Probing the role of the heme distal and proximal environment in ligand

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Probing the role of the heme distal and proximal environment in ligand dynamics in the signal transducer protein HemAT by timeresolved step-scan FTIR and resonance Raman spectroscopy Andrea Pavlou, Andreas Loullis, Hideaki Yoshimura, Shigetoshi Aono, and Eftychia Pinakoulaki Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00558 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017

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Probing the role of the heme distal and proximal environment in ligand dynamics in the signal transducer protein HemAT by time-resolved stepscan FTIR and resonance Raman spectroscopy Andrea Pavlou,¶,† Andreas Loullis, ¶ Hideaki Yoshimura, §,‡ Shigetoshi Aono, § Eftychia Pinakoulaki¶,* ¶

§

University of Cyprus, Department of Chemistry, P.O. Box 20537, 1678 Nicosia, Cyprus.

Institute for Molecular Science, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.

ACS Paragon Plus Environment

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ABSTRACT HemAT is a heme-containing oxygen sensor protein that controls aerotaxis. Time-resolved stepscan FTIR studies were performed on the isolated sensor domain and full-length HemAT proteins as well as on the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) mutants to elucidate the effect of the site-specific mutations on the ligand dynamics subsequent to CO photolysis. The mutations aimed to perturb H-bonding and electrostatic interactions near the heme Fe bound gaseous ligand (CO) and the heme proximal environment. Rebinding of CO to the heme Fe is biphasic in the sensor domain and full length HemAT as well as in the mutants, with the exception of the Y133F mutant protein. The monophasic rebinding of CO in the Y133F suggests that in the absence of the H-bond between Y133 and the heme proximal H123 residue the ligand rebinding process is significantly affected. The role of the proximal environment is also probed by resonance Raman photodissociation experiments, in which the Fe-His mode of the photoproduct of the sensor domain HemAT-CO is detected at higher frequency than the deoxy form in the difference resonance Raman spectra. The role of the conformational changes of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in regulating ligand dynamics in the heme pocket are discussed.


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Introduction HemAT is one of the heme-containing oxygen sensor proteins with a globin fold that control aerotaxis of certain bacteria and archaea, consisting of a sensor and a signalling domain in the Nand C-terminal regions, respectively.1-6 YddV, HemDGC, BpeGReg, AvGReg, GsGCS and AfGcHK are the other heme-based globin-coupled sensor proteins, in which the activation or inhibition of a regulated domain is controlled by recognition and discrimination of a specific gaseous molecule.1-6 The structures and signal transduction mechanisms of globin-coupled sensor proteins are distinct and vary from those of the heme-based sensor proteins that contain a heme bound PAS fold domain such as FixL and EcDOS. The HemAT sensor domain subunit has a 15% sequence similarity to sperm whale Mb and 25% similarity to Hb from V. stercoraria, and the crystal structure has revealed a homodimeric protein in which the dimerization interface forms a four helical bundle as a core.4-5 The heme proximal histidine ligand to the heme Fe (His123 in HemAT) is conserved in all three proteins. The biological activity of heme-based sensor proteins is accompanied by rapid structural changes and spectroscopic studies have suggested that in HemAT the B-and G-helices are involved in the signaling pathway.7-14 As a first step, O2 binding to the heme Fe results in conformational changes in the heme pocket that are ultimately transferred in the signaling domain of HemAT, stimulating the signaling process. Based on the observed changes in the kinetics of overall O2 and CO rebinding to wild type HemAT and point-mutants it has been suggested that asymmetry and negative cooperativity play an important role in the signal transduction pathway.6 However, little is thus far understood about the protein dynamics in the communication pathway from the sensor to the signalling domain by the transmembrane chemoreceptors.


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The potential pathways, through which the conformational changes induced upon ligand binding in the heme Fe are communicated to the protein, have been reported for HemAT.7-16 In the heme distal environment Y70 and T95 residues are present and suggested to be key contributors for ligand recognition and discrimination and thus, for specific sensing of gases (Figure 1). Open and closed six-coordinate O2-bound HemAT-Bs forms have been characterized by resonance Raman spectroscopy, revealing the involvement of the T95 in regulating O2 binding and of L92 in controlling the concerted movement of T95 and Y70 for the formation of the different forms of the O2-bound HemAT.8 Furthermore, multiple conformations have been detected in the CO- and NO-bound forms of HemAT.15 The comparative analysis of the O2, NO and CO data has demonstrated that Thr95 and Y70 are essential for ligand recognition and discrimination and L92 for directing the conformational changes of the Thr95 and Y70 residues upon ligand-binding.8,15 Both the wild type sensor and full length HemAT assume two different CO-bound state conformations, A0 and A1 that can be distinguished by their different CO bands at 1967 (A0, open form) and 1925 (A1, closed form) cm-1 and have been assigned to the neutral and strong H-bonding conformers of the heme Fe-CO complex originating from different orientations of Y70 in the distal pocket.7,15 Transient absorption data of photo-dissociation experiments on the HemAT-CO complex revealed monophasic rebinding that occurs on a millisecond time scale, while biphasic rebinding was reported for the corresponding HemAT-O2 complexes.6


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Figure 1. Structural representation of the unliganded heme site of the isolated sensor domain HemAT-Bs (PDB ID 1OR6). Resonance Raman (RR) and FTIR spectroscopy have been employed to monitor the distal and proximal environment of the heme Fe in the O2-, CO- and NO-bound forms and the structural dynamics of the protein were investigated by UV and visible time-resolved (TR3) Raman spectroscopy.7-16 The visible TR3 experiments revealed the formation of a H-bond between Y133 and H123 in the CO-bound form and demonstrated the relaxation of the Fe-His123 in hundreds of picoseconds.11 The UV-TR3 experiments revealed significant information regarding the communication pathway from the sensor to the signaling domain of HemAT.14 Based on the Raman intensities of W, Y, and F residues the presence of two to three phases of conformational changes in the nanosecond to microsecond range subsequent to CO photolysis was reported.14 It was proposed that the heme structural changes are communicated to specific sites in the sensor domain and a continuous conformational relaxation occurs in the ligand-free reduced form of the protein. Based on the UV-TR3 experiments the role of Y133 in the G-helix in the signaling mechanism has been proposed.14 Similar conclusions were drawn from our previous work on


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HemAT, where evidence for a ligand accommodation cavity in the proximal heme site near Y133 residue of the G-helix was provided.16 Time-resolved step-scan (TRS2) FTIR spectroscopy has been established as a very potent technique that shows the distinction of different species with fractional population in the percent range.17-21 This is extremely useful when it comes to finding key processes such as information on the energy barriers governing internal migration and rebinding of the photodissociated ligand. Both A0 and A1 states in the CO bound complex of HemAT are photolabile ensuring that ligand binding reactions can be investigated by TRS2-FTIR spectroscopy. Intramolecular rebinding to the heme Fe is controlled by ligand migration within the protein matrix which can give rise to a sequence of resolved kinetic features.20-22 Because of the importance of signaling mechanism and the growing interest in understanding the connectivity between the distal heme binding site and the proximal site, it is important to investigate the properties of the globin-coupled heme-based oxygen sensor proteins. Moreover, the dynamics of signal transduction by transmembrane chemoreceptors and the role of the distal and proximal environments in regulating the binding of the specific gaseous molecules (O2, CO, NO) are yet inadequately understood. Determination of the structural dynamics associated with the ligand binding/photodissociation to/from the heme Fe is an essential step towards our understanding the initial events in the signaling mechanism. The purpose of the present work is to understand the role of the residues in the heme distal and proximal site by probing the rebinding of CO to the heme Fe by TRS2-FTIR spectroscopy and to probe the proximal environment of the heme Fe by RR spectroscopy. All mutant proteins examined have similar photoproduct yields at 6 (or 8 µs) subsequent to CO photodissociation (92-95 %) with the exception of L92A (70%) and CO rebinding is biphasic in the wild type full length and sensor domain HemAT as well as in most mutant proteins. An unusual behavior is


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observed in the Y133F protein where monophasic rebinding of CO is detected. The RR results reveal the structural relaxation of the heme in response to the ligand photodissociation and rebinding. Although photoproduct structure determination is a difficult pursuit, it is successful if the photoproduct species can be populated to a significant fraction. The photoproduct RR spectrum of HemAT-CO has been studied by Soret and UV-Raman excitations to which we relate our RR and TRS2-FTIR experiments.11,14 The 405 nm spectrum of the photoproduct species obtained in this work displays a peak/trough feature in the region of the Fe-His mode in the difference spectrum (photoproduct minus deoxy form) that is similar to that obtained by TR3 spectroscopy.11 This feature is present in the sensor domain protein, but not in the full length protein, whereas the opposite was observed in the TR3 experiments.11 The TRS2-FTIR data presented here outline the role of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in controlling the ligand dynamics in the heme pocket. Materials and Methods The isolated sensor domain and full length HemAT as well as the distal mutants Y70F, T95A, and L92A and proximal Y133F mutants were expressed and purified according to previously published procedures.15,16 The protein concentration of the samples for FTIR experiments was ∼1.0 mM in 50 mM Tris buffer, pD 8. The protein samples were reduced by addition of dithionite solution under anaerobic conditions and exposed to 1 atm of CO for the formation of the CO-bound HemAT adducts. The HemAT-CO samples were placed in a tightly sealed FTIR cell with 6 µm path-length. CO gas was purchased from Linde. The protein concentration of samples used for RR measurements was 50 µM in 50 mM Tris buffer. The experimental setup and procedure for the TRS2-FTIR experiments has been described in detail in previous work.17 Briefly, a Vertex 80V FTIR spectrometer equipped with a photovoltaic


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MCT detector (Kolmar Technologies KV100-1B-7/190) and a 180-kHz, 24-bit, analog-to-digital converter (ADC) was used to perform the measurements, and the second harmonic at 532 nm from a Continuum Minilite II Nd:YAG laser (5 ns pulse width , 10 Hz) was used to photodissociate CO from the equilibrium HemAT-CO adducts. Triggering the FTIR and synchronization with the Nd:YAG laser was achieved by a digital delay pulse generator (Quantum Composers, 9524). The experimental setup was placed on an optical vibration isolation table (Newport VH) to eliminate environmental noise interferences. The spectra were aquired as single-sided, at 4 cm-1 spectral resolution, 6 or 8 µs time resolution, with 10 coadditions per data point. Ten to fifteen individual measurements were collected and averaged. For the Fourier transformation the Blackman-Harris three-term apodization function and the Mertz phase correction algorithm were used. The absorbance ratio of the CO mode in the TRS2FTIR difference spectrum at 6/8 µs to that of the corresponding equilibrium HemAT-CO adduct was used to determine the photoproduct yield at 6/8 µs. The Raman experiments were performed at room temperature with a 640 mm focal length Czerny-Turner spectrograph (Horiba, T64000 operated in single stage). Measurements were taken using 1800 g/mm holographic grating and Horiba Symphony BIUV1024x256 CCD detector. The excitation wavelength was provided by an Ondax SureLock LM-405 laser with a CleanLine ASE filter. The protein samples were transferred in a spinning cell to reduce local heating, and the scattering was collected in a 90o geometry. Rayleigh scattering was rejected by a Semrock StopLine 405 nm single-notch filter. The power incident on the sample was 5 mW for the reduced proteins and CO-photoproducts and 0.4 mW for the CO-adducts. For the COphotoproducts the samples were held stationary with no spinning. The accumulation time for each spectrum was 10-40 min. Toluene was used for calibration and spectra were processed and


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analysed using Origin software. The stability of the samples was confirmed by UV/Vis absorbance spectra (Shimadzu UV1700 spectrometer) before and after TRS2-FTIR and Raman measurements. Results Time-resolved step-scan FTIR (TRS2-FTIR): In an effort to investigate the heme Fe-CO properties we present in Figure 2 the TRS2-FTIR spectra of the sensor (panel A) and full length (panel B) HemAT-CO subsequent to CO photolysis at pD 8 and at room temperature. The observed negative peaks at 1967 and 1925 cm-1, which have been assigned to the neutral and strong H-bonded conformers of the heme Fe-CO complex, respectively, are both photolabile. The final spectra at 3.4 ms (panel A) and 6.2 ms (panel B) demonstrate that CO rebinding to the heme Fe is complete at the end of the measurement and there is no irreversible light-induced effect. Measuring the decreasing intensity of the CO modes associated with the heme Fe over the 8 µs-7 ms time scale enables the detection and quantification of ligand rebinding to heme Fe, and is depicted in Figure 2C and 2D. The intensities of Fe2+-CO bands at 1967 cm-1 bands versus time were measured and the rates of CO recombination to the heme Fe in the full length and sensor domain proteins at room temperature were determined. The red curves shown in Figure 2C and 2D are bi-exponential fits to the experimental data and the rate constants for CO rebinding were calculated according to pseudo first order kinetics for each phase. Both the wild type and isolated sensor domain HemAT exhibit biphasic kinetics for CO rebinding in contrast to previous work that indicted monophasic rebinding for CO and monophasic rebinding for O2.6 The rate constant of k1=24x103 s-1 for the first phase of CO rebinding in the sensor domain HemAT is very similar to that observed for the full length protein (k1=22x103 s-1), while the second phase of CO rebinding is faster in the sensor domain HemAT. Table 1 presents the time


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and rate constants for CO rebinding in the full length and sensor domain HemAT and mutant proteins along with the amplitudes of each phase.

Figure 2. TRS2-FTIR difference spectra of the isolated sensor domain HemAT-CO adduct from 8 to 3432 µs subsequent to CO photolysis (panel A) and the corresponding plot of the ∆Α of the 1967 cm-1 mode versus time on a logarithmic scale (panel C). TRS2-FTIR difference spectra of the full length HemAT-CO adduct from 8 to 6232 µs subsequent to CO photolysis (panel B) and the corresponding plot of the ∆Α of the CO mode at 1967 cm-1 versus time on a logarithmic scale (panel D). The red lines in panels C and D correspond to the exponential fitting of the experimental data.


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Table 1: Calculated time and rate constant for CO rebinding in HemAT sensor domain and full length wild type and mutant proteins. The amplitudes for each phase of rebinding are included. t1 (µs)

t2 (µs)

k1 (s-1)

k2 (s-1)

%1st phase

%2nd phase

WT sensor

29 ± 11

722 ± 33

24x103

960

18

82

Y70F sensor

19 ± 8

904 ± 18

36x103

767

17

83

L92A sensor

26 ± 8

243 ±12

26x103

2850

30

70

T95A sensor

18 ± 7

2154 ± 121 37x103

322

15

85

Y133F sensor

1028 ± 23

WT FL

32 ± 13

1327 ± 56

22x103

522

18

82

Y70F FL

31 ± 6

1133 ± 49

22x103

611

20

80

L92A FL

29 ± 4

168 ± 19

24x103

4122

41

59

T95A FL

14 ± 6

2327 ± 160 51x103

298

12

88

Y133F FL 1222 ± 34

674

100

567

100

Figures 3 shows the TRS2-FTIR difference spectra (of Y70F (panel A), L92A (panel B), T95A (panel C) and Y133F (panel D) mutant proteins of the sensor domain HemAT at the indicated timescales subsequent to CO photolysis. Panels E-H present the intensity of the CO bands versus time and the red lines represent the fits to the experimental data. The rebinding rates for the fast phase (k1) are in the 26x103 to 37x103 s-1 range and similar to those obtained for the wild type sensor and full length HemAT with the striking exception of the Y133F sensor protein, where k=674 s-1 and the rebinding of CO is monophasic. The rate constants of the slow phase (k2) in the


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sensor domain Y70F and T95A mutants are similar to that obtained for the wild type sensor protein, whereas that of the L92A is 2850 s-1. It should be noted that the photoproduct yield at 8 µs for the L92A mutant is 70%, significantly lower compared to the 92-95% observed for the wild type HemAT and other mutants. Moreover the amplitudes of the first and second phases are different in the L92A mutant.

Figure 3. TRS2-FTIR difference spectra of the isolated sensor domain Y70F (panel A), L92A (panel B), T95A (panel C) and Y133F (panel D) mutant HemAT-CO adducts subsequent to CO photolysis. Plot of the ∆A of the corresponding CO modes versus time on a logarithmic scale (panels E-H).


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Figure 4. TRS2-FTIR difference spectra of the full length Y70F (panel A), L92A (panel B), T95A (panel C) and Y133F (panel D) mutant HemAT-CO adducts subsequent to CO photolysis. Plot of the ∆A of the corresponding CO modes versus time on a logarithmic scale (panels E-H). The corresponding TRS2-FTIR difference spectra and the rebinding curves of the full length HemAT are shown in Figure 4. There are no significant differences in the frequency of the C-O band between the mutant sensor and full length proteins and also in the kinetics of the fast (k1) and slow (k2) phases. It is noteworthy to mention that the kinetics for CO rebinding also exhibit monophasic behaviour in the full length Y133F mutant, as it was observed for the corresponding


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sensor domain protein. Similar behaviour is also noted for the L92A full length and sensor domain mutants, albeit with a variation in the amplitudes of the two phases of rebinding (see Table 1). Resonance Raman photoproduct of HemAT: The high-frequency Raman spectra of reduced, CO-bound and of the photoproduct subsequent to CO photolysis in the 1100-1700 cm-1 region of the sensor and full length HemAT are shown in Figure 5A and 5B, respectively. The oxidation state marker, ν4, is located at 1355 cm-1 in the spectrum of the dithionite-reduced protein, characteristic of heme Fe in the ferrous state.7,9 The location of the ν2 and ν3 coresensitive bands at 1560 cm-1 and 1471 cm-1, respectively, are distinctive of five-coordinate highspin heme Fe, and thus the binding of CO to the ferrous heme Fe can occur without the displacement of an endogenous distal ligand of the heme. In the spectra of the CO-bound form the ν4 is shifted to 1371, the ν3 is observed 1497 cm-1 and the ν2 is located at 1579 cm-1. The frequency of ν4 is the same as that observed in the O2 bound form indicating that the electron delocalization of π electrons from the eg orbital of porphyrin to the π* of the bound ligand though the dπ orbital of Fe is the same and thus the heme Fe alone it is not capable in ligand discrimination.7-9 In the spectra of the photolytic species, the intensity of the ν4 mode at 1355 cm-1 demonstrates the formation of the 5-coordinate ferrous heme, while the shoulder at 1371 cm-1 indicates that the CO-bound species is not fully photolabile under our experimental conditions. The increased intensity of the ν3 band in the photoproduct without, however, any shift to lower frequency indicates that the heme core does not expand upon CO photolysis. The behavior of both the ν4 and ν3 modes is similar to that observed in the transient spectra of various hemoglobins, but is different from that of heme a3 of cytochrome c oxidase (CcO).22-26 There are no significant differences in the high frequency Raman spectra between the full length and the


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sensor HemAT of the deoxy, the CO bound and the photoproduct species, with the exception of a higher yield of photoproduct species in the full length protein as indicated by the weaker intensity of the 6-coordinate ν4 at 1371 cm-1 compared to the sensor domain protein.

Figure 5. High frequency resonance Raman spectra of reduced HemAT (trace a), the HemATCO adduct (trace b) and the HemAT-CO photoproduct (trace c) of the isolated sensor domain protein (panel A) and the full length protein (panel B) at pH 7.5. The excitation wavelength was 405 nm and the power incident on the sample was 5 mW for traces a and c, and 400 µW for traces b. The low-frequency region of the corresponding to the high frequency spectra of reduced, CObound and of the photoproduct sensor domain and full length HemAT are shown in Figure 6A-D. In the spectrum of the reduced sensor domain HemAT Fig. 6A (trace a) the peaks at 228, 318, 340, 365 and 409 cm-1 are assigned to ν(Fe2+-His), γ7 (CaCm) an out of plane mode, ν8, δ(CβCcCd) involving deformation of the propionate methylene group, and δ(CβCaCb).11 All the


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above mentioned vibrations are sensitive to the CO-binding as indicated by the reduced intensities shown in CO-bound form (trace b) and in agreement with previous RR results.7 The 496 cm-1 mode has been assigned to ν(Fe2+-CO) and is partially photolyzed under laser power conditions, at which the Mb-CO complex is fully photolabile. The low-frequency region of reduced, CO-bound and of the photoproduct Y133F sensor domain and full length HemAT mutants are shown in panels A and B, respectively, of Figure S1 in Supporting Information. The Fe-His stretch is located at 226 cm-1 in the reduced and photoproduct forms of the Y133F sensor domain mutant and at 224 cm-1 in the corresponding spectra of the Y133F full length protein, demonstrating a 2 cm-1 downshift compared to the wild type HemAT proteins, in agreement to previous work.11 The CO photodissociation yield in the Y133F mutants is lower compared to the WT proteins under the resonance Raman experimental conditions, resulting in reduced intensity of the Fe-His mode in the photoproduct compared to the deoxy form, with no observable frequency shift.


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Figure 6. Low frequency resonance Raman spectra of reduced HemAT (trace a), the HemATCO adduct (trace b) and the HemAT-CO photoproduct (trace c) of the isolated sensor domain protein at pH 7.5 (panel A) and at pH 9.0 (panel B), and of the full length protein at pH 7.5 (panel C) and at pH 9.0 (panel D). The difference c-a spectrum multiplied by a factor of 3 is included in each panel. The excitation wavelength was 405 nm and the power incident on the sample was 5 mW for traces a and c, and 400 µW for traces b. There is no significant change in the spectra between pH 7.5 and pH 9 (Fig. 6B) or between the spectrum of the sensor and the full length protein, except for a slight downshift of the Fe-His mode from 228 cm-1 in the sensor domain to 226 cm-1 in the full length HemAT (Fig.6 C-D). In addition, there is no obvious change in the frequency of the Fe-His mode upon photodissociation of CO, but a small change of the δ(CβCcCd) that involves deformation of the propionate


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methylene group. This observation suggests that the heme structure is not fully relaxed to the deoxy form. The investigation of protein dynamics of human adult hemoglobin (HbA) subsequent to CO and O2 photolysis revealed that both photoproducts exhibit differences from the deoxy form in the Fe-His and the methine wagging γ7 frequencies and in the pyrrole stretching and substituent bending (ν8) mode intensities.23 The difference spectrum of the photoproduct minus the deoxy form of sensor domain HemAT at pH 7.5 (trace c-a) revealed the presence of a peak/trough at 244/228 cm-1 that provides additional evidence that the photoproduct spectrum differs from that of the deoxy form. The frequency of the Fe-His vibration appears in the 200-260 cm-1 region in five-coordinate high spin His-ligated hemes, and its value reflects the extent of histidine anionic character. In globins and CcO the Fe-His mode is located between 200 and 230 cm-1 and is affected by the strength of H-bonding to the His NδH.23-27 The frequency of the Fe-His mode at 228 cm-1 in the deoxy form of HemAT indicates that the proximal His is H-bonded and is slightly perturbed by the Y133F mutation. The TR3 experiments have suggested that Y133 is the residue forming a H-bond with the proximal H123 upon CO binding.11 The 228 cm-1 mode in the deoxy protein shifts to higher frequency in the photoproduct, as indicated by the peak/trough feature in the difference spectrum of the sensor domain HemAT. Similar transient Fe-His behavior was observed in the TR3 experiments for the full length protein.11 Noteworthy, analogous Fe-His mode frequency shift has been detected subsequent to O2 photodissociation from the heme a3 of CcO during the CcO/O2 reaction.25 Discussion The potential pathways, through which the conformational changes induced to the heme Fe upon ligand binding are transmitted from the sensor to the signalling domain, require understanding of the ligand interactions and effects to the distal and proximal heme


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environments. The Fe-His mode is a probe for the heme/proximal environment interactions. In general, the frequency changes of the Fe-His mode have been attributed to the strength of the Hbond of the proximal His-ligand to protein residues.23-27 The lower frequencies for the Fe-His vibration are expected for weak (or absent) H-bond to the proximal His, reflecting the weaker Fe-His bond. In HemAT the 2 cm-1 difference in the frequency of the Fe-His vibration between the sensor domain and the full length protein indicates a slight variation in the strength of the Hbond of the proximal histidine in the presence of the signaling domain. TR3 experiments have revealed that relaxation of the proximal H123 is ligand dependent.11,12 In the CO photolysis experiments relaxation occurred in less than 10 ns in the full length HemAT and less than 100 ps in the Y133F mutant indicating the role of Y133 residue in the heme relaxation. On the other hand, although noticeable intensity changes were observed in the difference spectra, it was reported that in the sensor domain relaxation occurs in less than 100 ps.11 In the O2 photodissociation experiments, relaxation to the deoxy form occurred in the ns to µs timescale demonstrating strong ligand-dependent (CO vs O2) relaxation of the heme.11,12 It was also reported that upon O2 photodissociation the structural dynamics for the full length HemAT are faster than those observed for the sensor domain protein. This observation was interpreted as evidence for the involvement of the proximal structural dynamics upon O2 dissociation in the signal transduction of HemAT.12 The transient absorption data at 436 nm subsequent to CO photolysis indicated, in contrast to the visible TR3 experiments, an unrelaxed heme pocket and monophasic CO rebinding in the millisecond time scale.6,11,12 The UV TR3 experiments indicated that the conformational motions of the protein towards the equilibrium ligand-free structure occurred in hundreds of microseconds.14 Our TRS2-FTIR data clearly indicate biphasic rebinding kinetics on the


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microsecond to millisecond timescale and thus coupling of the rebinding process to the conformational rearrangements of the protein and the heme domain. In addition, the Raman data revealed that pH sensitive structural changes take place in the CO photoproduct of the sensor domain HemAT. Moreover, the distinct behavior of Fe-His mode associated with dissociation of CO for the full-length HemAT and the sensor domain protein indicates that the properties of the heme pocket are affected by the intra-protein interactions of the sensor and signaling domains. The Y133 residue that forms a H-bond with H123 in the CO-bound form11 in the proximal environment is a significant regulator in the process of CO recombination to the heme Fe, as demonstrated by the TRS2-FTIR experiment. The rebinding process is monophasic in the Y133F mutant, in contrast to the wild type and all other mutant proteins, where biphasic rebinding is observed (see below). As it has been demonstrated by time-resolved spectroscopic and crystallographic studies of heme proteins, after dissociation from the heme iron, ligands either rebind geminately from within the distal heme pocket or escape, assisted by protein fluctuations that transiently open exit/entry channels. In order to rebind to the heme, the ligands have to overcome a series of energy barriers.21-22,28 In an attempt to probe the main pathway for ligand movement into HemAT, specific mutations in the distal and proximal sites were designed to probe if residues at specific positions contribute to the energy barrier for ligand movement into and out of the heme site in HemAT. If the residue being targeted is positioned in the pathway for ligand movement into the protein, such mutations would alter the recombination k1 and k2 rates. There is no significant effect on the k1 recombination rate between the sensor and full length protein, but a measurable effect is noted on k2 demonstrating that ligands migrate away from the heme group and population of different protein sites occurs between the sensor domain and full length


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protein. A close inspection of Table 1 reveals that there are small differences between the sensor and full length mutant proteins in the k1 and k2 rates compared to the wild type, with the exception of the L92A mutants that show a significant change in the relative amplitudes of the two phases of rebinding and a large effect on the k2 rate, which is significantly decreased. A noticeable effect in the k2 rate also appears in T95A mutants, but in this case the k2 rate is decreased. In the Y133F sensor domain and full length mutants CO rebinding is monophasic and the recombination rate is comparable to the k2 rates of wild type and other mutants of HemAT. These observations indicate that Y70F (B-helix) is not a major part of the barrier to ligand movement into and out of the protein. On the other hand, Y133 (G-helix) and L92/T95 (E-helix) play a crucial role in ligand rebinding by acting as open/close gates in the ligand exit/entry channel. The ligand moves in the distal pocket once the L92/T95 gate is open and resides inside sufficient time to be captured when the gate closes. The experimental data suggest that Y133 (Ghelix) is situated along a pathway for ligand movement into the protein and transient motions of the protein permit access to the heme Fe that is controlled by L92 and T95 and net dissociation to the surroundings. Combining the results above with those previously published,7-16 the following points emerge. Based on the crystal structure5 and on the open and closed O2-bound forms8 the active site contains a unique distal heme pocket surrounded by Y70, L92, T95 and a H2O molecule. The T95 and Y70 distal site residues produce distinct conformations upon O2, CO, and NO binding to the heme Fe, demonstrating that they are the key players in ligand recognition and discrimination.8,15 The role of the L92 is equally important, since its presence in the distal heme site is essential to induce structural changes to T95 and Y70 for the formation of ligand (O2-, CO- and NO-) bound conformations that display strong interaction with the distal residues


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(closed forms) and the conformations exhibiting weak or no interaction (open forms).8,15 The rebinding of CO to the full-length, sensor domain, Y70F and T95A mutant proteins is markedly biphasic with a large slow phase. These results imply a fast equilibration of CO with subunit A, followed by slow conformational changes that result in further CO binding to the remaining population in the dimer. The slow conformational changes are not caused by interconversion of the open (A0, non H-bonded) and closed (A1, H-bonded) conformations of the active site, since both conformers were observed in TRS2-FTIR experiments in the rebinding process in the wild type, T95A and Y70F mutants. These observations indicate that the heterogeneous CO binding is due to different interactions in the asymmetric subunits of the HemAT homodimer in a way similar to that previously described in the biphasic binding of O2.6 In the O2 bound form of HemAT the H-bonding interaction of the bound O2 with T95 (E-helix) and H86 (CE-loop) with the heme propionate have been suggested to play a role in the sensing and signal transduction of HemAT.10 Although the sequential formation of these H-bonds of T95 and H86 upon O2 binding in the distal pocket causes substantial conformational alteration of the protein matrix in the E helix, the data reported herein indicate that ligand rebinding is greatly influenced not only by the L92 and T95 residues which are located on the E-helix in the distal heme site, but also by the Y133 residue which is located in the proximal heme site in G helix. In the homodimer of the sensor domain of HemAT the two antiparallel G and H helices form a four-helix bundle, and the H helix is continuous with the helical structure of the signaling domain defining a potential pathway for communicating conformational changes from the sensor domain to the signaling domain.5 Our results indicate the presence of a pathway connecting the heme binding site and G-helix through the His123-Y133 interaction.


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Conclusions The identification of the T95, L92 (E-helix) and Y133 (G-helix) sites in HemAT as significant regulators of ligand dynamics provides a framework for the analysis of their functional role in ligand discrimination and signaling. The E-helix L92 and T95 residues act as open/close gates for ligand entry in the heme distal site. The TRS2-FTIR results of the Y133F mutant, in conjunction with the pH-dependent behavior of the Fe-His mode observed in the RR photodissociation experiments, indicate that alterations in the hydrogen bonding interaction of H123–Y133 is significant for ligand dynamics. Furthermore, the data indicate the presence of an electrostatic field or a H-bonding network surrounding the ligand in the H123–Y133 site, suggesting that conformational changes at the proximal environment of HemAT can provide a “switch on” or “off” for conformational motion to its kinase domain in response to changes in ligand tension. The protein environment near Y133 imposes constrains on the released CO, inhibiting its fast rebinding to heme Fe. The overall results suggest the presence of a pathway that connects the heme binding site and G-helix.

ASSOCIATED CONTENT Supporting Information. Resonance Raman spectra of the Y133F sensor domain and full length HemAT mutants.

AUTHOR INFORMATION Corresponding Author *to whom correspondence should be addressed. E-mail: [email protected]


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Present Addresses †

Molecular Biomimetics, Department of Chemistry-Ångström Laboratory, Uppsala University, Sweden. ‡The University of Tokyo, Department of Chemistry, Japan.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors acknowledge the financial support from the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation (ANAVATHMISI/PAGIO/0308/14 and PENEK/0609/41) and University of Cyprus internal grant to EP. Notes The authors declare no competing financial interest.

ABBREVIATIONS HemAT-Bs, heme-based aerotactic transducer from Bacillus subtilis; TRS2-FTIR, time-resolved step-scan Fourier transform infrared; RR, resonance Raman; TR3, time-resolved resonance Raman; Mb, myoglobin; Hb, hemoglobin


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REFERENCES (1) Shimizu, T., Huang, D., Yan, F., Stranava, M., Bartosova, M., Fojtíková, V., and Martínková, M. (2015) Gaseous O2, NO, and CO in signal transduction: Structure and function relationships of heme-based gas sensors and heme-redox sensors, Chem. Rev. 115, 6491–6533. (2) Aono, S. (2008) Metal-containing sensor proteins sensing diatomic gas molecules, Dalton Trans. 24, 3137-3146. (3) Martínková, M., Kitanishi, K., and Shimizu, T. (2013) Heme-based globin-coupled oxygen sensors: Linking oxygen to functional regulation of diguanylate cyclase, histidine kinase, and methyl-accepting chemotaxis, J. Biol. Chem. 288, 27702-27711. (4) Hou, S., Larsen, R. W., Boudko, D., Riley, C. W., Karatan, E., Zimmer, M., Ordal, G. W., and Alam, M. (2000) Myoglobin-like aerotaxis transducers in Archaea and Bacteria, Nature 403, 540-547. (5) Zhang W., and Phillips, G. N. Jr. (2003) Structure of the oxygen sensor in Bacillus subtilis: Signal transduction of chemotaxis by control of symmetry, Structure 11, 1097-1110. (6) Zhang, W., Olson, J. S., and Phillips, G. N. Jr. (2005) Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis, Biophys. J. 88, 28012814. (7) Aono, S., Kato, T., Matsuki, M., Nakajima, H., Ohta, T., Uchida, T., and Kitagawa, T. (2002) Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis, J. Biol. Chem. 277, 13528-13538.


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(8) Ohta, T., Yoshimura, H., Yoshioka, S., Aono, S., and Kitagawa, T. (2004) Oxygen sensing mechanism of HemAT from Bacillus subtilis: A resonance Raman spectroscopic study, J. Am. Chem. Soc. 126, 15000-15001. (9) Ohta, T., and Kitagawa, T. (2005) Resonance Raman investigation of the specific sensing mechanism of a target molecule by gas sensory proteins, Inorg. Chem. 44, 758-769. (10) Yoshimura, H., Yoshioka, S., Kobayashi, K., Ohta, T., Uchida, T., Kubo, M., Kitagawa T., and Aono, S. (2006) Specific hydrogen-bonding networks responsible for selective O2 sensing of the oxygen sensor protein HemAT from Bacillus subtilis, Biochemistry 45, 83018307. (11) Yoshimura, H., Yoshioka, S., Mizutani, Y., and Aono, S. (2007) The formation of hydrogen bond in the proximal heme pocket of HemAT-Bs upon ligand binding, Biochem. Biophys. Res. Commun. 357, 1053-1057. (12) Yoshida, Y., Ishikawa, H., Aono, S., and Mizutani, Y. (2012) Structural dynamics of proximal heme pocket in HemAT-Bs associated with oxygen dissociation, Biochim. Biophys. Acta – Proteins and Proteomics 1824, 866-872. (13) El-Mashtoly, S. F., Gu, Y., Yoshimura, H., Yoshioka, S., Aono, S., and Kitagawa, T. (2008) Protein conformational changes of HemAT-Bs upon ligand binding probed by ultraviolet resonance Raman spectroscopy, J. Biol. Chem. 283, 6942-6949. (14) El-Mashtoly, S. F., Kubo, M., Gu, Y., Sawai, H., Nakashima, S., Ogura, T., Aono, S., and Kitagawa, T. (2012) Site-specific protein dynamics in communication pathway from sensor to signaling domain of oxygen sensor protein HemAT-Bs, J. Biol. Chem. 287, 19973-19984.


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(15) Pinakoulaki, E., Yoshimura, H., Yoshioka, S., Aono, S., and Varotsis, C. (2006) Recognition and discrimination of gases by the oxygen-sensing signal transducer protein HemAT as revealed by FTIR spectroscopy, Biochemistry 45, 7763-7766. (16) Pinakoulaki, E., Yoshimura, H., Daskalakis, V., Yoshioka, S., Aono, S., and Varotsis, C. (2006) Two ligand binding sites in the O2-sensing signal transducer HemAT: Implications for ligand recognition/discrimination and signaling, Proc. Natl. Acad. Sci. USA 103, 14796-14801. (17) Pavlou, A., Martínková, M., Shimizu, T., Kitanishi, K., Stranava, M., Loullis, A., and Pinakoulaki, E. (2015) Probing the ligand recognition and discrimination environment of the globin-coupled oxygen sensor protein YddV by FTIR and time-resolved step-scan FTIR spectroscopy, Phys. Chem. Chem. Phys. 17, 17007-17015. (18) Koutsoupakis, C., Pinakoulaki, E., Stavrakis, S., Daskalakis, V., and Varotsis, C. (2004) Time-resolved step-scan Fourier transform infrared investigation of heme-copper oxidases: Implications for O2 input and H2O/H+ output channels, Biochim. Biophys. Acta – Bioenergetics 1655, 347-352. (19) Gerwert, K. (2001) Time-resolved FT-IR difference spectroscopy: A tool to monitor molecular reaction mechanisms of proteins, in Infrared and Raman Spectroscopy of Biological Materials (Gremlich, H.-U., and Yan, B. Ed.) pp193-231, Marcel Dekker, New York. (20) Heibrink, D., Sigurdson, H., Bolwien, C., Brzezinski, P., and Heberle, J. (2002) Transient binding of CO to CuB in cytochrome c oxidase is dynamically linked to structural changes around a carboxyl group: A time-resolved step-scan Fourier transform infrared investigation, Biophys. J. 82, 1-10.


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Biochemistry

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(21) Koutsoupakis, C., Soulimane, T., and Varotsis, C. (2003) Ligand binding in a docking site of cytochrome c oxidase: A time-resolved step-scan Fourier transform infrared study, J. Am. Chem. Soc. 125, 14728. (22) Anfinrud, P. A., Han, C., and Hochstrasser, R. M. (1989) Direct observations of ligand dynamics in hemoglobin by subpicosecond infrared spectroscopy, Proc. Natl. Acad. Sci. USA 86, 8387-8391. (23) Murakawa, Y., Nagai, M., and Mizutani, Y. (2012) Differences between protein dynamics of hemoglobin upon dissociation of oxygen and carbon Monoxide, J. Am. Chem. Soc. 134, 14341437. (24) Schelvis, J. P. M., Deinum, G. Varotsis, C. A., Ferguson-Miller, S., and Babcock, G. T. (1997) Low-power picosecond resonance Raman evidence for histidine ligation to heme a3 after photodissociation of CO from cytochrome c oxidase, J. Am. Chem. Soc. 119, 8409-8416 (25) Varotsis, C. A., and Babcock, G. T. (1995) Photolytic activity of early intermediates in dioxygen activation and reduction by cytochrome oxidase, J. Am. Chem. Soc. 117, 11260-11269. (26) Pinakoulaki, E., and Varotsis, C. (2003) Time-resolved resonance Raman and timeresolved step-scan FTIR studies of nitric oxide reductase from Paracoccus denitrificans: Comparison of the heme b3-FeB Site to that of the heme-CuB in oxidases, Biochemistry 42, 14856-14861. (27) Pavlou, A., Soulimane, T., and Pinakoulaki, E. (2011) Evidence for the presence of two conformations of the heme a3-CuB pocket of cytochrome caa3 from Thermus thermophilus, J. Phys. Chem. B 115, 11455-11461.


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(28) Ostermann, A., Waschipky, R., Parak, F. G., and Nienhaus, G.U. (2000) Ligand binding and conformational motions in myoglobin, Nature 404, 205-208.


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For Table of Contents Use Only


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Probing the role of the heme distal and proximal environment in ligand

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