ARTICLE pubs.acs.org/JPCB
Histidine E7 Dynamics Modulates Ligand Exchange between Distal Pocket and Solvent in AHb1 from Arabidopsis thaliana Francesca Spyrakis,†,‡ Serena Faggiano,§ Stefania Abbruzzetti,||,^ Paola Dominici,^ Elena Cacciatori,^ Alessandra Astegno,^ Enrica Droghetti,# Alessandro Feis,# Giulietta Smulevich,# Stefano Bruno,§ Andrea Mozzarelli,‡,§ Pietro Cozzini,†,‡ Cristiano Viappiani,*,|| A. Bidon-Chanal,3 and F. Javier Luque3 †
Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Universita degli Studi di Parma, Italy INBB, Istituto Nazionale Biostrutture e Biosistemi, Consorzio Interuniversitario, Roma, Italia § Dipartimento di Biochimica e Biologia Molecolare, Universita degli Studi di Parma, Italy Dipartimento di Fisica, Universita degli Studi di Parma, NEST, Istituto Nanoscienze-CNR, Italy ^ Dipartimento di Biotecnologie, Universita degli Studi di Verona, Italy # Dipartimento di Chimica “Ugo Schiff”, Universita degli Studi di Firenze, Italy 3 Departament de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain
)
‡
bS Supporting Information ABSTRACT: The distal His residue in type 1 nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana plays a fundamental role in stabilizing the bound ligand. This residue might also be important in regulating the accessibility to the distal cavity. The feasibility of this functional role has been examined using a combination of experimental and computational methods. We show that the exchange of CO between the solvent and the reaction site is modulated by a swinging motion of the distal His, which opens a channel that connects directly the distal heme pocket with the solvent. The nearby PheB10 aids the distal His in the stabilization of the bound ligand by providing additional protection against solvation. Overall, these findings provide evidence supporting the functional implications of the conformational rearrangement found for the distal His in AHb1, which mimics the gating role proposed for the same residue in myoglobin.
’ INTRODUCTION Plants contain three types of hemoglobins which are not associated with nitrogen fixing bacteria and have been accordingly termed nonsymbiotic hemoglobins (nsHbs).1,2 Class 1 nsHbs display very high oxygen affinity, with dissociation constants in the nanomolar range. They are expressed under hypoxia, osmotic stress, nutrient deprivation, cold stress, rhizobial infection, nitric oxide exposure, and fungal infection and may represent some of the defense strategies against stress with yet partly unknown mechanisms.1,35 Class 1 nsHbs are part of a metabolic pathway involving nitric oxide (NO), which may provide an alternative type of respiration to mitochondrial electron transport under limiting oxygen concentrations.1 It was suggested that class 1 nsHbs under hypoxic conditions act as part of a soluble, terminal, NO dioxygenase system, yielding nitrate from the reaction of oxyHb with NO.6 Class 1 nsHb from Arabidopsis thaliana (AHb1) was reported to have a NO dioxygenase activity in vitro, a property which may reduce levels of NO under hypoxic stress also in vivo.7,8 Rapid nitrate accumulation is accompanied by NO-dependent oxidation (oxygenated to ferric) of AHb1. In the presence of reducing agents such as NADPH, the Fe3þ heme can be reduced to allow continuous nitrate accumulation in the presence of excess NO. r 2011 American Chemical Society
Oxidized nsHb may be reduced by a mixture of NADH and FAD or by a methemoglobin reductase, whose identity remains so far elusive.9 As for other class 1 nsHbs, AHb1 combines its extremely high O2 affinity with an internal hexacoordination of the distal histidine HisE7.10,11 Previous resonance Raman (RR) studies on carbon monoxide complexes of Fe2þ AHb1 have shown that the ligand undergoes polar interactions with distal pocket residues. 11 This finding, which was attributed to hydrogen bonding with HisE7, was supported by the upshift/downshift in the νCO/ νFe-CO frequencies observed for the HisfLeu mutant.12 RR studies also showed that mutation of PheB10, which is close to HisE7, to Leu reduces this interaction, increasing the population of the conformer with a less polar environment.12 Moreover, the equilibrium constant between the bishistidyl hexa- and the penta-coordinated species (KH) was lower for the PheB10fLeu mutant (KH ≈ 0.5) than for wt AHb1 (KH ≈ 1.6), showing that PheB10 has a stabilizing effect on hexacoordination.12 Received: November 12, 2010 Revised: February 2, 2011 Published: March 23, 2011 4138
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Scheme 1. Chemical Equilibria for the Reaction of CO with wt and Mutated AHb1 (Hb) to Form the CO Complex (HbCO)a
a Penta- and hexacoordinated species are indicated by the suffix p and h, respectively. (Hbp:CO) indicates primary docking sites with CO still inside the distal pocket, while (Hbp:CO)1 indicates a site in which the photodissociated ligand is docked into an internal hydrophobic cavity, accessible from the primary docking site.
The distal HisE7 imposes a relevant barrier to rebinding, and laser flash photolysis experiments consistently showed that geminate recombination is very limited and that the photodissociated ligand is easily released to the solvent at near room temperature. Molecular dynamics (MD) simulations allowed us to explain these findings through the formation of a tunnel connecting the distal pocket with the exterior of the protein.12 Finally, similar studies for the PheB10fLeu mutant revealed an increased geminate rebinding, which was interpreted by a reshaping of the internal cavities in the protein. The preceding findings raise the challenging question of whether a distal His gate mechanism is operative in AHb1 that assists ligand exchange between the distal heme pocket and the solvent. Already in 1966, Perutz and Matthews proposed that the pathway for ligand entry and exit into mammalian hemoglobins and myoglobins involves rotation of the distal histidine to form a short and direct channel between the heme pocket and solvent.13 To address this issue, it is convenient to investigate the structural and functional properties of this protein under conditions where HisE7 might modulate ligand exchange. The ionization properties of this residue, with a standard pKa slightly below neutrality, offer a specific advantage to explore the involvement of a gating mechanism. In fact, it has been observed that protonation of HisE7 leads to the rotation of the imidazole ring out of the distal cavity in CO-bound myoglobin (Mb).14 The idea that the exchange of the ligand between the distal pocket and the solvent is gated by the distal His64 (E7) in myoglobin was supported by a body of experimental evidence which includes mutagenesis mapping studies,15,16 and crystallographic investigations.14,17,18 More recent computational studies have suggested that, when thermal fluctuation and protein dynamics are taken into account, additional exchange pathways other than the His gate become active for Mb19 and other related globins.20 These findings are also supported by limited experimental evidence.21,22 The conformational change of the distal His—in the case it occurs in AHb1 at acidic pH—would (i) alter the steric hindrance at the binding site, (ii) change the accessibility of the escape route connecting the distal pocket with the solvent, and (iii) weaken the polar interactions between distal residues and bound CO. We report herein a combined experimental and theoretical study of AHb1 to determine the feasibility of a HisE7 gate mechanism and to examine the implications of the HisE7 conformational change on the structural features of the distal
Figure 1. pH-dependent RR spectra of the CO complexes of wt AHb1 and the PheB10fLeu mutant. Experimental conditions: (wt AHb1), 2 mW laser power at the sample, and 140 min integration time (pH 8.0); 1 mW laser power at the sample and 100 min integration time (pH 5.0). (PheB10fLeu mutant), 2 mW laser power at the sample; 40 (low frequency) and 180 min (high frequency); integration times (pH 7.0); 120 (low frequency) and 150 min (high-frequency region) integration times (pH 5.0).
Table 1. Vibrational Frequencies (cm1) of the FeCO Unit of wt AHb1 and Its Mutants ν(FeCO)
ν(CO)
533a
1923a 1921 (89%), 1959 (7%), 1968 (4%)d
wt AHb1, acid form
500c
1921 (53%), 1957 (40%), 1969 (7%)e
PheB10fLeu
519b
1923b
PheB10fLeu, acid form
493c
1965c
b
1964b
wt AHb1
1919 (40%), 1950 (7%), 1964 (53%)f HisE7fLeu a
501 b
Data from ref 11. Data from ref 12. c This work. d FTIR data at pH = 7.8 from ref 31. e FTIR data at pH = 6.0 from ref 31. f FTIR data at pH = 7.5 from ref 31.
cavity. To this end, RR has been used to explore the interactions in the distal cavity for CO complexes of both wild type (wt) AHb1 and its PheB10fLeu mutant at acidic and neutral pH. Moreover, information about the influence of pH on the reactivity with CO and ligand rebinding kinetics has been gained from laser flash photolysis. Finally, MD simulations have been performed to gain insight into the structural changes induced upon protonation of HisE7 in the distal cavity. The results allow us to provide a more comprehensive understanding of the molecular mechanisms that mediate ligand migration pathways in AHb1 and more generally of nsHbs in plants.
’ MATERIALS AND METHODS Recombinant Protein Production and Purification. The cDNA encoding AHb1 was inserted into pET11a (Novagen) and used to transform Escherichia coli BL21(DE3). The expression of recombinant proteins was carried out in the presence of 30 μM hemine chloride at 24 C. Recombinant AHb1 was purified by chromatography on a Q-Sepharose Fast Flow (GE Healthcare) column eluted with a 100 mM Tris buffer at pH 7.2. The protein was then loaded on a Q-Sepharose High Performance (GE Healthcare) column, and a linear gradient of NaCl from 0 to 0.1 M in 20 mM Tris buffer at pH 8.5 was used for elution. The coding sequence for AHb1, cloned in the vector pGEM-T Easy (Promega), was used as a template to introduce mutations Phe35B10fLeu and His68E7fLeu by means of the 4139
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Figure 2. a,a0 : RR spectra of the CO complexes of wt AHb1 and the PheB10fLeu mutant at pH 8.0. b,b0 : CO complex prepared at pH 5.0 and diluted in 100 mM Tris buffer to pH 8.0. c,c0 : pH 5.0. Experimental conditions: wt AHb1, (a,c) see Caption of Figure 1. (b) 1 mW laser power at the sample, 100 min integration time. PheB10fLeu mutant, 2 mW laser power at the sample. (a0 ) 60 min integration time; (b0 ) 50 min integration time; (c0 ) 30 min integration time.
Quik-Change II mutagenesis kit (Stratagene) following the manufacturer’s protocol. The mutants were expressed and purified as described for the wild type (wt) protein. The yield of mutant proteins was comparable with that of wt AHb1. Sample Preparation. The deoxy wt AHb1 and its mutant were prepared for flash photolysis experiments by diluting the concentrated stock of proteins with a deoxygenated buffer containing 100 mM sodium phosphate, 1 mM EDTA pH 7.0, or 100 mM MES, 1 mM sodium EDTA pH 5.5, to a final concentration ranging from 60 to 70 μM. Before the experiment, solutions were equilibrated with nitrogen/CO mixtures of known CO partial pressure, and sodium dithionite was added to a final concentration of 2 mM. CO complexes for RR spectroscopy were prepared by first flushing the protein solutions (30 μM) with nitrogen, then flushing with CO (Rivoira), and finally adding dithionite (Fluka Chemicals) to reach a final 20 mM concentration. Resonance Raman. RR spectra were obtained at room temperature with excitation from the 413.1 nm line of a Krþ laser (Coherent). The backscattered light from a slowly rotating NMR tube was collected and focused into a triple spectrometer (consisting of two Acton Research SpectraPro 2300i and a SpectraPro 2500i in the final stage with a 1800 or 3600 grooves/nm grating) working in the subtractive mode, equipped with a liquid nitrogen-cooled CCD detector (Roper Scientific Princeton Instruments). The spectra were calibrated to an accuracy of 1 cm1 for intense isolated bands with indene, acetone, acetonitrile, and CCl4 as standards. The grating was 3600 grooves/mm, with a spectral resolution of 1 cm1, for the low-frequency region and 1800 grooves/mm, with a spectral resolution of 3 cm1, for the high-frequency region. Kinetic Studies. Flash photolysis was carried out with the circularly polarized second harmonic (532 nm) of a Q-switched Nd:YAG laser and a CW Xe arc lamp as a probe source. The transient absorbance traces were measured at 436 nm through a 0.25 m spectrograph with a five-stage photomultiplier. The experimental setup was as described.11,23 The cuvette had an optical path length of 2 mm. The repetition rate was about 0.3 Hz to allow for full sample recovery between laser flashes. A maximum entropy method (MEM)24,25 was used to retrieve model-independent lifetime distributions, as described previously.23,26 We have followed the minimal model previously proposed for CO rebinding to AHb1 solutions11,23,27 and sketched in Scheme 1 to describe the rebinding kinetics. The differential equations
Figure 3. Top: Electronic absorption spectra of deoxy (solid lines) and carboxy (dotted lines) wt AHb1 at pH 7.0 (black), 5.5 (red), and 3.8 (green). Bottom: Far-UV circular dichroism of deoxy wt AHb1 at pH 7.0 (black), 5.5 (red), and 3.6 (green).
associated with Scheme 1 were solved numerically, and the rate constants appearing in the equilibrium were optimized to obtain a best fit to the experimental data. Numerical solutions to the set of coupled differential equations corresponding to Scheme 1 were determined by using the function ODE15s within Matlab 7.0 (The MathWorks, Inc.). Fitting of the numerical solution to experimental data (and optimization of microscopic rate constants) was obtained with a Matlab version of the optimization package Minuit (CERN). Autoxidation Rate Measurements. The rate of autoxidation of oxyAHb1 was measured in 0.1 M buffer in the presence of 1 mM EDTA at room temperature over a pH range of 412 for wt AHb1 and 512 for the mutant protein. The reaction was initiated by adding AHb1 to a 0.2 mL solution containing the appropriate buffer, to a final concentration of 60 mM; the changes in the absorption spectrum were recorded at measured intervals of time over 450700 nm; the rapid reaction was also followed by the absorbance change at 576 nm (R-peak). For the final state of reaction, the oxyform was completely converted to met-form by the addition of potassium ferricyanide. The rate constant observed at a given pH for the autoxidation of oxy-form to met-form (kobs) was determined from the slope of each straight line in the first-order plot. The curve fittings for a plot of kobs vs pH were made by an iterative least-squares method. The buffers used were acetate, MES, Tris HCl, MOPS, PIPES, TAPS, and CAPS. Absorption spectra were recorded in a Jasco double beam spectrophotometer (model V-560). Molecular Dynamics. To examine the conformational flexibility of the distal His residue, MD was run for wt AHb1 with neutral 4140
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Figure 4. (A) Analysis of CO rebinding kinetics after photolysis of CO complexes with wt AHb1 (left) and its PheB10fLeu mutant (right) in solution at 10 C and 1 atm CO. The fits (yellow lines) obtained using Scheme 1 are superimposed to the experimental data (red circles: pH = 5.5; black circles: pH = 7). (B) Lifetime distributions retrieved from the MEM analysis for the corresponding traces in panels A (same color code).
Table 2. Comparison between Microscopic Rate Constants for CO Rebinding to wt AHb1 and the PheB10fLeu Mutant at pH = 7.0 and pH = 5.5a k1 (106 s1)
a
k2 (107 s1)
k2 (107 M1 s1)
kb (s1)
kb (s1)
kc (107 s1)
kc (107 s1)
10 2.3
25 23.5
17 14.5
7.0 2.1
0.45 0.25
8.0
0.82
1.5
7.5
wt
pH = 5.5 pH = 7.0
5.0 5.1
20 9
PheB10fLeu
pH = 5.5
5.0
4.0
5.9
pH = 7.0
5.2
2.6
3.1
179
645
44.2
79.6
T = 20 C.
(HIE; NεH tautomer) and protonated HisE7 using the parmm99SB28 force field and the Amber-9 package.28 Starting models were taken from the structures considered in our previous study.12 For HisF8, which occupies the fifth coordination position of the heme iron, the NδH tautomeric form was used. Assignment of the most adequate ionization state for ionizable residues in the mild acidic conditions used in experimental studies (pH 5.5) was performed using a PROPKA algorithm.28 Calculations were performed for 10 snapshots taken evenly distributed during the last 40 ns of the trajectory run at neutral pH. In particular, all the titratable Asp and Glu residues were assigned the standard ionization state since the highest predicted pKa's were 3.9 and 4.6 for Asp and Glu, respectively. Besides HisF8 and HisE7, two other histidines (His113, His147) exist in AHb1, whose ionization state was further checked upon visual inspection of the local environment and solvent accessibility of the residues due to the larger uncertainty of PROPKA in predicting the pKa values of histidines. His113 is partially exposed to the solvent; however, it is surrounded by apolar residues Met49, Val109, and Val110, and the imidazole ring is hydrogen bonded to the backbone NH unit of Val110. Similar features are found for His147, which mainly resides in a pocket containing Leu84, Val90, and Ala151, and the imidazole ring is hydrogen-bonded to the carbonyl unit of Gly88. Accordingly, they were also modeled in the nonionized form considering the mild acidic experimental conditions. The starting structures were immersed in a preequilibrated octahedral box of TIP3P29 water molecules. The final systems contained around 10 100 waters and a chlorine ion to keep electrical neutrality. The SHAKE algorithm was used to keep bonds involving hydrogen atoms at their equilibrium length, in conjunction with a 1 fs time step for the integration of the Newton equations. Trajectories were collected in the NPT
(1 atm, 298 K) ensemble using periodic boundary conditions and Ewald sums (grid spacing of 1 Å) for long-range electrostatic interactions. The systems were minimized using a multistep protocol, involving first the adjustment of hydrogens, then the refinement of water molecules, and finally the minimization of the whole system. The equilibration was performed by heating from 100 to 298 K in four 100 ps steps at 150, 200, 250, and 298 K. Finally, for each simulated system 50 ns production trajectories were run, collecting frames at 1 ps intervals. The FPOCKET program30 was used to detect cavities in the protein matrix. Calculations were performed for two sets of 500 snapshots taken regularly in the 2030 ns and 4050 ns windows of the trajectory. The identified cavities were superposed in time and space, and a density map was generated from this superposition. High-density cavities correspond to stable cavities found during the trajectory, while low- density cavities are transient or nearly nonexistent in the MD simulation.
’ RESULTS AND DISCUSSION Resonance Raman Spectroscopy. Figure 1 shows the pHdependent RR spectra of CO complexes of both wild type (wt) AHb1 and its PheB10fLeu mutant at near physiological pH and at mildly acidic conditions. The RR spectra of wt AHb1 at pH 5.0 are characterized by an intensity decrease of the ν(FeCO) band at 533 cm1 and a concomitant occurrence of a new ν(FeCO) at 500 cm1. This result is in agreement with the previous FTIR spectra collected at pH 6 and 4 K, which showed that the population of the conformer with ν(CΟ) at 1957 cm1 increased at the expense of that of the conformer with ν(CO) at 1921 cm1, whereas a minor contribution from the conformer at ν(CO) = 1969 (1968) cm1 was found to be independent of pH (see Table 1).31 In fact, lower ν(FeCO) frequencies are correlated with higher ν(CO) frequencies 4141
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Figure 5. Time (ns) evolution of the torsional angles (degrees) that define the orientation of the side chain for the distal HisE7 in neutral (A) and protonated (B) states (black, NCRCβCγ; red,: CRCβCγNδ). Representative structures of conformational fluctuations of the distal HisE7 in neutral (C) and protonated (D) states.
and are observed for those CO complexes in which the Fe-bound CO ligand interacts weakly with the surrounding polar amino acids. This is a consequence of the relative FeCO π back-bonding decrease, occurring when the polarity of the environment around the bound CO is low.32 Therefore, the intensity increase at pH 5.0 of the ν(FeCO) band at 500 cm1 (Figure 1) is correlated with that of the ν(CO) band at 1957 cm1 (Table 1), and the concomitant intensity decrease of the ν(FeCO) band at 533 cm1 (Figure 1) is correlated with that of the ν(CO) band at 1921 cm1 (Table 1). The dramatic increase in autoxidation rate observed when pH is lowered below 5 prevents achieving experimental conditions where the less polar conformer is expected to prevail (see below). Figure 1 also shows that a single ν(FeCO) band at 493 cm1 is observed for the PheB10fLeu mutant at pH 5, with the concomitant disappearance of the band at 519 cm1 . Accordingly, an intensity increase of the ν(FeCO) band at 1965 cm1 is observed in both RR and FTIR spectra.31 The comparison between the RR spectra of Figure 1 indicates that the pKa for the interconversion from a more polar conformer to a less polar conformer is shifted to a much higher value for the mutant.
The finding of a less polar conformer for both wt AHb1 and the PheB10fLeu mutant at acid pH cannot be ascribed to protein denaturation. In fact, the RR spectra of CO complexes which were either directly prepared at pH 8 or at pH 8 following acidification at pH 5.0 (Figure 2) do not show any relevant differences. This indicates that no major changes in the distal cavities of the proteins occur, in agreement with the far-UV circular dichroism and electronic absorption spectra of deoxy and carboxy wt AHb1 taken at pH 7.0 and 5.5. Acid denaturation occurs below pH = 4.5 for both proteins and is easily detected by changes in the absorption spectra (Figure 3). Similar spectra are observed also for the PheB10fLeu mutant (data not shown). Acid denaturation due to secondary structure loss occurs below pH = 4.5 for both proteins and is easily detected by changes in the absorption spectra and circular dichroism spectra (see representative far-UV circular dichroism spectrum at pH 3.6 and electronic absorption spectra at 3.8). Ligand Binding Kinetics. To probe the involvement of HisE7 in the modulation of heme reactivity, ligand rebinding kinetics was measured at pH 5.5, where the less polar conformer is populated. Lowering the pH to 5.5 leads to a decrease in 4142
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Figure 6. Time (ns) evolution of the distance (Å) from (A) the HisE7 Nδ to propionate oxygens O1A (black) and O2A (red), (B) the guanidinium moiety of ArgF3 to propionate oxygens O1A (black) and O2A (red), and (C) the HisE7 Nδ to propionate oxygens O1D (black) and O2D (red) for the protein with protonated distal HisE7.
geminate recombination for both the wt and PheB10fLeu proteins (see Figure 4), which suggests an overall reduction of the probability for direct rebinding over other competitive processes. This is likely due to opening of an additional escape route when the polar interactions with the bound ligand are weakened. The effect is strikingly evident also from the lifetime distributions obtained from the MEM25 analysis. Table 2 summarizes the microscopic rate constants obtained from the analysis of kinetic data with the reaction in Scheme 1.
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While k1 remains constant, an appreciable increase in k2 (by around a factor of 2) is observed on lowering the pH for wt AHb1. On the other hand, an even higher enhancement (by a factor of 4.3) is found for the bimolecular rebinding rate k2. According to these parameters, kON for wt AHb1 increases from 1.2 106 M1 s1 at pH = 7 to 2.4 106 M1 s1 at pH = 5.5 at 20 C. The effect of pH on kON for PheB10fLeu AHb1 is less pronounced (5.1 106 M1 s1 at pH = 7, 6.55 106 M1 s1 at pH = 5.5), suggesting that accessibility of the distal cavity is less influenced by the pH change. This last finding is not surprising since vibrational spectroscopy results revealed for PheB10fLeu AHb1 a more open distal pocket with less interaction between the distal His and the bound CO. On the basis of the preceding results, it can be concluded that, when the pH is lowered below neutrality, an easily accessible exchange channel between the distal pocket and the solvent phase is opened, through which ligands can migrate in and out. We propose that the opening of the channel is triggered by the protonation of the distal His. In addition, from pH 7 to 5.5 the equilibrium constant for formation of the hexacoordinated bis-histidinic species (KH = kb/ kb) decreases from 1.6 to 1.4 for the wt AHb1 and from 0.6 to 0.3 for PheB10fLeu AHb1, respectively. Therefore, a small, but consistent, effect on the equilibrium between hexa- and pentacoordinated species in both wt and PheB10fLeu proteins further facilitates ligand exchange. This trend is not unexpected since protonation of the distal His reduces its affinity for the heme Fe. Molecular Modeling. To confirm the hypothesis that a new escape channel at acidic pH originates from protonation of the distal His, a 50 ns MD simulation was run for deoxy wt AHb1 with protonated HisE7 following the same protocol adopted in previous studies.12 The profiles determined for the time evolution of the potential energy and root-mean square deviation (rmsd) along the trajectories supported the stability of the simulation (see Supporting Information). The structural analysis of the heme cavity reveals the larger conformational flexibility of HisE7 upon protonation (see Figure 5). In the neutral form the torsional angle NCRCβCγ is close to 180 (Figure 5A). Accordingly, the side chain of HisE7 is oriented pointing toward the interior of the heme cavity during the whole trajectory. Thermal fluctuations simply give rise to conformational rearrangements of the imidazole ring, as noted in the transitions of the dihedral angle CRCβCγNδ from values close to 90 and 270 (Figure 5A and 5C). In contrast to the limited conformational flexibility found for the neutral HisE7, protonation of the distal HisE7 leads to enhanced conformational flexibility, as noted in the time evolution of the torsional angles that define the orientation of the side chain (Figure 5B). Thus, the dihedral angle NCRCβCγ adopts values close to 180 in the period 1035 ns and around 300 during the first 7 ns and the last 14 ns of the trajectory. In the former case, HisE7 resides inside the heme cavity, as found for the neutral state, but the conformational change from 180 to 300 reorients the side chain of HisE7 in the distal cavity, exposing the imidazole ring to the aqueous solvent (see Figure 5D). The conformational reorientation of the distal HisE7 seems to be modulated by the breaking of the interaction between the protonated imidazole ring and one of the heme propionate groups (oxygens O1A and O2A). This is reflected in the correlation found between the time evolution of the torsional 4143
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Figure 7. Representation of the internal cavities identified with FPOCKET in the interior of wt AHb1 with protonated HisE7. Calculations were performed for 200 snapshots taken in the windows (A) 2030 ns and (B) 4050 ns. HisE7, ArgF3, and the heme are shown in sticks.
angle (NCRCβCγ) of the HisE7 side chain and the distance between the imidazole ring and the carboxylate moiety, as noted in the comparison of the profiles shown in Figure 5B and Figure 6A. Thus, when HisE7 adopts the conformational state defined by the dihedral angle NCRCβCγ close to 300 and becomes exposed to the aqueous solvent, there is no remarkable interaction between the imidazole and carboxylate units. However, when HisE7 is located in the interior of the distal cavity, a close interaction (around 3.4 Å as averaged for the windows 1027 ns and 3136 ns) is found between those fragments. As expected, the analysis of the trajectory run for the protein with neutral HisE7 reveals that the distance between the imidazole ring and the carboxylate moiety is much larger (around 8 Å; see Figure S3A in Supporting Information) along the whole trajectory. The disruption of the ionic interaction between the protonated imidazole ring and the propionate unit appears to be influenced by the formation of a salt bridge between the propionate group and the guanidium moiety of ArgF3 (compare Figures 6A and 6B). Thus, the formation of the salt bridge between carboxylate and guanidinium groups (in periods 2831 and 3850 ns) is accompanied by the breaking of the ionic interaction between the protonated imidazole and the carboxylate moiety. Transient interactions between the propionate unit and ArgF3 are also found in the simulation run for AHb1 with neutral HisE7 (see Figure S3B in Supporting Information), thus reflecting the balance between the Coulombic interaction between those charged groups and the hydration by water molecules. Nevertheless, protonation of HisE7 enables the interaction with the propionate unit of the heme, and the electrostatic competition with the guanidinium unit of ArgF3 permits the sequestration of the propionate unit, which in turn facilitates the conformational change that causes HisE7 to be exposed to the solvent. Finally, it is also worth noting that the solvent-exposed conformation of HisE7 is found to be further stabilized by the interaction of the protonated imidazole with the carboxylate unit of the other propionate group (oxygens O1D and O2D), as noted in the close interaction found between those units at the end of the trajectory (see Figure 6C). The conformational rearrangement of the distal HisE7 is associated with the formation of an escape channel connecting the interior of the heme cavity with the protein surface. This finding is clearly reflected in the shape of the cavities detected
Figure 8. Representation of selected water molecules that access the distal cavity through the exchange channel opened by the gating motion of protonated HisE7.
with the FPOCKET program in the protein matrix (see Figure 7). In particular, when HisE7 is in the neutral form, the distal cavity protrudes into the interior of the protein, and another cleft, delineated by residues Cys77, Cys78, Ala81, Trp141, Ala144, Tyr145, and Leu148, is also visible.12 Differently, in the protonated model (Figure 7), the reorientation of the HisE7 side chain opens a channel directly connecting the distal cavity with the surface of the protein, playing a functionally relevant role in facilitating the access of small ligands. Moreover, the inspection of representative snapshots revealed the occurrence of water molecules accessing the distal cavity through the exchange channel (Figure 8). Overall, these findings suggest that, compared to the protein with neutral HisE7, protonation of the distal His enhances the conformational flexibility and facilitates a gating mechanism, arising from electrostatic stabilization afforded by the interaction with the surrounding water molecules in the solvent-exposed conformation Autoxidation Rate Measurements. In view of the putative NO dioxygenase activity of AHb1, which likely involves an oxyFe2þ intermediate that reacts with NO, stability of the oxyferrous complex toward autoxidation33 is a fundamental property. Interaction of the distal His with the bound ligand appears to provide at the same time the necessary stabilization and shielding from 4144
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rotation of the side chain of HisE7 creates a direct channel from the solvent to the iron atom. Finally, they are also in agreement with the transient rotation of the distal His observed in timeresolved crystallography upon photolysis of MbCO.41
’ ASSOCIATED CONTENT
bS
Supporting Information. Complete details of experimental and computational methods, analysis of autoxidation kinetics, and additional analysis of MD simulations. This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 9. pH dependence of the autoxidation rate of oxygenated wt AHb1 (black squares) and PheB10 f Leu (red squares). 0.1 M buffer, 1 mM EDTA at 25 C. The fit obtained using the acid-catalyzed twostate model proposed by Shikama is superimposed to the experimental data (see Supporting Information and ref 37).
the solvent. At neutral pH, the observed first-order rate constant for wt AHb1 oxidation is kobs = 2.6 102 h1, lower than the value determined for the related wt rice Hb1 (kobs = 8 102 h1 at 20 C).34 The autoxidation rate for AHb1 appears to be intermediate between the high values observed for murine and human neuroglobin (19 and 5.4 h1, respectively, at 37 C)35 and the low rates observed for O2 transport proteins such as Mb (0.5 102 h1 for sperm whale MbO2 and 0.72 102 h1 for bovine heart MbO2 at 25 C).36 When the interaction with HisE7 is released, either by lowering the pH or by introducing the PheB10fLeu mutation, the autoxidation rate increases substantially, as demonstrated by the data reported in Figure 9 (see also Supporting Information for a quantitative analysis). Thus, PheB10 appears to help waterproof the distal pocket and hence stabilize the oxygenated protein. Furthermore, the analysis of the autoxidation profile reported in Figure 9 (see Supporting Information) strongly supports the protecting role of the distal His. From the application of the acidcatalyzed two-state model proposed by Shikama,37 it can be concluded that most of the autoxidation of oxygenated AHb1 is catalyzed by protons, relying on a weakly acidic group, easily identified as the distal His, with pK1 = 6.3 in the case of wt AHb1 and pK1 = 5.3 in the case of PheB10fLeu AHb1. These pKa values are consistent with the pH dependence of the RR spectra shown in Figure 1. Moreover, accessibility of the heme pocket to OH appears to be 100-fold higher for the PheB10fLeu mutant than for the wt protein. A more open distal pocket is in agreement with the reduced polar interactions that are demonstrated by vibrational spectroscopies for the CO complexes of the mutant.
’ CONCLUSIONS Present results support the involvement of the distal HisE7 residue of AHb1 in a gating mechanism that reorients this residue toward the solvent, leaving a channel accessible from the protein surface that might be functionally relevant for ligand exchange. This finding explains the decrease in geminate recombination observed for both the wt AHb1 and PheB10fLeu proteins upon lowering the pH to 5.5. These trends mimic the effect observed earlier for Mb in rebinding kinetics.14,38 Moreover, together with outward displacement of the distal His in the crystal structures of imidazole, phenylhydrazine, and ethyl isocyanide complexes of Mb17,18,39 and of Mb at low pH,14 these findings provide evidence supporting the idea, first put forward by Perutz,40 that
’ AUTHOR INFORMATION Corresponding Author
*Tel.: þ390521905208. Fax: þ390521905223. E-mail: cristiano. viappiani@fis.unipr.it.
’ ACKNOWLEDGMENT The authors acknowledge the Italian Ministero dell’Istruzione, dell’Universita e della Ricerca (PRIN2004 2004052135, PRIN 2008, 2008BFJ34R, and Azioni Integrate Italia Spagna 2009 IT10L1M59M), the Spanish Ministerio de Innovacion y Ciencia (SAF2008-05595, IT2009-0010), the Generalitat de Catalunya (SGR2009-294 and XRQTC), and the EU (FP7 Framework Program; project NOstress) for financial support. The Barcelona Supercomputer Center is acknowledged for computational facilities. ’ REFERENCES (1) Dordas, C. Plant Sci. 2009, 176, 433. (2) 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.; Appleby, C. A. Biopolymers 2009, 91, 1083. (3) Trevaskis, B.; Watts, R. A.; Andersson, C. R.; Llewellyn, D. J.; Hargrove, M. S.; Olson, J. S.; Dennis, E. S.; Peacock, W. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12230. (4) Wang, R.; Guegler, K.; LaBrie, S. T.; Crawford, N. M. Plant Cell 2000, 12, 1491. (5) Nie, X.; Hill, R. D. Plant Physiol. 1997, 114, 835. (6) Smagghe, B. J.; Trent, J. T., III; Hargrove, M. S. PLoS ONE 2008, 3, e2039. (7) Perazzolli, M.; Dominici, P.; Puertas, M. C. R.; Zago, E.; Zeier, J.; Sonoda, M.; Lamb, C.; Delledonne, M. Plant Cell 2004, 16, 2785. (8) Belenghi, B.; Romero-Puertas, M. C.; Vercammen, D.; Brackenier, A.; Inze, D.; Delledonne, M.; Breusegem, F. V. J. Biol. Chem. 2007, 282, 1352. (9) Perazzolli, M.; Romero-Puertas, M. C.; Delledonne, M. J. Exp. Bot. 2006, 57, 479. (10) Arredondo-Peter, R.; Hargrove, M. S.; Moran, J. F.; Sarath, G.; Klucas, R. V. Plant Physiol. 1998, 118, 1121. (11) Bruno, S.; Faggiano, S.; Spyrakis, F.; Mozzarelli, A.; Abbruzzetti, S.; Grandi, E.; Viappiani, C.; Feis, A.; Mackowiak, S.; Smulevich, G.; Cacciatori, E.; Dominici, P. J. Am. Chem. Soc. 2007, 129, 2880. (12) Faggiano, S.; Abbruzzetti, S.; Spyrakis, F.; Grandi, E.; Viappiani, C.; Bruno, S.; Mozzarelli, A.; Cozzini, P.; Astegno, A.; Dominici, P.; Brogioni, S.; Feis, A.; Smulevich, G.; Carrillo, O.; Schmidtke, P.; BidonChanal, A.; Luque, F. J. J. Phys. Chem. B 2009, 113, 16028. (13) Perutz, M. F.; Matthews, F. S. J. Mol. Biol. 1966, 21, 199. (14) Yang, F.; Phillips, G. N., Jr. J. Mol. Biol. 1996, 256, 762. (15) Scott, E. E.; Gibson, Q. H. Biochemistry 1997, 36, 11909. (16) Scott, E. E.; Gibson, Q. H.; Olson, J. S. J. Biol. Chem. 2001, 276, 5177. 4145
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