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Hydrogen-bonding network in heme active site regulates the hydrolysis activity of myoglobin
Journal of Molecular Catalysis B: Enzymatic 111 (2015) 9–15
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Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Hydrogen-bonding network in heme active site regulates the hydrolysis activity of myoglobin Jin Zeng a , Yuan Zhao a , Wei Li b , Xiangshi Tan b , Ge-Bo Wen c , Ying-Wu Lin a,c,∗ a b c
School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China Department of Chemistry & Institute of Biomedical Science, Fudan University, Shanghai 200433, China Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China
a r t i c l e
i n f o
Article history: Received 8 August 2014 Received in revised form 23 November 2014 Accepted 23 November 2014 Available online 28 November 2014 Keywords: Heme proteins Protein design Hydrogen-bonding Hydrolysis Kinetics
a b s t r a c t Although heme proteins are extensively studied, the structure and hydrolysis activity relationship of heme proteins is still not well understood. We herein made a comparison of the heme active site of myoglobin (Mb) and its two distal mutants, L29H Mb and L29E Mb, and found that the heme distal hydrogen-bonding network regulates the hydrolysis activity, with L29E Mb being more reactive. Fluoride competitive binding studies revealed that L29E Mb has an axial water molecule readily replaced by fluoride anion, whereas L29H Mb has a much more stable axial water molecule. pH titration studies showed that L29E Mb exhibits a lower pKa and L29H Mb exhibits a higher pKa of the heme coordinated water molecule compared to that of Mb, rationalizing the relative hydrolysis activities. Moreover, pHjump induced unfolding studies suggested that the heme distal hydrogen-bonding network, especially in L29H Mb, plays a key role in stabilization of the protein. This study thus provides valuable information for understanding the structure and hydrolysis activity relationship of heme proteins, as well as for rational protein design with the knowledge of structural and functional diversity of heme proteins. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Heme proteins are key metalloproteins in biological systems and perform diverse important functions, such as oxygen transport (Hemoglobin, Hb and Myoglobin, Mb, etc), electron transfer (Cytochrome c, Cyt c and cytochrome b5 , cyt b5 , etc), catalysis (horseradish peroxidase, HRP and Cytochrome P450, CYP450, etc), and small gas signaling (NO sensor soluble guanylate cyclase, sGC, and CO sensor CooA, etc) [1–4]. Moreover, the same heme protein may display various functions in different environmental conditions. For example, Mb, an oxygen carrier, shows nitrite reductase (NIR) activity during hypoxia by reducing nitrite (NO2 − ) to nitric oxide (NO), and regulates the mitochondrial function [5,6]. Furthermore, Mb shows peroxidase activity by metabolizing hydrogen peroxide (H2 O2 ), a major endogenous reactive oxygen species (ROS) [7], and protects cells from oxidative damage subsequent to hypoxia [8]. In addition to these functions, hydrolysis activity has been observed for Mb for more than half a century [9], as well as for
∗ Corresponding author at: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China. Tel.: +86 734 8282375; fax: +86 734 8282133. E-mail addresses: [email protected], [email protected] (Y.-W. Lin). http://dx.doi.org/10.1016/j.molcatb.2014.11.008 1381-1177/© 2014 Elsevier B.V. All rights reserved.
Hb [10,11]. Meanwhile, the physiological significance of hydrolysis activity of Mb and Hb is not clear yet, and the structure and hydrolysis activity relationship of heme proteins is not well understood, as it received little attention compared to other biological functions. On the other hand, we recently found that the heme axial mutants of cyt b5 , an electron transfer protein, H39Q cyt b5 and H39S cyt b5 , exhibit enhanced hydrolysis activity by catalyzing the hydrolysis of 4-nitrophenyl acetate (4NPA), although these two mutants were originally designed by Huang and co-workers to improve their peroxidase activity [12]. It showed that both a heme active site and a substrate-binding pocket are required for the hydrolysis activity. The heme distal Gln39 or Ser39 likely plays a role in promoting the hydrolysis by hydrogen-bonding interactions with the heme axially coordinated water molecule. With these information, we were further interested in probing the structure and hydrolysis activity relationship of heme proteins based on Mb. Due to the small size (153 amino acids) with a single heme group, the ease of recombinant overexpression and crystallization, Mb has been shown to be an ideal protein model for revealing the structure-function relationship of heme proteins by rational protein design [13–15]. In recent studies of the functional diversity of Mb [16–20], we designed several Mb mutants with alterations in the heme active site, such as L29H Mb and L29E Mb, and solved their X-ray crystal structures (Fig. 1). These progresses
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Fig. 1. Crystal structure of WT Mb (A), L29H Mb (B) and L29E Mb (C), showing the heme distal hydrogen-bonding network (dotted lines) and distal cavities (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lay down the groundwork for us to study the hydrolysis activity of Mbs with a different heme active site, and to probe their structure and hydrolysis activity relationship. In this study, we made a comparison of the heme active site of wild-type (WT) Mb and its mutants, L29H Mb and L29E Mb, as well as their hydrolysis activities, and found that the hydrogen-bonding network in heme active site regulates the hydrolysis activity of Mb, which is also associated with ligand binding and protein unfolding. 2. Materials and methods
absence of Mbs in buffer. The initial rates (kobs ) were determined from linear fits of the absorbance versus time using an extinction coefficient of ε400 = 18.3 mM−1 cm−1 [24]. The kinetic parameters (kcat and Km ) were determined by fitting the curves of initial rates versus substrate concentration to the Michaelis–Menten equation [25] (Eq. (1)), which were corrected by the spontaneous hydrolysis of 4NPA in each case.
[Protein]
=
kcat · [4NPA] Km + [4NPA]
(1)
2.1. Protein preparation
2.4. Protein cavities analysis
WT sperm whale Mb was expressed in BL21(DE3) cells using the Mb gene of pMbt7-7 and purified using the procedure described previously [21]. The L29H Mb and L29E Mb gene were constructed by using the QuickChange Site Directed Mutagenesis Kit (Stratagene) with primers, 5 -GGT CAG GAC ATC CAC ATT CGA CTG TTC-3 (L29H) and 5 -GGT CAG GAC ATC GAG ATT CGA CTG TTC-3 (L29E), and their reverse complements. The L29H and L29E mutations were confirmed by DNA sequencing assay. L29H Mb was expressed and purified using the same procedure as that for WT Mb. L29E Mb was expressed in inclusion bodies and purified using the procedure described previously for L29E/F43H Mb [22].
The X-ray crystal structure of WT Mb (PDB code 1JP6 [26]) and its mutants, L29H Mb (PDB code 4IT8 [17]) and L29E Mb (PDB code 4PQ6 [20]), were submitted to CASTp (Computed Atlas of Surface Topography of proteins) server (http://cast.engr.uic.edu) [27] to ˚ The identify internal cavities with a default probe radius of 1.4 A. protein pocked was visualized by program PyMOL 0.99rc6 [28].
2.2. EPR spectroscopy Electron paramagnetic resonance (EPR) spectra of WT Mb, L29H Mb and L29E Mb were recorded on a Bruker A300 spectrometer (Xband) equipped with Bruker ER4141VTM liquid nitrogen system. The protein sample (0.5 mM in 100 mM KH2 PO4 , pH 7.0) was transferred into an EPR tube with a volume of 300 L. The spectra were measured at 100 K, with frequency of 9.43 GHz, center field 2200 G and sweep width 3600 G, microwave power 0.595 mW and modulation amplitude 3.0 G. Protein concentration was determined with an extinction coefficient of ε409 = 157 mM−1 cm−1 for WT Mb [21], ε409 = 155 mM−1 cm−1 for L29H Mb [17], and ε413 = 135 mM−1 cm−1 for L29E Mb, as calculated using the standard hemochromagen method [23]. 2.3. Hydrolysis activity assay The hydrolysis activities of WT Mb, L29H Mb and L29E Mb were evaluated by using 4-nitrophenyl acetate (4NPA) as a substrate. Kinetic experiments were initiated by the addition of 4NPA to a solution containing 1.0 M protein in 2 mL of 10 mM Tris–HCl, pH 7.0, at 25 ◦ C, with a final concentration of 0.5–7.5 mM. The hydrolysis product of 4-nitrophenoxide was monitored by absorption increase at 400 nm with an Agilent 8453 diode array spectrophotometer. Spontaneous hydrolysis of 4NPA was also monitored in the
2.5. Fluoride titration study Mbs (10 M) were dissolved in 50 mM Tris–HCl, pH 7.0 and titrated with fluoride anions (NaF) at 25 ◦ C. The UV–vis spectra were recorded in a range of 300–700 nm with dropwise addition of NaF to a final concentration of 5 mM for L29E Mb, 25 mM for WT Mb and 500 mM for L29H Mb, respectively. To determine the dissociation constant (KD ) of fluoride binding, we referenced to Kosowicz and Boon [29]. The dependency of the charge-transfer (CT1) band change (A) on concentrations of fluoride was analyzed by the following equation (Eq. (2)): A = Amax
{CP + CL + KD − [(CP + CL + KD )2 − 4CP CL ] 2Cp
1/2
}
(2)
here, A is the absorbance difference for the CT1 band; Amax is the maximum absorbance difference with ferric heme fully occupied by fluoride ion; CP and CL are the total protein and total fluoride concentration, respectively; and KD is the equilibrium dissociation constant. 2.6. pH titration study Mbs (10 M) were dissolved in 10 mM Tris–HCl and titrated with increasing amounts of 10 M NaOH at 25 ◦ C. The pH values were measured directly in the cuvette using a microelectrode (type LE422) connected to a Mettler Toledo pH meter (type FE20). The acid–alkaline equilibrium was determined with an Agilent 8453 diode array spectrophotometer. The pKa values were calculated by
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fitting the absorbance of Soret band versus pH to the Boltzmann function (Eq. (3)). A = A2 +
A1 − A2 1 + e(pH−pKa )/dpH
(3)
here, A is the absorbance of Soret band; A1 and A2 are the initial and final absorbance of Soret band, respectively. 2.7. pH-jump induced unfolding study Kinetic determinants for pH-jump induced unfolding of WT Mb, L29H Mb and L29E Mb with were performed with a SF-61DX2 Hi-Tech KinetAsystTM dual mixing stopped-flow spectrophotometer. Typically, one syringe contains 10 M of protein in 10 mM Tris–HCl, pH 7.0, and the second syringe contains HCl, pH 2.5. The pH-jump induced unfolding was stated with mixing of equal volume of solutions from both syringes. 100 time-dependent spectra were collected over 5 s from 350 to 700 nm at 25 ◦ C. The observed rate constants (kobs , s−1 ) were calculated by fitting the absorbance of Soret band versus time to a single-exponential decay function (Eq. (4)). y = y0 + a e−kt
(4)
3. Results and discussion 3.1. Heme active site of Mbs With structural information available for WT Mb (PDB code 1JP6 [26]) and its mutants, L29H Mb (PDB code 4IT8 [17]) and L29E Mb (PDB code 4PQ6 [20]), we have a chance to compare the heme active site. In contrast to WT Mb that contains one axial water molecule (Fig. 1A), L29H Mb contains two water molecules in the distal pocket, one coordinates to the heme iron as in WT Mb, and the other forms two hydrogen bonds with the axial water and distal His29 (Fig. 1B) [17]. Interestingly, our recent X-ray crystallographic study revealed that L29E Mb contains three distal water molecules, where Glu29 interacts with two of them, and His64 interacts with one of them instead of the axial water, forming a unique hydrogenbonding network in the heme active site (Fig. 1C) [20]. Although two distal water molecules were observed in the X-ray structures of other Mb mutants, such as F43H Mb (PDB code 4PQC [20]) and L29H/F43H Mb (PDB code 4FWZ [30]), three distal water molecules in Mb mutants were not observed previously. We considered that the distal hydrogen-bonding network is able to regulate the diverse functions of Mb. For example, we recently showed that distal His29 in L29H Mb decreases the NIR activity of Mb [17], whereas increases its peroxidase activity [18]. To further provide structural information for the heme active site, we performed EPR spectroscopic studies of WT Mb, L29H Mb and L29E Mb in ferric state. As shown in Fig. 2, the EPR spectra exhibit similar high-spin heme signals for both WT Mb (g ∼ 5.94, 1.99) and its two mutants (L29H Mb, g ∼ 5.98, 1.99, and L29E Mb, g ∼ 5.96, 1.99), which agree with previous observations for ferric Mb [31]. The results suggest that with mutation in heme distal pocket, the heme coordination state in these Mbs remains the same, i.e., His93/water, whereas the distal micro-environment might be slightly altered by formation of different distal hydrogen-bonding networks. 3.2. Hydrolysis activity of Mbs To gain insight into the structure and hydrolysis activity relationship of Mb, we evaluated the hydrolysis activities of WT Mb, L29H Mb and L29E Mb, using 4NPA as a typical substrate and monitoring the absorption increase at 400 nm of the hydrolysis product,
Fig. 2. EPR spectrum of WT Mb (a), L29H Mb (b) and L29E Mb (c). Spectra were collected in 100 mM KH2 PO4 , pH 7.0 at 100 K, 0.595 mW power and 9.43 GHz.
Fig. 3. Michaelis–Menten kinetics for the hydrolysis of 4NPA catalyzed by WT Mb, L29H Mb and L29E Mb (1.0 M protein in 10 mM Tris–HCl, pH 7.0, 25 ◦ C). Spontaneous hydrolysis of 4NPA under the same condition was shown for comparison.
4-nitrophenoxide. Fig. 3 shows the plots of kobs versus the concentration of 4NPA, with the spontaneous hydrolysis of 4NPA in buffer, Tris–HCl (10 mM, pH 7.0), shown for comparison. By correction of the spontaneous hydrolysis effect, the kinetic parameters determined are summarized in Table 1. L29H Mb and L29E Mb were found to have a kcat value of 2.78 × 10−3 s−1 and 11.44 × 10−3 s−1 , respectively, which is ∼1.9-fold decrease and ∼2.2-fold increase, respectively, compared to that of WT Mb (5.26 × 10−3 s−1 ). Since the X-ray structure showed that both L29H Mb and L29E Mb have almost identical surface to that of WT Mb, we attributed the difference in hydrolysis activity to the effect of heme active site, although Table 1 Kinetic parameters of 4NPA hydrolysis catalyzed by WT Mb, L29H Mb and L29E Mb. Mbs
kcat [s−1 ] (×10−3 )
Km [mM]
kcat /Km [M−1 s−1 ]
WT Mb L29H Mb L29E Mb
5.26 ± 0.69 2.78 ± 0.41 11.44 ± 0.43
2.68 ± 0.83 1.35 ± 0.62 1.45 ± 0.16
1.96 2.06 7.89
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Table 2 CT1 band maxima and fluoride binding dissociation constant (KD ) for the Mbsfluoride complexes, the pKa value of the heme coordinated water molecule, and the observed rate constants (kobs ) upon pH-jump induced unfolding of WT Mb, L29H Mb and L29E Mb. Mbs
CT1 (nm)
KD (mM)
pKa
kobs [s−1 ]
WT Mb L29H Mb L29E Mb
607 ND 609
5.57 ± 0.56 ND 0.68 ± 0.07
8.81 12.07 7.86
0.37 ± 0.01 2.04 ± 0.02 1.74 ± 0.02
ND, not detected.
the catalysis might be performed at the protein surface where several histidines located. Our recent study also demonstrated that the hydrolysis activity of cyt b5 axial mutants is closely associated with the heme active site [12]. Therefore, the hydrolysis activity of L29H Mb and L29E Mb different from WT Mb suggests that distal His29 inhibits whereas Glu29 promotes the catalytic hydrolysis of 4NPA. It is likely due to the fact that His29 and Glu29 form different distal hydrogen-bonding network, which might regulate the heme axial water molecule in L29H Mb to be inactive, whereas in L29E Mb to be more active compared to that in WT Mb. On the other hand, the Km values of L29H Mb and L29E Mb were found to be 1.35 mM and 1.45 mM, respectively, similar to that for an artificial esterase designed in trimeric peptide (1.7 mM) [32]. The values are ∼2-fold decreased compared to that of WT Mb (2.68 mM), indicating that these two mutants have a higher affinity for the substrate of 4NPA. Protein cavities analysis revealed that L29H Mb has a similar distal pocket volume (48 A˚ 3 ) to that of Mb (49 A˚ 3 ), and L29E Mb has a slightly larger distal pocket volume (65 A˚ 3 ) (Fig. 1), which suggests that the enhanced affinity for both L29H Mb and L29E Mb is not due to the difference in accessible volume for the substrate, instead, is likely due to the stabilization by hydrogen-bonding interactions. Based on the kcat and Km values, L29H Mb shows an overall catalytic activity (kcat /Km ) of 2.06 M−1 s−1 , which is similar to that of WT Mb (1.96 M−1 s−1 ). Meanwhile, L29E Mb exhibits ∼4fold increased catalytic activity (7.98 M−1 s−1 ), in a similar range of H39Q cyt b5 (12.5 M−1 s−1 ) and H39S cyt b5 (5.13 M−1 s−1 ) [12], which indicates that the hydrolysis activity of heme proteins is tunable by a distal hydrogen-bonding network. 3.3. Fluoride binding to Mbs To probe the property of hydrogen-bonding interactions in the heme distal pocket of WT Mb, L29H Mb and L29E Mb, we used fluoride for competitive ligand binding to the heme center, which has been shown to be an effective probe for hydrogen-bonding interactions in the distal cavity of heme proteins [29,33–37]. It showed that upon titration of fluoride anion, the Soret band of WT Mb decreased in intensity and shifted from 409 to 407 nm, with a decrease of 504 nm absorption and concomitant increase of a charge-transfer (CT1) band at 607 nm (Fig. 4A), suggesting formation of WT Mbfluoride complex, consistent with previous observations [37]. The KD (F- ) was determined to be 5.57 mM for WT Mb (Table 2), a typical dissociation constant for a ferric heme protein in reaction with fluoride [29]. The titration of L29E Mb showed that the Soret band shifted from 413 to 408 nm (Fig. 4B), indicating fluoride binding to the heme iron by competition with the axial ligand. At the same time, CT1 band was observed at 609 nm, which is 2-nm red-shift compared to that of WT Mb-fluoride complex (Table 2), suggesting the presence of stronger hydrogen-bonding interactions between fluoride and distal residues, likely donated by both His64 and Glu29, as well as distal water molecules, instead of only distal His64 and a water molecule in WT Mb [33]. Consequently, L29E Mb was found to have a KD (F- ) value (0.68 mM) ∼8-fold decrease compared to that of
Fig. 4. UV–vis spectra of WT Mb (A) and L29E Mb (B) upon titration with fluoride anion (10 M protein in 50 mM Tris–HCl, pH 7.0, 25 ◦ C). The fitting of the intensity changes of Soret band versus fluoride concentrations is shown as an inset; (C) UV–vis spectra of L29H Mb in the absence and presence of 500 mM NaF. The changes of visible bands are shown as insets in (A–C).
WT Mb, further indicating stronger binding of fluoride by multiple distal hydrogen-bonding interactions. Surprisingly, the UV–vis spectrum of L29H Mb remained almost the same in titration of fluoride anion, even with a final concentration as high as 500 mM (Fig. 4C), which indicates that fluoride anion can hardly bind to the heme center due to the strong coordination of axial water molecule. This observation further suggests that hydrogen-bonding interaction network in the distal pocket of L29H Mb is much more stable compared to that in both WT Mb and L29E Mb, which might rationalize the lower kcat value determined for L29H Mb. 3.4. Acid–alkaline equilibrium of Mbs To further probe the stability of distal hydrogen-bonding interactions involving the heme axial water molecule, we measured the acid–alkaline transition of WT Mb, L29H Mb and L29E Mb in
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the Soret region. As shown in Fig. 5A, the Soret band of WT Mb decreased and shifted from 409 to 413 nm with increasing pH values. By fitting of the intensity changes of Soret band versus pH values (Fig. 5A, inset), the pKa of the transition was calculated to be 8.81 (Table 2), which agrees with the previously reported value for sperm whale Mb (8.9) [38]. Similar spectra changes were observed for L29E Mb (Fig. 5B), which slightly decreased and shifted from 413 to 416 nm. Data fitting showed that L29E Mb has a pKa value (7.86) almost one pH unit lower than that of WT Mb, suggesting that the heme coordinated water molecule is less stable compared to that in WT Mb. On the other hand, L29H Mb has an extremely high stability in basic conditions, and its Soret band decreased slightly at pH as high as 11. At more higher pH values (pH > 11), the Soret band decreases dramatically (Fig. 5C), indicating the dissociation of heme from protein matrix. Data fitting showed that L29H Mb has a super high pKa value of 12.07, which suggesting that the water molecule coordinated to heme iron was stabilized by the distal hydrogenbonding interactions. Similar to this observation, previous study [39] showed that Aplysia limacina Mb lacking a distal hydrogenbonding network has a lower pKa value (7.5) than the mutants with a distal hydrogen-bonding network, such as V(E7)H/R(E10)T Mb (pKa = 10.9). Moreover, Osmia bicornis Mb with a stronger distal hydrogen-bonding interaction exhibits a higher pKa value (9.27) than that for sperm whale Mb and other Mbs [40]. The relative stabilities of the heme axial water molecule for WT Mb, L29E Mb and L29H Mb are consistent with the results of fluoride binding studies, which thus rationalizes the relative hydrolysis activities of these Mbs. Based on these results, we proposed a hydrolysis mechanism of 4NPA catalyzed by Mbs. As shown in Scheme 1, the L29H mutation results in a more stabilized distal hydrogen-bonding network with a higher pKa value for the coordinated heme axial water, and thus a lower hydrolysis activity. By contrast, the L29E mutation weakens the coordination of axial water to the heme iron, leading to a lower pKa value and an enhanced hydrolysis activity. 3.5. pH-jump induced unfolding of Mbs
Fig. 5. UV–vis spectra of WT Mb (A), L29E Mb (B) and L29H Mb (C) upon pH titration. The fitting of the intensity changes of Soret band versus pH values was shown as an inset.
Since WT Mb, L29H Mb and L29E Mb show different stabilities in basic conditions, we also interested in their stabilities in acidic conditions, which can provide additional information for the role of distal hydrogen-bonding interactions in protein stabilization. Therefore, we performed pH-jump induced unfolding studies by rapidly mixing the protein with pH 2.5 HCl solution. Stoppedflow spectra showed that the protein Soret bands rapidly decreased in intensity and shifted to that of free heme (∼370 nm). The timedependant changes of the Soret band are shown in Fig. 6A. The observed rate constant (kobs ) was determined to be 0.37 s−1 for WT Mb, which is ∼5-fold slower than that for L29H Mb (2.04 s−1 )
Scheme 1. Proposed mechanism of 4NPA hydrolysis catalyzed by WT Mb and its L29H and L29E mutants.
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binding and pH titration studies revealed that the L29H mutation results in a much more stabilized distal hydrogen-bonding network with a higher pKa value and a decreased hydrolysis activity. By contrast, the L29E mutation weakens the coordination of axial water to the heme iron, which lowers the pKa value and enhances the hydrolysis activity. Moreover, pH-jump induced unfolding studies suggested that the heme distal hydrogen-bonding network, especially in L29H Mb, plays a key role in stabilization of the protein. This study shows that the heme distal hydrogen-bonding network regulates the hydrolysis activity of Mb, and provides insights into the structure and hydrolysis activity relationship of heme proteins, which is valuable for rational protein design by fine-tuning the structure and function of heme proteins.
Acknowledgements It is a pleasure to acknowledge Prof. S. G. Sligar and Prof. Y. Lu at University of Illinois at Urbana-Champaign, for their kind gift of the swMb gene. This work was supported by the National Science Foundation of China, NSFC (No. 31370812) and Hunan Provincial Natural Science Foundation (No. 2015JJ1012).
References
Fig. 6. (A) Time-dependent changes of Soret band for WT Mb, L29H Mb and L29E Mb upon pH-jump induced unfolding; (B) UV–vis spectra changes of L29H Mb upon pHjump induced unfolding. Time-dependent changes of 527 nm and the visible region were shown as insets.
and L29E Mb (1.74 s−1 ) (Table 2). These values suggest that both mutants have a lower stability upon proton attacking, which is likely due to the introduction of hydrophilic residue in the heme distal pocket that is readily protonated, resulting in rapid disruption of the distal hydrogen-bonding network. Moreover, it is interesting to observe that in pH-jump induced unfolding of L29H Mb, the Soret band shifted from 409 to 411 nm in the first 0.2 s, then shifted to ∼370 nm. Concurrently, the visible band increased at 527 nm in the first 0.2 s, then decreased over time (Fig. 6B). The spectrum of the intermediate (Soret band, 411 nm, visible band, 527 nm) resembles that of heme proteins with a bis-His coordination in ferric state, such as the double mutant of H64V/V68H Mb with His68/His93 coordination (Soret band, 412 nm, visible band, 525 nm) [41], which indicates that one distal His coordinated to the heme iron transitionally in the first stage of unfolding. Note that there was no intermediate was observed for both WT Mb and L29E Mb during their unfolding processes under the same conditions, agreeing with a two-state nature of transition [42]. These observations suggest that distal His29 plays a role in altering acid-induced unfolding, implying that His29 is important for stabilization of L29H Mb by the distal hydrogen-bonding network. 4. Conclusion In summary, we compared the structure and hydrolysis activity of WT Mb and its two distal mutants, L29H Mb and L29E Mb, which contain one to three water molecules in the heme active site forming a distinct hydrogen-bonding network. Fluoride competitive
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Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Hydrogen-bonding network in heme active site regulates the hydrolysis activity of myoglobin Jin Zeng a , Yuan Zhao a , Wei Li b , Xiangshi Tan b , Ge-Bo Wen c , Ying-Wu Lin a,c,∗ a b c
School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China Department of Chemistry & Institute of Biomedical Science, Fudan University, Shanghai 200433, China Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China
a r t i c l e
i n f o
Article history: Received 8 August 2014 Received in revised form 23 November 2014 Accepted 23 November 2014 Available online 28 November 2014 Keywords: Heme proteins Protein design Hydrogen-bonding Hydrolysis Kinetics
a b s t r a c t Although heme proteins are extensively studied, the structure and hydrolysis activity relationship of heme proteins is still not well understood. We herein made a comparison of the heme active site of myoglobin (Mb) and its two distal mutants, L29H Mb and L29E Mb, and found that the heme distal hydrogen-bonding network regulates the hydrolysis activity, with L29E Mb being more reactive. Fluoride competitive binding studies revealed that L29E Mb has an axial water molecule readily replaced by fluoride anion, whereas L29H Mb has a much more stable axial water molecule. pH titration studies showed that L29E Mb exhibits a lower pKa and L29H Mb exhibits a higher pKa of the heme coordinated water molecule compared to that of Mb, rationalizing the relative hydrolysis activities. Moreover, pHjump induced unfolding studies suggested that the heme distal hydrogen-bonding network, especially in L29H Mb, plays a key role in stabilization of the protein. This study thus provides valuable information for understanding the structure and hydrolysis activity relationship of heme proteins, as well as for rational protein design with the knowledge of structural and functional diversity of heme proteins. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Heme proteins are key metalloproteins in biological systems and perform diverse important functions, such as oxygen transport (Hemoglobin, Hb and Myoglobin, Mb, etc), electron transfer (Cytochrome c, Cyt c and cytochrome b5 , cyt b5 , etc), catalysis (horseradish peroxidase, HRP and Cytochrome P450, CYP450, etc), and small gas signaling (NO sensor soluble guanylate cyclase, sGC, and CO sensor CooA, etc) [1–4]. Moreover, the same heme protein may display various functions in different environmental conditions. For example, Mb, an oxygen carrier, shows nitrite reductase (NIR) activity during hypoxia by reducing nitrite (NO2 − ) to nitric oxide (NO), and regulates the mitochondrial function [5,6]. Furthermore, Mb shows peroxidase activity by metabolizing hydrogen peroxide (H2 O2 ), a major endogenous reactive oxygen species (ROS) [7], and protects cells from oxidative damage subsequent to hypoxia [8]. In addition to these functions, hydrolysis activity has been observed for Mb for more than half a century [9], as well as for
∗ Corresponding author at: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China. Tel.: +86 734 8282375; fax: +86 734 8282133. E-mail addresses: [email protected], [email protected] (Y.-W. Lin). http://dx.doi.org/10.1016/j.molcatb.2014.11.008 1381-1177/© 2014 Elsevier B.V. All rights reserved.
Hb [10,11]. Meanwhile, the physiological significance of hydrolysis activity of Mb and Hb is not clear yet, and the structure and hydrolysis activity relationship of heme proteins is not well understood, as it received little attention compared to other biological functions. On the other hand, we recently found that the heme axial mutants of cyt b5 , an electron transfer protein, H39Q cyt b5 and H39S cyt b5 , exhibit enhanced hydrolysis activity by catalyzing the hydrolysis of 4-nitrophenyl acetate (4NPA), although these two mutants were originally designed by Huang and co-workers to improve their peroxidase activity [12]. It showed that both a heme active site and a substrate-binding pocket are required for the hydrolysis activity. The heme distal Gln39 or Ser39 likely plays a role in promoting the hydrolysis by hydrogen-bonding interactions with the heme axially coordinated water molecule. With these information, we were further interested in probing the structure and hydrolysis activity relationship of heme proteins based on Mb. Due to the small size (153 amino acids) with a single heme group, the ease of recombinant overexpression and crystallization, Mb has been shown to be an ideal protein model for revealing the structure-function relationship of heme proteins by rational protein design [13–15]. In recent studies of the functional diversity of Mb [16–20], we designed several Mb mutants with alterations in the heme active site, such as L29H Mb and L29E Mb, and solved their X-ray crystal structures (Fig. 1). These progresses
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Fig. 1. Crystal structure of WT Mb (A), L29H Mb (B) and L29E Mb (C), showing the heme distal hydrogen-bonding network (dotted lines) and distal cavities (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lay down the groundwork for us to study the hydrolysis activity of Mbs with a different heme active site, and to probe their structure and hydrolysis activity relationship. In this study, we made a comparison of the heme active site of wild-type (WT) Mb and its mutants, L29H Mb and L29E Mb, as well as their hydrolysis activities, and found that the hydrogen-bonding network in heme active site regulates the hydrolysis activity of Mb, which is also associated with ligand binding and protein unfolding. 2. Materials and methods
absence of Mbs in buffer. The initial rates (kobs ) were determined from linear fits of the absorbance versus time using an extinction coefficient of ε400 = 18.3 mM−1 cm−1 [24]. The kinetic parameters (kcat and Km ) were determined by fitting the curves of initial rates versus substrate concentration to the Michaelis–Menten equation [25] (Eq. (1)), which were corrected by the spontaneous hydrolysis of 4NPA in each case.
[Protein]
=
kcat · [4NPA] Km + [4NPA]
(1)
2.1. Protein preparation
2.4. Protein cavities analysis
WT sperm whale Mb was expressed in BL21(DE3) cells using the Mb gene of pMbt7-7 and purified using the procedure described previously [21]. The L29H Mb and L29E Mb gene were constructed by using the QuickChange Site Directed Mutagenesis Kit (Stratagene) with primers, 5 -GGT CAG GAC ATC CAC ATT CGA CTG TTC-3 (L29H) and 5 -GGT CAG GAC ATC GAG ATT CGA CTG TTC-3 (L29E), and their reverse complements. The L29H and L29E mutations were confirmed by DNA sequencing assay. L29H Mb was expressed and purified using the same procedure as that for WT Mb. L29E Mb was expressed in inclusion bodies and purified using the procedure described previously for L29E/F43H Mb [22].
The X-ray crystal structure of WT Mb (PDB code 1JP6 [26]) and its mutants, L29H Mb (PDB code 4IT8 [17]) and L29E Mb (PDB code 4PQ6 [20]), were submitted to CASTp (Computed Atlas of Surface Topography of proteins) server (http://cast.engr.uic.edu) [27] to ˚ The identify internal cavities with a default probe radius of 1.4 A. protein pocked was visualized by program PyMOL 0.99rc6 [28].
2.2. EPR spectroscopy Electron paramagnetic resonance (EPR) spectra of WT Mb, L29H Mb and L29E Mb were recorded on a Bruker A300 spectrometer (Xband) equipped with Bruker ER4141VTM liquid nitrogen system. The protein sample (0.5 mM in 100 mM KH2 PO4 , pH 7.0) was transferred into an EPR tube with a volume of 300 L. The spectra were measured at 100 K, with frequency of 9.43 GHz, center field 2200 G and sweep width 3600 G, microwave power 0.595 mW and modulation amplitude 3.0 G. Protein concentration was determined with an extinction coefficient of ε409 = 157 mM−1 cm−1 for WT Mb [21], ε409 = 155 mM−1 cm−1 for L29H Mb [17], and ε413 = 135 mM−1 cm−1 for L29E Mb, as calculated using the standard hemochromagen method [23]. 2.3. Hydrolysis activity assay The hydrolysis activities of WT Mb, L29H Mb and L29E Mb were evaluated by using 4-nitrophenyl acetate (4NPA) as a substrate. Kinetic experiments were initiated by the addition of 4NPA to a solution containing 1.0 M protein in 2 mL of 10 mM Tris–HCl, pH 7.0, at 25 ◦ C, with a final concentration of 0.5–7.5 mM. The hydrolysis product of 4-nitrophenoxide was monitored by absorption increase at 400 nm with an Agilent 8453 diode array spectrophotometer. Spontaneous hydrolysis of 4NPA was also monitored in the
2.5. Fluoride titration study Mbs (10 M) were dissolved in 50 mM Tris–HCl, pH 7.0 and titrated with fluoride anions (NaF) at 25 ◦ C. The UV–vis spectra were recorded in a range of 300–700 nm with dropwise addition of NaF to a final concentration of 5 mM for L29E Mb, 25 mM for WT Mb and 500 mM for L29H Mb, respectively. To determine the dissociation constant (KD ) of fluoride binding, we referenced to Kosowicz and Boon [29]. The dependency of the charge-transfer (CT1) band change (A) on concentrations of fluoride was analyzed by the following equation (Eq. (2)): A = Amax
{CP + CL + KD − [(CP + CL + KD )2 − 4CP CL ] 2Cp
1/2
}
(2)
here, A is the absorbance difference for the CT1 band; Amax is the maximum absorbance difference with ferric heme fully occupied by fluoride ion; CP and CL are the total protein and total fluoride concentration, respectively; and KD is the equilibrium dissociation constant. 2.6. pH titration study Mbs (10 M) were dissolved in 10 mM Tris–HCl and titrated with increasing amounts of 10 M NaOH at 25 ◦ C. The pH values were measured directly in the cuvette using a microelectrode (type LE422) connected to a Mettler Toledo pH meter (type FE20). The acid–alkaline equilibrium was determined with an Agilent 8453 diode array spectrophotometer. The pKa values were calculated by
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fitting the absorbance of Soret band versus pH to the Boltzmann function (Eq. (3)). A = A2 +
A1 − A2 1 + e(pH−pKa )/dpH
(3)
here, A is the absorbance of Soret band; A1 and A2 are the initial and final absorbance of Soret band, respectively. 2.7. pH-jump induced unfolding study Kinetic determinants for pH-jump induced unfolding of WT Mb, L29H Mb and L29E Mb with were performed with a SF-61DX2 Hi-Tech KinetAsystTM dual mixing stopped-flow spectrophotometer. Typically, one syringe contains 10 M of protein in 10 mM Tris–HCl, pH 7.0, and the second syringe contains HCl, pH 2.5. The pH-jump induced unfolding was stated with mixing of equal volume of solutions from both syringes. 100 time-dependent spectra were collected over 5 s from 350 to 700 nm at 25 ◦ C. The observed rate constants (kobs , s−1 ) were calculated by fitting the absorbance of Soret band versus time to a single-exponential decay function (Eq. (4)). y = y0 + a e−kt
(4)
3. Results and discussion 3.1. Heme active site of Mbs With structural information available for WT Mb (PDB code 1JP6 [26]) and its mutants, L29H Mb (PDB code 4IT8 [17]) and L29E Mb (PDB code 4PQ6 [20]), we have a chance to compare the heme active site. In contrast to WT Mb that contains one axial water molecule (Fig. 1A), L29H Mb contains two water molecules in the distal pocket, one coordinates to the heme iron as in WT Mb, and the other forms two hydrogen bonds with the axial water and distal His29 (Fig. 1B) [17]. Interestingly, our recent X-ray crystallographic study revealed that L29E Mb contains three distal water molecules, where Glu29 interacts with two of them, and His64 interacts with one of them instead of the axial water, forming a unique hydrogenbonding network in the heme active site (Fig. 1C) [20]. Although two distal water molecules were observed in the X-ray structures of other Mb mutants, such as F43H Mb (PDB code 4PQC [20]) and L29H/F43H Mb (PDB code 4FWZ [30]), three distal water molecules in Mb mutants were not observed previously. We considered that the distal hydrogen-bonding network is able to regulate the diverse functions of Mb. For example, we recently showed that distal His29 in L29H Mb decreases the NIR activity of Mb [17], whereas increases its peroxidase activity [18]. To further provide structural information for the heme active site, we performed EPR spectroscopic studies of WT Mb, L29H Mb and L29E Mb in ferric state. As shown in Fig. 2, the EPR spectra exhibit similar high-spin heme signals for both WT Mb (g ∼ 5.94, 1.99) and its two mutants (L29H Mb, g ∼ 5.98, 1.99, and L29E Mb, g ∼ 5.96, 1.99), which agree with previous observations for ferric Mb [31]. The results suggest that with mutation in heme distal pocket, the heme coordination state in these Mbs remains the same, i.e., His93/water, whereas the distal micro-environment might be slightly altered by formation of different distal hydrogen-bonding networks. 3.2. Hydrolysis activity of Mbs To gain insight into the structure and hydrolysis activity relationship of Mb, we evaluated the hydrolysis activities of WT Mb, L29H Mb and L29E Mb, using 4NPA as a typical substrate and monitoring the absorption increase at 400 nm of the hydrolysis product,
Fig. 2. EPR spectrum of WT Mb (a), L29H Mb (b) and L29E Mb (c). Spectra were collected in 100 mM KH2 PO4 , pH 7.0 at 100 K, 0.595 mW power and 9.43 GHz.
Fig. 3. Michaelis–Menten kinetics for the hydrolysis of 4NPA catalyzed by WT Mb, L29H Mb and L29E Mb (1.0 M protein in 10 mM Tris–HCl, pH 7.0, 25 ◦ C). Spontaneous hydrolysis of 4NPA under the same condition was shown for comparison.
4-nitrophenoxide. Fig. 3 shows the plots of kobs versus the concentration of 4NPA, with the spontaneous hydrolysis of 4NPA in buffer, Tris–HCl (10 mM, pH 7.0), shown for comparison. By correction of the spontaneous hydrolysis effect, the kinetic parameters determined are summarized in Table 1. L29H Mb and L29E Mb were found to have a kcat value of 2.78 × 10−3 s−1 and 11.44 × 10−3 s−1 , respectively, which is ∼1.9-fold decrease and ∼2.2-fold increase, respectively, compared to that of WT Mb (5.26 × 10−3 s−1 ). Since the X-ray structure showed that both L29H Mb and L29E Mb have almost identical surface to that of WT Mb, we attributed the difference in hydrolysis activity to the effect of heme active site, although Table 1 Kinetic parameters of 4NPA hydrolysis catalyzed by WT Mb, L29H Mb and L29E Mb. Mbs
kcat [s−1 ] (×10−3 )
Km [mM]
kcat /Km [M−1 s−1 ]
WT Mb L29H Mb L29E Mb
5.26 ± 0.69 2.78 ± 0.41 11.44 ± 0.43
2.68 ± 0.83 1.35 ± 0.62 1.45 ± 0.16
1.96 2.06 7.89
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Table 2 CT1 band maxima and fluoride binding dissociation constant (KD ) for the Mbsfluoride complexes, the pKa value of the heme coordinated water molecule, and the observed rate constants (kobs ) upon pH-jump induced unfolding of WT Mb, L29H Mb and L29E Mb. Mbs
CT1 (nm)
KD (mM)
pKa
kobs [s−1 ]
WT Mb L29H Mb L29E Mb
607 ND 609
5.57 ± 0.56 ND 0.68 ± 0.07
8.81 12.07 7.86
0.37 ± 0.01 2.04 ± 0.02 1.74 ± 0.02
ND, not detected.
the catalysis might be performed at the protein surface where several histidines located. Our recent study also demonstrated that the hydrolysis activity of cyt b5 axial mutants is closely associated with the heme active site [12]. Therefore, the hydrolysis activity of L29H Mb and L29E Mb different from WT Mb suggests that distal His29 inhibits whereas Glu29 promotes the catalytic hydrolysis of 4NPA. It is likely due to the fact that His29 and Glu29 form different distal hydrogen-bonding network, which might regulate the heme axial water molecule in L29H Mb to be inactive, whereas in L29E Mb to be more active compared to that in WT Mb. On the other hand, the Km values of L29H Mb and L29E Mb were found to be 1.35 mM and 1.45 mM, respectively, similar to that for an artificial esterase designed in trimeric peptide (1.7 mM) [32]. The values are ∼2-fold decreased compared to that of WT Mb (2.68 mM), indicating that these two mutants have a higher affinity for the substrate of 4NPA. Protein cavities analysis revealed that L29H Mb has a similar distal pocket volume (48 A˚ 3 ) to that of Mb (49 A˚ 3 ), and L29E Mb has a slightly larger distal pocket volume (65 A˚ 3 ) (Fig. 1), which suggests that the enhanced affinity for both L29H Mb and L29E Mb is not due to the difference in accessible volume for the substrate, instead, is likely due to the stabilization by hydrogen-bonding interactions. Based on the kcat and Km values, L29H Mb shows an overall catalytic activity (kcat /Km ) of 2.06 M−1 s−1 , which is similar to that of WT Mb (1.96 M−1 s−1 ). Meanwhile, L29E Mb exhibits ∼4fold increased catalytic activity (7.98 M−1 s−1 ), in a similar range of H39Q cyt b5 (12.5 M−1 s−1 ) and H39S cyt b5 (5.13 M−1 s−1 ) [12], which indicates that the hydrolysis activity of heme proteins is tunable by a distal hydrogen-bonding network. 3.3. Fluoride binding to Mbs To probe the property of hydrogen-bonding interactions in the heme distal pocket of WT Mb, L29H Mb and L29E Mb, we used fluoride for competitive ligand binding to the heme center, which has been shown to be an effective probe for hydrogen-bonding interactions in the distal cavity of heme proteins [29,33–37]. It showed that upon titration of fluoride anion, the Soret band of WT Mb decreased in intensity and shifted from 409 to 407 nm, with a decrease of 504 nm absorption and concomitant increase of a charge-transfer (CT1) band at 607 nm (Fig. 4A), suggesting formation of WT Mbfluoride complex, consistent with previous observations [37]. The KD (F- ) was determined to be 5.57 mM for WT Mb (Table 2), a typical dissociation constant for a ferric heme protein in reaction with fluoride [29]. The titration of L29E Mb showed that the Soret band shifted from 413 to 408 nm (Fig. 4B), indicating fluoride binding to the heme iron by competition with the axial ligand. At the same time, CT1 band was observed at 609 nm, which is 2-nm red-shift compared to that of WT Mb-fluoride complex (Table 2), suggesting the presence of stronger hydrogen-bonding interactions between fluoride and distal residues, likely donated by both His64 and Glu29, as well as distal water molecules, instead of only distal His64 and a water molecule in WT Mb [33]. Consequently, L29E Mb was found to have a KD (F- ) value (0.68 mM) ∼8-fold decrease compared to that of
Fig. 4. UV–vis spectra of WT Mb (A) and L29E Mb (B) upon titration with fluoride anion (10 M protein in 50 mM Tris–HCl, pH 7.0, 25 ◦ C). The fitting of the intensity changes of Soret band versus fluoride concentrations is shown as an inset; (C) UV–vis spectra of L29H Mb in the absence and presence of 500 mM NaF. The changes of visible bands are shown as insets in (A–C).
WT Mb, further indicating stronger binding of fluoride by multiple distal hydrogen-bonding interactions. Surprisingly, the UV–vis spectrum of L29H Mb remained almost the same in titration of fluoride anion, even with a final concentration as high as 500 mM (Fig. 4C), which indicates that fluoride anion can hardly bind to the heme center due to the strong coordination of axial water molecule. This observation further suggests that hydrogen-bonding interaction network in the distal pocket of L29H Mb is much more stable compared to that in both WT Mb and L29E Mb, which might rationalize the lower kcat value determined for L29H Mb. 3.4. Acid–alkaline equilibrium of Mbs To further probe the stability of distal hydrogen-bonding interactions involving the heme axial water molecule, we measured the acid–alkaline transition of WT Mb, L29H Mb and L29E Mb in
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the Soret region. As shown in Fig. 5A, the Soret band of WT Mb decreased and shifted from 409 to 413 nm with increasing pH values. By fitting of the intensity changes of Soret band versus pH values (Fig. 5A, inset), the pKa of the transition was calculated to be 8.81 (Table 2), which agrees with the previously reported value for sperm whale Mb (8.9) [38]. Similar spectra changes were observed for L29E Mb (Fig. 5B), which slightly decreased and shifted from 413 to 416 nm. Data fitting showed that L29E Mb has a pKa value (7.86) almost one pH unit lower than that of WT Mb, suggesting that the heme coordinated water molecule is less stable compared to that in WT Mb. On the other hand, L29H Mb has an extremely high stability in basic conditions, and its Soret band decreased slightly at pH as high as 11. At more higher pH values (pH > 11), the Soret band decreases dramatically (Fig. 5C), indicating the dissociation of heme from protein matrix. Data fitting showed that L29H Mb has a super high pKa value of 12.07, which suggesting that the water molecule coordinated to heme iron was stabilized by the distal hydrogenbonding interactions. Similar to this observation, previous study [39] showed that Aplysia limacina Mb lacking a distal hydrogenbonding network has a lower pKa value (7.5) than the mutants with a distal hydrogen-bonding network, such as V(E7)H/R(E10)T Mb (pKa = 10.9). Moreover, Osmia bicornis Mb with a stronger distal hydrogen-bonding interaction exhibits a higher pKa value (9.27) than that for sperm whale Mb and other Mbs [40]. The relative stabilities of the heme axial water molecule for WT Mb, L29E Mb and L29H Mb are consistent with the results of fluoride binding studies, which thus rationalizes the relative hydrolysis activities of these Mbs. Based on these results, we proposed a hydrolysis mechanism of 4NPA catalyzed by Mbs. As shown in Scheme 1, the L29H mutation results in a more stabilized distal hydrogen-bonding network with a higher pKa value for the coordinated heme axial water, and thus a lower hydrolysis activity. By contrast, the L29E mutation weakens the coordination of axial water to the heme iron, leading to a lower pKa value and an enhanced hydrolysis activity. 3.5. pH-jump induced unfolding of Mbs
Fig. 5. UV–vis spectra of WT Mb (A), L29E Mb (B) and L29H Mb (C) upon pH titration. The fitting of the intensity changes of Soret band versus pH values was shown as an inset.
Since WT Mb, L29H Mb and L29E Mb show different stabilities in basic conditions, we also interested in their stabilities in acidic conditions, which can provide additional information for the role of distal hydrogen-bonding interactions in protein stabilization. Therefore, we performed pH-jump induced unfolding studies by rapidly mixing the protein with pH 2.5 HCl solution. Stoppedflow spectra showed that the protein Soret bands rapidly decreased in intensity and shifted to that of free heme (∼370 nm). The timedependant changes of the Soret band are shown in Fig. 6A. The observed rate constant (kobs ) was determined to be 0.37 s−1 for WT Mb, which is ∼5-fold slower than that for L29H Mb (2.04 s−1 )
Scheme 1. Proposed mechanism of 4NPA hydrolysis catalyzed by WT Mb and its L29H and L29E mutants.
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binding and pH titration studies revealed that the L29H mutation results in a much more stabilized distal hydrogen-bonding network with a higher pKa value and a decreased hydrolysis activity. By contrast, the L29E mutation weakens the coordination of axial water to the heme iron, which lowers the pKa value and enhances the hydrolysis activity. Moreover, pH-jump induced unfolding studies suggested that the heme distal hydrogen-bonding network, especially in L29H Mb, plays a key role in stabilization of the protein. This study shows that the heme distal hydrogen-bonding network regulates the hydrolysis activity of Mb, and provides insights into the structure and hydrolysis activity relationship of heme proteins, which is valuable for rational protein design by fine-tuning the structure and function of heme proteins.
Acknowledgements It is a pleasure to acknowledge Prof. S. G. Sligar and Prof. Y. Lu at University of Illinois at Urbana-Champaign, for their kind gift of the swMb gene. This work was supported by the National Science Foundation of China, NSFC (No. 31370812) and Hunan Provincial Natural Science Foundation (No. 2015JJ1012).
References
Fig. 6. (A) Time-dependent changes of Soret band for WT Mb, L29H Mb and L29E Mb upon pH-jump induced unfolding; (B) UV–vis spectra changes of L29H Mb upon pHjump induced unfolding. Time-dependent changes of 527 nm and the visible region were shown as insets.
and L29E Mb (1.74 s−1 ) (Table 2). These values suggest that both mutants have a lower stability upon proton attacking, which is likely due to the introduction of hydrophilic residue in the heme distal pocket that is readily protonated, resulting in rapid disruption of the distal hydrogen-bonding network. Moreover, it is interesting to observe that in pH-jump induced unfolding of L29H Mb, the Soret band shifted from 409 to 411 nm in the first 0.2 s, then shifted to ∼370 nm. Concurrently, the visible band increased at 527 nm in the first 0.2 s, then decreased over time (Fig. 6B). The spectrum of the intermediate (Soret band, 411 nm, visible band, 527 nm) resembles that of heme proteins with a bis-His coordination in ferric state, such as the double mutant of H64V/V68H Mb with His68/His93 coordination (Soret band, 412 nm, visible band, 525 nm) [41], which indicates that one distal His coordinated to the heme iron transitionally in the first stage of unfolding. Note that there was no intermediate was observed for both WT Mb and L29E Mb during their unfolding processes under the same conditions, agreeing with a two-state nature of transition [42]. These observations suggest that distal His29 plays a role in altering acid-induced unfolding, implying that His29 is important for stabilization of L29H Mb by the distal hydrogen-bonding network. 4. Conclusion In summary, we compared the structure and hydrolysis activity of WT Mb and its two distal mutants, L29H Mb and L29E Mb, which contain one to three water molecules in the heme active site forming a distinct hydrogen-bonding network. Fluoride competitive
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