

ferric (Fe3+) ferrous (Fe2+) Fe2+-CO Fe3+-O2 k′CO (μM−1 s−1) k′O2(μM−1 s−1) kO2

(s−1) KO2(μM−1)

AHb3 wt 408 nm 430 nm 421 nm 411 nm 0.013 (0.014a) 0.28 (0.2a) 0.35 (0.3a) 0.8AHb3-Δ25N 411 nm 426 nm 421 nm 412 nm 0.016 0.28 0.40 0.7AHb3-Δ25C 413 nm 424 nm 418 nm 411 nm 0.014 0. 27 0.31 0.87myoglobin 408 nm 434 nm 417 nm 416 nm 0.51 17 15 1.1soybean Lba 408 nm 434 nm 417 nm 417 nm 16b 130b 5.6b 23b

AHb1 411 nm 424 nm 417 nm 416 nm 0.55a 74a 0.12a 616AHb2 412 nm 424 nm 417 nm 416 nm 22a 86a 0.14a 614rice Hb1 409 nm 430 nm 421 nm 412 nm 72c 68c 0.04c 1800

Azide Binding

Soret peak (nm) Q band (nm) Soret peak (nm) Q band (nm)

ferric (Fe3+) Fe3+-azide k′azide (mM−1 s−1)

AHb3 wt 408 542/580 419 540/570 8AHb3-Δ25N 411 540/578 417 541/568 15AHb3-Δ25C 413 540/570 414 538/562 0.06myoglobin 408 503 and minor peaks 421 540/568 3AHb1 411 542/578 418 540/571 0.03AHb2 412 540/578 418 540/571 0.04rice Hb1 409 532/580 411 542/557 0.11

aReported kinetic value from ref 11. bReported kinetic value from ref 75. cReported kinetic value from ref 76.

Figure 5. Spectroscopic properties of AHb3 and its truncated mutants. Overlay of Fe3+ (red), Fe2+ (blue), and Fe2+-CO (green) absorbance spectraof (A) wild-type AHb3, (B) AHb3-Δ25C, and (C) AHb3-Δ25N displaying a single broad Q-band at 554 nm in the ferrous state for wild-type AHb3and AHb3-Δ25C while split Q-bands (535 and 561 nm) for AHb3-Δ25N. The spectral characteristics indicate potential hexacoordination of hemeiron by external/internal ligand in the N-terminal deletion mutant in the ferrous form. pH titration curves displaying the acid−alkaline transition in(D) AHb3-Δ25C and (E) AHb3-Δ25N, with an apparent pK of ∼8.2.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1733

influence of the extended N- and C-termini, if any, on theoverall structural integrity and folding of the protein, CDspectra for the wild type and its mutants were recorded in thefar-UV region (data not shown). Similar CD spectra for thewild-type and mutants proteins, characteristic of all α-helicalfolds, preclude the role of both N- and C-terminal extension inthe overall structural integrity or folding of the protein.Influence of the Mutants on Autoxidation and Heme

Dissociation Rates. The influence of the terminal deletions on

the heme−polypeptide interaction was assessed by measuringautoxidation and heme dissociation rates for AHb3 and itsmutants. All the mutants displayed autoxidation rates (AHb3-Δ25C, 0.91 h−1; AHb3-Δ25N, 0.96 h−1) comparable to that ofthe wild-type protein (0.92 h−1) and much higher than that ofwild-type Mb (0.141 h−1),28 indicating that AHb3 might notplay a role in oxygen transport or storage (SupplementaryFigure 3A). Both AHb3-Δ25N and AHb3-Δ25C readily loseheme in the upper organic layer, which is similar to the case for

Figure 6. Structural features of MD-simulated AHb3 and its mutant (AHb3-ΔN25). (A) Superposition of the backbone for snapshots collectedacross the trajectories, with the wild-type crystal structure (dark blue) as the starting point. Salt bridges stabilizing the pre-A-helix to the protein corein (B) the crystal structure of AHb3 and (C) the MD-simulated structure remain unaffected. (D) Stereoview of the superposed crystal structure(dark blue) and snapshots of MD-simulated trajectories of pre-A-helix-deleted AHb3. The similar backbone conformations indicated that thedeletion of the N-terminus had no significant influence on the overall globin fold or tertiary structure.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1734

the wild-type protein when a heme dissociation assay wasperformed (data not shown). The mutants displayeddissociation rates similar to that of the wild-type protein(Supplementary Figure 3B), indicating that the truncations didnot significantly alter the heme−polypeptide interaction.Stability Studies. The influence of the terminal truncations

on the conformational stability of the globin fold was evaluatedby exposing the wild-type protein and its derivatives to differentdenaturing conditions such as pH and temperature. The changein the Soret peak in response to different pHs ranging from 2 to11 was monitored, and no significant difference was observed intheir profiles or midpoints of transitions (Supplementray Figure4). Similarly, CD measurements at 222 nm suggested that pHinfluenced the changes in secondary structure to the sameextent in all three proteins (Supplementray Figure 4). Thermalstability studies of the wild type and its deleted variantsdisplayed a similar thermal denaturation profile as well whenprobed by CD (Supplementray Figure 4), indicating that thesedeletions did not affect the overall stability of the protein.UV−Vis Spectroscopy. UV−visible absorbance spectra for

AHb3 and its derivatives were measured from 260 to 700 nm.Absorbance spectra of AHb3, AHb3-Δ25C, and AHb3-Δ25Nshowed Soret peak at 408, 409, and 412 nm, respectively(Table 3 and Figure 5). The Q-bands for AHb3 and AHb3-Δ25C were similar, displaying a single asymmetric absorbancepeak at 556 nm in ferrous form and a weak and broadabsorbance band near 635 nm and between 500 and 550 nmfor the ferric protein, typical of pentacoordinate Hbs (Figure5A,B). On the other hand, AHb3-Δ25N showed distinctsplitting in the visible region Q-bands in ferrous form (535 and561 nm), suggesting the presence of some internal or externalligand occupying the active site of ferrous protein making theheme iron hexacoordinated (Figure 5C). It is thus evident thatthe N-terminus of AHb3 helps the globin to attain itspentacoordinate heme iron state that is just sufficientlyhydrated by water molecules (Figure 2A), its absence givingrise to hexacoordinate heme iron. Interestingly, unlike that of

wild-type protein (Figure 2E), pH titration of ferric AHb3-ΔN25 and AHb3-ΔC25 (Figure 5 D,E) displayed a pKa of ∼8.2between the Fe3+-H2O and Fe3+-OH transition, indicating ahigher proportion of distal pocket hydration in these globins.

Polypeptide Terminal Deletions Do Not Influence theGaseous Ligand Binding Kinetics of AHb3 but AffectAzide Binding Kinetics. UV−vis absorption spectra of O2-and CO-bound forms of AHb3-Δ25N and AHb3-Δ25C weresimilar in nature to that of wild-type AHb3, suggesting thatthese termini did not affect equilibrium ligand binding. Toinvestigate the effect on the rate of ligand binding, kineticmeasurements of O2 and CO binding were also taken usingstopped-flow and laser flash photolysis. The rates determinedfor O2 and CO binding for AHb3, AHb3-Δ25N, and AHb3-Δ25C are listed in Table 3 along with the rates for several otherglobins. All the kinetic parameters determined for the mutantswere similar to those of the wild-type globin, indicating that thehexacoordination observed for AHb3-Δ25N was too weak toinfluence kinetics and was probably brought about by watermolecules in the pocket. Further kinetic investigation in thisregard is necessary to delineate such an influence, but it isobvious that the corresponding N- and C-terminal deletionshad no influence on the O2/CO binding rate constants ofAHb3. In fact, it is interesting to note that the kineticparameters of AHb3 and its mutants are unique compared to allothers (Table 3) in that both the association and dissociationrates constants are similar and quite low. The two rateconstants are also similar for Mb but much higher in numbers.Moreover, the oxygen binding affinity of AHb3 was also similarto that of Mb unlike those of the other plant globins, thoughthe low oxygen dissociation rate constant would preclude a rolein oxygen transport unlike Mb.On the other hand, the azide ligand binding kinetics

presented an interesting finding (Table 3). This ligand, theprototype for ferric globins, binds with AHb3 at rates similar tothat of Mb but unlike that of AHb1, AHb2, or rice Hb1, whichare hexacoordinate globins. The N-terminal mutant binds twice

Figure 7. Dynamics of the active site residues in MD-simulated structures of AHb3 and AHb3-ΔN25. (A) Structural dynamics of the residues in theactive site of the wild-type simulated structure (pink) (B) Pre-A-helix-deleted MD simulated trajectories (green), in comparison to the AHb3 crystalstructure (blue). It is evident that in the absence of the N-terminus, AHb3 displays significant changes in the orientations of the key side chains,some of which are indicated by arrows.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1735

as fast. However, the C-terminal mutant hardly binds to azide,as also observed for AHb1 and AHb2. This indicates that the C-terminal extension of AHb3 indeed plays a very significant rolein ligand−protein (and maybe protein−protein) interaction,especially in the ferric state.MD Simulation Studies for Investigating the Roles of

the N-Terminus of AHb3. To explore the role of the pre-A-helix in protein dynamics, extensive MD simulations of the wildtype (with 25 residues of the C-terminus deleted) and AHb3with a deleted pre-A-helix were conducted. The X-ray crystalstructure of AHb3 was used as a starting point for thesimulations. The stability of the trajectories was calculated incomparison to the crystal structure of AHb3. The rmsd of thelast snapshot of the trajectories and the X-ray crystallographicstructure varied from 1 to 1.3 Å for the wild type and from 1.9to 2.6 Å for AHb3-Δ25N. For wild-type simulations, analysis ofthe MD trajectories showed that the relative movement of theprotein backbone does not change significantly with a stableconformation of the pre-A-helix across the trajectories (Figure6A). Salt bridges between residues in the pre-A-helix and theH-helix remained unaffected throughout the simulations, whichmay contribute to its conformational rigidity (Figure 6B,C). Incomparison to the wild-type simulations, minor changes in theprotein backbone was observed when the pre-A-helix wasdeleted from the protein core (AHb3-Δ25N) (Figure 6D).Significant changes in the dynamics of protein structure wereobserved around E- and F-helices and CD and EF loop regions(Figure 6D). Fluctuations in these regions could be importantfor the regulation of access of the ligand to the heme pocket bymodulating ligand migration cavities and the heme pocketenvironment. The changes might even be of further significancein the quaternary interactions of the mutant protein becauseAHb3-Δ25N was observed experimentally to oligomerize andaggregate.To identify the local changes in the heme pocket architecture

through the course of simulations, series of snapshots collectedat different intervals for wild-type and AHb3-Δ25N simulationswere compared to that of the crystal structure of AHb3.Interestingly, deletion of the pre-A-helix introduced anoticeable change in the dynamics of residues shielding theheme pocket. Major fluctuations were observed for GlnE11,TyrB10, and PheE15 (Figure 7). In comparison to the wildtype, several transitions of open and closed events wereobserved for Phe36, Phe74 (E14), Phe75 (E15), and Phe143 in

AHb3-ΔN25, across the trajectories (Figure 7). Similar openingand closing events for PheE15 was previously reported for M.tuberculosis hemoglobin N, where it has been implicated toregulate access of the ligand to the heme pocket and in turn itsfunction.63

To further gain insights into the dynamics of ligandmigration cavities imposed by deleting the pre-A-helix inAHb3, extensive cavity analysis was conducted for both thesimulated structures in comparison to the crystal structure. Ouranalysis showed that the deletion of the pre-A-helix introducedsignificant alterations into the ligand diffusion pathway byproviding alternate pathways to the heme active site (Figure 8).Such dynamics in ligand migration cavities upon deletion of thepre-A-helix suggests its functional significance in regulatingligand entry and/or exit.

■ DISCUSSION

Ever since the discovery of nonsymbiotic hemoglobins inplants, a large fraction of research has been directed to gaindeeper insights into their physiological role. A plethora ofinformation has been generated for class I and class IInonsymbiotic hemoglobins; these have been implicated inperforming diverse physiological functions such as oxygensensing, NO detoxification, etc.16,64 Detailed crystallographicinvestigations of class I nonsymbiotic hemoglobins supportedNO dioxygenase activity.8 In contrast to class I and class IInonsymbiotic hemoglobins, research on plant truncatedhemoglobins is still in its infancy. Detailed structural andfunctional investigation for this class of protein is therefore theneed of the hour to understand their biological role. Ourcrystallographic investigation of truncated hemoglobin from themodel plant A. thaliana attempted to provide new insights intoits biological role from a structural perspective. The tertiarystructure of the protein displayed a bacterial-like 2/2 α-helicaltruncated globin fold with pentacoordinated heme. In unison,Reeder et al.26 in 2014 had also determined and reported thecrystal structure of Arabidopsis truncated hemoglobin at 1.7 Å.Detailed structural comparison of the two structures displayedidentical globin folds and active site architectures. In the case ofthe previous structure, however, the heme iron was coordinatedto a hydroxyl ligand, whose role in biological function or ligandbinding is not yet understood. Spectroscopically, however, weobserved AHb3 to be pentacoordinated in solution, and only

Figure 8. Structural illustration of the tunnel systems in MD-simulated trajectories. Representation of dynamic tunnels (green mesh) for (A) wild-type AHb3 and (B) AHb3-Δ25N. The absence of the pre-A-helix in the mutant changes the topology of tunnels.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1736

N-terminal deletion resulted in a spectrum typical ofhexacoordinated globin. The active site of AHb3 in the currentstructure was occupied by two water molecules stabilized by H-bonding and electrostatic interactions with GlnE11 inassistance with TrpG8 and TyrCD1. The spectroscopicproperty of the globin was thus more in tune with what weobserved in the structure presented here. The presence of suchloosely bound solvent molecules in the heme pocket, togetherwith the possibility of the presence of hydroxide as shown byReeder et al.,26 may rationalize the transient observations ofdifferent coordination states in the globin reported previously.11

Moreover, these observations indicate that the appearance ofabsorbance spectra typical of hexacoordinate globins may notalways relate to an internal amino acid side chain resulting inhexacoordination; it could simply arise due to the coordinationof extrinsic solvent molecules, as seen here for the N-terminalmutant of AHb3.Water molecules or hydroxyl ligand in the heme pocket of

AHb3, as described above, and their rearrangement andelectrostatic interaction with heme pocket side chains mightcontribute to the biological function of AHb3. Such watermolecules have been implicated in the regulation of ligandbinding in other heme proteins, including myoglobin, heme-oxygenase, and a few truncated hemoglobins.52 Comparativeanalysis of the crystal structure of AHb3 with bacterialtruncated hemoglobins in the investigation presented hereindeed displayed high structural conservation across the familywith well-conserved distal active site residues, suggesting asimilar evolutionary trail and a similar function. The bacterialtruncated hemoglobins were implicated as playing a role innitric oxide detoxification, oxygen or nitric oxide sensing, andlong-term ligand storage,10,65,66 and the same might be true forplant truncated hemoglobins, as well.In contrast to bacterial truncated hemoglobins, plant

truncated hemoglobins displayed extra N- and C-terminalappendages flanking the truncated globin core. The structuraland functional significance of these appendages in planthemoglobins in not known, though they have been implicatedin ligand sensing, nitric oxide scavenging, and interaction with apartner molecule in vivo in protoglobins and otherglobins.56,67−69 The N-terminal α-helical extension is highlyconserved across plant truncated hemoglobins (SupplementaryFigure 1), suggestive of its physiological significance. We haveshown that this extension mainly influences the quaternarystructure of the globin and its distal pocket hydration. This,thus, presents a unique feature common to all novel planthemoglobins. In the investigation presented here, the C-terminus was found to influence the ligand (azide) bindingproperties of ferric AHb3 to a larger extent than the N-terminus; however, MD simulations suggested the probablerole of the N-terminus in regulating NO detoxificationanalogous to M. tuberculosis truncated hemoglobin N.Computational analysis of the protein sequence of AHb3 andother plant truncated hemoglobins predicted both N- and C-termini to be structurally disordered, which could explain themissing density of C-termini in the crystal structure. Suchproteins with intrinsically disordered regions (IDRs) representa broad class of polypeptides, mostly inhabiting eukaryoticgenomes.27,70,71 These proteins are widespread in the plantkingdoms and are predicted to play important role in severalregulatory and environmental signals.27 The disordered C-terminus of AHb3 may be essential for the recognition andbinding of interacting protein partners as is known for several

intrinsically disordered proteins27,71 and predicted for otherglobins with such extensions. The survival of the C-terminus inAHb3 over the course of evolution suggests that it must havean important physiological role to play, though it seems to havea minimal influence on the general structural properties of theglobins. We show here that the C-terminus influences ligandbinding in the ferric state and hence indeed plays a role inligand−protein interaction. The floppy C-terminus may help inregulating the entry and/or exit to the open water cavity. Thefact that the C-terminus influences the monomer−dimerequilibrium of AHb3 also indicates its role in protein−proteininteraction. The fact that it protrudes out of the core of theglobin fold [modeled structure (Figures 1E and 3)] also lendssupport to its putative role as an interacting domain. Theseobservations present an immense scope of investigation inAHb3.Flanking IDRs of plant proteins are often correlated with the

presence of multiple PTM sites in these regions.27,72 Inagreement with these findings, in silico analysis of Arabidopsistruncated hemoglobins indeed displayed the occurrence ofacetylation and other post-translational modification sites inboth disordered N- and C-termini (data not shown). In fact,Johnson et al.73 recently showed the presence of acetylationsites at the N-terminal peptides of Chlamydomonas truncatedhemoglobin 1, further supporting the hypothesis, though thephysiological significance of these modifications is still not wellunderstood.We have performed MD simulations with supportive

experimental evidence to address the role of these extendedtermini in Arabidopsis truncated hemoglobin. A detailedbiochemical and biophysical fingerprint of AHb3-Δ25N andAHb3-Δ25C obliterated their role in maintaining the globinfold and structural integrity of the protein. Both the deletedmutants displayed similar gaseous ligand binding fingerprints,further precluding their role in gaseous ligand binding.Interestingly, AHb3-Δ25N displayed distinct splitting in thevisible region of Q-bands in the ferrous form, suggesting thepresence of some ligand occupying the active site of ferrousprotein forming a weak covalent bond. The kinetic inves-tigation, however, indicated that the active site was occupied byhydroxyl ligand or weakly coordinated water molecules and notany heme pocket side chain. In spite of the presence of an openheme pocket, the association rate constant for ligand bindingwas found to be low, which could be due to the tightly boundwater molecule in the active site of the protein acting as abarrier to ligand binding. A large number of potential aminoacid side chains in the distal pocket (Results) may help in suchstabilization. At the same time, once a ligand binds, because ofthe strong stabilization of the ligand by distal pocket residues,the dissociation rate constant of the bound ligand was alsofound to be low. These factors provide AHb3 with a uniquekinetic characteristic. Even though the association of the ligandin the ferric state was found to be low in the C-terminallydeleted mutant, the N- and C-terminally deleted mutants didnot influence ligand binding of the gaseous ligands. Thegaseous ligand binding may play a more significant role underabnormal conditions like stress. The C-terminus thus may playa role in preventing overhydration of the active site, probablyby blocking tunnels in the protein matrix that facilitate facilesolvent entry of certain ligands like azide that bind in the ferricstate.The most significant influence of the N-terminus was its

effect on the quaternary state of AHb3. In its absence, the

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1737

globin cannot maintain itself in the dimeric state and tends tobe polymeric in nature. The N-terminal extension thus mightplay a crucial role in dimeric interaction of the globin.Dimerization of globins has returned as a recurring theme formany of the novel globins and must be important for theirphysiological function and regulation.23,74

Comparative analysis of MD-simulated trajectories of wild-type AHb3 and AHb3 with deleted N-termini displayed majorfluctuations around E- and F-helices. A distinct change in theconformation of residues lining the heme pocket was observedwhen the pre-A-helix was deleted from the protein core. Severaltransitions of open and closed events were observed for Phe36,Phe74 (E14), Phe75 (E15), and Phe143. Similar shifting of theopen and closed conformations of these residues was previouslyreported for M. tuberculosis hemoglobin N, where these havebeen implicated in the regulation of access of the ligand to theheme pocket and in turn its function.63,69 The importance ofthe PheE15 gate in modulating the NOD function of MtrHbNis well-documented, in which it plays a key role in mediatingthe access of NO to the oxygenated protein. Analysis of ligandmigration dynamics across the simulated models displayedsignificant divergence in the migration routes that mayinfluence its biological role. In agreement to the previousleads from Mycobacterium trHbN, the pre-A-helix could beessential for maintaining NOD activity in AHb3, thoughdetailed experimental validation is required to further validatethis role.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.bio-chem.5b01013.

Multiple-sequence alignment of the N-terminal extensionof multiple plant truncated hemoglobins from differentspecies (Supplementary Figure 1), a cartoon representa-tion of the ligand migration protein matrix tunnels in afew 3-on-3 and 2-on-2 globins (Supplementary Figure 2),the effect of N- and C-terminal deletions on thephysicochemical properties of AHb3 determined byrecording the rate of change in autoxidation and hemedissociation of AHb3 and its deleted variants (Supple-mentary Figure 3), and pH- and temperature-dependentstability profile of wild-type AHb3 with respect to thedeleted mutants (Supplementary Figure 4) (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: suman.kundu@south.du.ac.in. Telephone: +91-11-24117460. Fax: +91-11-24115270.*E-mail: jagreet@south.du.ac.in.FundingFinancial aid from the University of Delhi and the Departmentof Science and Technology, Government of India, under thePURSE program [Dean(R)/2009/868] (J.K. and S.K.) isacknowledged. The University of Delhi is duly acknowledgedfor R&D funding (DRCH/R&D/2013-14/4155) (J.K. andS.K.). Fellowship support from UGC, Government of India, toN.M. and R.A. and CSIR, Government of India, to S.U. are alsoappreciated.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The Macromolecular Crystallography Unit (MCU), InstituteInstrumentation Centre, IIT Roorkee (PK), is acknowledgedfor X-ray diffraction experiments. The Central InstrumentationFacility at University of Delhi South Campus is appreciated forthe help with circular dichroism. Dr. Monica Sundd’s laboratoryat the National Institute of Immunology (New Delhi, India) isacknowledged for help with HPLC.

■ ABBREVIATIONS

Hb, hemoglobin; wt, wild type; nsHbs, nonsymbiotichemoglobins; trHbs, truncated hemoglobins; ptrHbs, planttruncated hemoglobins; 3/3Hb, 3-on-3 globin; 2/2Hb, 2-on-2globin; AHb1, Arabidopsis hemoglobin 1; AHb2, Arabidopsishemoglobin 2; AHb3, Arabidopsis hemoglobin 3; MD,molecular dynamics; CD, circular dichroism; HPLC, high-performance liquid chromatography; IDRs, intrinsically dis-ordered regions; NO dioxygenase, nitric oxide dioxygenase.

■ REFERENCES(1) Hardison, R. C. (1996) A brief history of hemoglobins: plant,animal, protist, and bacteria. Proc. Natl. Acad. Sci. U. S. A. 93, 5675−5679.(2) Hardison, R. (1998) Hemoglobins from bacteria to man:evolution of different patterns of gene expression. J. Exp Biol. 201,1099−1117.(3) Kundu, S., Trent, J. T., 3rd, and Hargrove, M. S. (2003) Plants,humans and hemoglobins. Trends Plant Sci. 8, 387−393.(4) Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G.,and Klucas, R. V. (1998) Plant hemoglobins. Plant Physiol 118, 1121−1125.(5) Sowa, A. W., Guy, P. A., Sowa, S., and Hill, R. D. (1999)Nonsymbiotic haemoglobins in plants. Acta Biochim Pol 46, 431−445.(6) Garrocho-Villegas, V., Gopalasubramaniam, S. K., andArredondo-Peter, R. (2007) Plant hemoglobins: what we know sixdecades after their discovery. Gene 398, 78−85.(7) Garrocho-Villegas, V., Bramaniam, S. K., and Arredondo-Peter, R.(2007) Plant Hemoglobins:What we know six decades after theirdiscovery. Gene 398, 78−85.(8) Mukhi, N., Dhindwal, S., Uppal, S., Kumar, P., Kaur, J., andKundu, S. (2013) X-ray crystallographic structural characteristics ofArabidopsis hemoglobin I and their functional implications. Biochim.Biophys. Acta, Proteins Proteomics 1834, 1944−1956.(9) Wittenberg, J. B., Bolognesi, M., Wittenberg, B. A., and Guertin,M. (2002) Truncated hemoglobins: a new family of hemoglobinswidely distributed in bacteria, unicellular eukaryotes, and plants. J. Biol.Chem. 277, 871−874.(10) Kumar, A., Nag, M., and Basak, S. (2013) Truncated or 2/2hemoglobins: A new class of globins with novel structure and function.Journal of proteins and proteomics 4, 45−64.(11) Watts, R. A., Hunt, P. W., Hvitved, A. N., Hargrove, M. S.,Peacock, W. J., and Dennis, E. S. (2001) A hemoglobin from plantshomologous to truncated hemoglobins of microorganisms. Proc. Natl.Acad. Sci. U. S. A. 98, 10119−10124.(12) Trevaskis, B., Watts, R. A., Andersson, C. R., Llewellyn, D. J.,Hargrove, M. S., Olson, J. S., Dennis, E. S., and Peacock, W. J. (1997)Two hemoglobin genes in Arabidopsis thaliana: the evolutionaryorigins of leghemoglobins. Proc. Natl. Acad. Sci. U. S. A. 94, 12230−12234.(13) Hoy, J. A., Robinson, H., Trent, J. T., 3rd, Kakar, S., Smagghe, B.J., and Hargrove, M. S. (2007) Plant hemoglobins: a molecular fossilrecord for the evolution of oxygen transport. J. Mol. Biol. 371, 168−179.(14) Hoy, J. A., and Hargrove, M. S. (2008) The structure andfunction of plant hemoglobins. Plant Physiol. Biochem. 46, 371−379.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1738

(15) Smagghe, B. J., Hoy, J. A., Percifield, R., Kundu, S., Hargrove, M.S., Sarath, G., Hilbert, J. L., Watts, R. A., Dennis, E. S., Peacock, W. J.,Dewilde, S., Moens, L., Blouin, G. C., Olson, J. S., and Appleby, C. A.(2009) Review: correlations between oxygen affinity and sequenceclassifications of plant hemoglobins. Biopolymers 91, 1083−1096.(16) Perazzolli, M., Dominici, P., Romero-Puertas, M. C., Zago, E.,Zeier, J., Sonoda, M., Lamb, C., and Delledonne, M. (2004)Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxidebioactivity. Plant Cell 16, 2785−2794.(17) Hebelstrup, K. H., van Zanten, M., Mandon, J., Voesenek, L. A.,Harren, F. J., Cristescu, S. M., Moller, I. M., and Mur, L. A. (2012)Haemoglobin modulates NO emission and hyponasty under hypoxia-related stress in Arabidopsis thaliana. J. Exp. Bot. 63, 5581−5591.(18) Hebelstrup, K. H., and Jensen, E. O. (2008) Expression of NOscavenging hemoglobin is involved in the timing of bolting inArabidopsis thaliana. Planta 227, 917−927.(19) Spyrakis, F., Bruno, S., Bidon-Chanal, A., Luque, F. J.,Abbruzzetti, S., Viappiani, C., Dominici, P., and Mozzarelli, A.(2011) Oxygen binding to Arabidopsis thaliana AHb2 nonsymbiotichemoglobin: evidence for a role in oxygen transport. IUBMB Life 63,355−362.(20) Wang, Y., Elhiti, M., Hebelstrup, K. H., Hill, R. D., and Stasolla,C. (2011) Manipulation of hemoglobin expression affects Arabidopsisshoot organogenesis. Plant Physiol. Biochem. 49, 1108−1116.(21) Vigeolas, H., Huhn, D., and Geigenberger, P. (2011)Nonsymbiotic hemoglobin-2 leads to an elevated energy state and toa combined increase in polyunsaturated fatty acids and total oilcontent when overexpressed in developing seeds of transgenicArabidopsis plants. Plant Physiol. 155, 1435−1444.(22) Hebelstrup, K. H., Hunt, P., Dennis, E., Jensen, S. B., andJensen, E. O. (2006) Hemoglobin is essential for normal growth ofArabidopsis organs. Physiol. Plant. 127, 157−166.(23) Kakar, S., Sturms, R., Tiffany, A., Nix, J. C., DiSpirito, A. A., andHargrove, M. S. (2011) Crystal structures of Parasponia and Tremahemoglobins: differential heme coordination is linked to quaternarystructure. Biochemistry 50, 4273−4280.(24) Goodman, M. D., and Hargrove, M. S. (2001) Quaternarystructure of rice nonsymbiotic hemoglobin. J. Biol. Chem. 276, 6834−6839.(25) Hargrove, M. S., Brucker, E. A., Stec, B., Sarath, G., Arredondo-Peter, R., Klucas, R. V., Olson, J. S., and Phillips, G. N. (2000) CrystalStructure of a nonsymbiotic plant hemoglobin. Structure 8, 1005−1014.(26) Reeder, B. J., and Hough, M. A. (2014) The structure of a class3 nonsymbiotic plant haemoglobin from Arabidopsis thaliana reveals anovel N-terminal helical extension. Acta Crystallogr., Sect. D: Biol.Crystallogr. 70, 1411−1418.(27) Kurotani, A., Tokmakov, A. A., Kuroda, Y., Fukami, Y.,Shinozaki, K., and Sakurai, T. (2014) Correlations between predictedprotein disorder and post-translational modifications in plants.Bioinformatics 30, 1095.(28) Uppal, S., Salhotra, S., Mukhi, N., Zaidi, F. K., Seal, M., Dey, S.G., Bhat, R., and Kundu, S. (2015) Significantly enhanced hemeretention ability of myoglobin engineered to mimic the third covalentlinkage by nonaxial histidine to heme (vinyl) in synechocystishemoglobin. J. Biol. Chem. 290, 1979−1993.(29) Otwinowski, Z., and Minor, W. (1997) Processing of X-raydiffraction data collected in oscillation mode. Methods Enzymol. 276,307−326.(30) Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K., andNoble, M. (2002) The CCP4 molecular-graphics project. ActaCrystallogr., Sect. D: Biol. Crystallogr. 58, 1955−1957.(31) Collaborative Computational Project, No. 4 (1994) The CCP4suite: programs for protein crystallography. Acta Crystallogr., Sect. D:Biol. Crystallogr. 50, 760−763.(32) Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S.,Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A.(2011) REFMAC5 for the refinement of macromolecular crystalstructures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67, 355−367.

(33) Winn, M. D., Murshudov, G. N., and Papiz, M. Z. (2003)Macromolecular TLS refinement in REFMAC at moderate reso-lutions. Methods Enzymol. 374, 300−321.(34) Emsley, P., and Cowtan, K. (2004) Coot: model-building toolsfor molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60,2126−2132.(35) DeLano, W. L. (2002) The PyMOL molecular graphics system,DeLano Scientific, San Carlos, CA.(36) Bernstein, F. C., Koetzle, T. F., Williams, G. J., Meyer, E. F., Jr.,Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., andTasumi, M. (1977) The Protein Data Bank: a computer-based archivalfile for macromolecular structures. J. Mol. Biol. 112, 535−542.(37) Olson, J. S. (1981) Stopped-flow, rapid mixing measurements ofligand binding to hemoglobin and red cells. Methods Enzymol. 76,631−651.(38) Hargrove, M. S., and Olson, J. S. (1996) The stability ofholomyoglobin is determined by heme affinity. Biochemistry 35,11310−11318.(39) Hargrove, M. S. (2000) A flash photolysis method tocharacterize hexacoordinate hemoglobin kinetics. Biophys. J. 79,2733−2738.(40) Draghi, F., Miele, A. E., Travaglini-Allocatelli, C., Vallone, B.,Brunori, M., Gibson, Q. H., and Olson, J. S. (2002) Controlling ligandbinding in myoglobin by mutagenesis. J. Biol. Chem. 277, 7509−7519.(41) Kundu, S., Snyder, B., Das, K., Chowdhury, P., Park, J., Petrich,J. W., and Hargrove, M. S. (2002) The leghemoglobin proximal hemepocket directs oxygen dissociation and stabilizes bound heme. Proteins:Struct., Funct., Genet. 46, 268−277.(42) Kundu, S., and Hargrove, M. S. (2003) Distal heme pocketregulation of ligand binding and stability in soybean leghemoglobin.Proteins: Struct., Funct., Genet. 50, 239−248.(43) Teale, F. W. (1959) Cleavage of the haem-protein link by acidmethylethylketone. Biochim. Biophys. Acta 35, 543.(44) Hargrove, M. S., Singleton, E. W., Quillin, M. L., Ortiz, L. A.,Phillips, G. N., Jr., Olson, J. S., and Mathews, A. J. (1994) His64(E7)–> Tyr apomyoglobin as a reagent for measuring rates of hemindissociation. J. Biol. Chem. 269, 4207−4214.(45) Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., and Zhang, Y.(2014) The I-TASSER Suite: protein structure and functionprediction. Nat. Methods 12, 7−8.(46) Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E.,Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005)Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781−1802.(47) Brooks, B. R., Brooks, C. L., 3rd, Mackerell, A. D., Jr., Nilsson,L., Petrella, R. J., Roux, B., Won, Y., Archontis, G., Bartels, C., Boresch,S., Caflisch, A., Caves, L., Cui, Q., Dinner, A. R., Feig, M., Fischer, S.,Gao, J., Hodoscek, M., Im, W., Kuczera, K., Lazaridis, T., Ma, J.,Ovchinnikov, V., Paci, E., Pastor, R. W., Post, C. B., Pu, J. Z., Schaefer,M., Tidor, B., Venable, R. M., Woodcock, H. L., Wu, X., Yang, W.,York, D. M., and Karplus, M. (2009) CHARMM: the biomolecularsimulation program. J. Comput. Chem. 30, 1545−1614.(48) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visualmolecular dynamics. J. Mol. Graphics 14, 27−38.(49) Sehnal, D., Svobodova Varekova, R., Berka, K., Pravda, L.,Navratilova, V., Banas, P., Ionescu, C. M., Otyepka, M., and Koca, J.(2013) MOLE 2.0: advanced approach for analysis of biomacromo-lecular channels. J. Cheminf. 5, 39.(50) Schmidtke, P., Bidon-Chanal, A., Luque, F. J., and Barril, X.(2011) MDpocket: open-source cavity detection and characterizationon molecular dynamics trajectories. Bioinformatics 27, 3276−3285.(51) Droghetti, E., Nicoletti, F. P., Bonamore, A., Boechi, L., ArroyoManez, P., Estrin, D. A., Boffi, A., Smulevich, G., and Feis, A. (2010)Heme pocket structural properties of a bacterial truncated hemoglobinfrom Thermobifida fusca. Biochemistry 49, 10394−10402.(52) Ouellet, Y. H., Daigle, R., Lague, P., Dantsker, D., Milani, M.,Bolognesi, M., Friedman, J. M., and Guertin, M. (2008) Ligandbinding to truncated hemoglobin N from Mycobacterium tuberculosis

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1739

is strongly modulated by the interplay between the distal heme pocketresidues and internal water. J. Biol. Chem. 283, 27270−27278.(53) Ouellet, H., Milani, M., LaBarre, M., Bolognesi, M., Couture, M.,and Guertin, M. (2007) The roles of Tyr(CD1) and Trp(G8) inMycobacterium tuberculosis truncated hemoglobin O in ligandbinding and on the heme distal site architecture. Biochemistry 46,11440−11450.(54) Ilari, A., Kjelgaard, P., von Wachenfeldt, C., Catacchio, B.,Chiancone, E., and Boffi, A. (2007) Crystal structure and ligandbinding properties of the truncated hemoglobin from Geobacillusstearothermophilus. Arch. Biochem. Biophys. 457, 85−94.(55) Ouellet, Y., Milani, M., Couture, M., Bolognesi, M., and Guertin,M. (2006) Ligand interactions in the distal heme pocket ofMycobacterium tuberculosis truncated hemoglobin N: roles ofTyrB10 and GlnE11 residues. Biochemistry 45, 8770−8781.(56) Jamil, F., Teh, A. H., Schadich, E., Saito, J. A., Najimudin, N.,and Alam, M. (2014) Crystal structure of truncated haemoglobin froman extremely thermophilic and acidophilic bacterium. J. Biochem. 156,97−106.(57) Ciaccio, C., Pesce, A., Tundo, G. R., Tilleman, L., Bertolacci, L.,Dewilde, S., Moens, L., Ascenzi, P., Bolognesi, M., Nardini, M., andColetta, M. (2013) Functional and structural roles of the N-terminalextension in Methanosarcina acetivorans protoglobin. Biochim. Biophys.Acta, Proteins Proteomics 1834, 1813−1823.(58) Milani, M., Pesce, A., Ouellet, Y., Ascenzi, P., Guertin, M., andBolognesi, M. (2001) Mycobacterium tuberculosis hemoglobin Ndisplays a protein tunnel suited for O2 diffusion to the heme. EMBO J.20, 3902−3909.(59) Boechi, L., Marti, M. A., Milani, M., Bolognesi, M., Luque, F. J.,and Estrin, D. A. (2008) Structural determinants of ligand migration inMycobacterium tuberculosis truncated hemoglobin O. Proteins: Struct.,Funct., Genet. 73, 372−379.(60) Pesce, A., Milani, M., Nardini, M., and Bolognesi, M. (2008)Mapping heme-ligand tunnels in group I truncated(2/2) hemoglobins.Methods Enzymol. 436, 303−315.(61) Krissinel, E., and Henrick, K. (2007) Inference of macro-molecular assemblies from crystalline state. J. Mol. Biol. 372, 774−797.(62) Fadouloglou, V. E., Kokkinidis, M., and Glykos, N. M. (2008)Determination of protein oligomerization state: two approaches basedon glutaraldehyde crosslinking. Anal. Biochem. 373, 404−406.(63) Oliveira, A., Singh, S., Bidon-Chanal, A., Forti, F., Marti, M. A.,Boechi, L., Estrin, D. A., Dikshit, K. L., and Luque, F. J. (2012) Role ofPheE15 gate in ligand entry and nitric oxide detoxification function ofmycobacterium tuberculosis truncated hemoglobin N. PLoS One 7,e49291.(64) Seregelyes, C., and Dudits, D. (2003) Phytoglobins and nitricoxide: new partners in an old signalling system in plants. Acta Biol.Hung. 54, 15−25.(65) Couture, M., Yeh, S. R., Wittenberg, B. A., Wittenberg, J. B.,Ouellet, Y., Rousseau, D. L., and Guertin, M. (1999) A cooperativeoxygen-binding hemoglobin from Mycobacterium tuberculosis. Proc.Natl. Acad. Sci. U. S. A. 96, 11223−11228.(66) Ouellet, H., Ouellet, Y., Richard, C., Labarre, M., Wittenberg, B.,Wittenberg, J., and Guertin, M. (2002) Truncated hemoglobin HbNprotects Mycobacterium bovis from nitric oxide. Proc. Natl. Acad. Sci.U. S. A. 99, 5902−5907.(67) Freitas, T. A., Saito, J. A., Hou, S., and Alam, M. (2005) Globin-coupled sensors, protoglobins, and the last universal commonancestor. J. Inorg. Biochem. 99, 23−33.(68) Saito, J. A., Wan, X., Lee, K. S., Hou, S., and Alam, M. (2008)Globin-coupled sensors and protoglobins share a common signalingmechanism. FEBS Lett. 582, 1840−1846.(69) Lama, A., Pawaria, S., Bidon-Chanal, A., Anand, A., Gelpi, J. L.,Arya, S., Marti, M., Estrin, D. A., Luque, F. J., and Dikshit, K. L. (2009)Role of Pre-A motif in nitric oxide scavenging by truncatedhemoglobin, HbN, of Mycobacterium tuberculosis. J. Biol. Chem.284, 14457−14468.

(70) Oldfield, C. J., and Dunker, A. K. (2014) Intrinsically disorderedproteins and intrinsically disordered protein regions. Annu. Rev.Biochem. 83, 553−584.(71) van der Lee, R., Buljan, M., Lang, B., Weatheritt, R. J.,Daughdrill, G. W., Dunker, A. K., Fuxreiter, M., Gough, J., Gsponer, J.,Jones, D. T., Kim, P. M., Kriwacki, R. W., Oldfield, C. J., Pappu, R. V.,Tompa, P., Uversky, V. N., Wright, P. E., and Babu, M. M. (2014)Classification of intrinsically disordered regions and proteins. Chem.Rev. 114, 6589−6631.(72) Kurotani, A., Yamada, Y., Shinozaki, K., Kuroda, Y., and Sakurai,T. (2015) Plant-PrAS: a database of physicochemical and structuralproperties and novel functional regions in plant proteomes. Plant CellPhysiol. 56, e11.(73) Johnson, E. A., Rice, S. L., Preimesberger, M. R., Nye, D. B.,Gilevicius, L., Wenke, B. B., Brown, J. M., Witman, G. B., and Lecomte,J. T. (2014) Characterization of THB1, a Chlamydomonas reinhardtiitruncated hemoglobin: linkage to nitrogen metabolism and identi-fication of lysine as the distal heme ligand. Biochemistry 53, 4573−4589.(74) Boffi, A., and Chiancone, E. (2004) Evaluating cooperativity indimeric hemoglobins. Methods Enzymol. 379, 55−64.(75) Hargrove, M. S., Barry, J. K., Brucker, E. A., Berry, M. B.,Phillips, G. N., Jr., Olson, J. S., Arredondo-Peter, R., Dean, J. M.,Klucas, R. V., and Sarath, G. (1997) Characterization of recombinantsoybean leghemoglobin a and apolar distal histidine mutants. J. Mol.Biol. 266, 1032−1042.(76) Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F.,Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Rice hemoglobins.Gene cloning, analysis, and O2-binding kinetics of a recombinantprotein synthesized in Escherichia coli. Plant Physiol 115, 1259−1266.

Biochemistry Article

DOI: 10.1021/acs.biochem.5b01013Biochemistry 2016, 55, 1724−1740

1740