Ultraviolet Resonance Raman Observations of the Structural

Aug 13, 2013 - Dynamics of Rhizobial Oxygen Sensor FixL on Ligand Recognition ... the sensor domain of FixL upon O2 dissociation indicated that struct...
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Ultraviolet Resonance Raman Observations of the Structural Dynamics of Rhizobial Oxygen Sensor FixL on Ligand Recognition Shinji Yano,† Haruto Ishikawa,† Misao Mizuno,† Hiro Nakamura,‡ Yoshitsugu Shiro,‡ and Yasuhisa Mizutani*,† †

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan

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ABSTRACT: FixL is a heme-based oxygen-sensing histidine kinase that induces expression of nitrogen fixation genes under hypoxic conditions. Oxygen binding to heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, inactivating autophosphorylation activity. Although FixL also can bind other diatomic ligands such as CO, the CO-bound FixL represents incomplete inhibition of kinase activity. Ultraviolet resonance Raman (UVRR) spectra revealed that oxygen binding to the truncated sensor domain of FixL markedly decreased the intensity of the Y8a band arising from Fα-10 Tyr. In contrast, no appreciable change in intensity of the Y8a band occurred after CO binding, and time-resolved UVRR spectra of the sensor domain of FixL upon O2 dissociation indicated that structural changes near Fα-10 Tyr occurred at ∼0.1 μs. These results suggest that O2 dissociation from FixL changes the protein conformation near the Fα-10 Tyr residue within a microsecond. The conformational changes of FixL upon O2 dissociation and the underlying sensing mechanism also are discussed.

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located close to the bond ligand (Figure 1). The FG loop is anchored between the ends of Fα and Gβ. The heme-bound O2 induces an inward movement of Gβ-2 Arg (corresponding to Arg214 in SmFixL and Arg220 in BjFixL), which is supposed to be involved in the structural changes in the kinase domain.9,11,14 The side chain of Gβ-2 Arg forms a hydrogen

ensor proteins allow regulation of signaling pathways in response to environmental stimuli. Heme-containing proteins have emerged as critical sensors for detecting gaseous molecules on the cellular level.1−4 In general, these proteins are composed of a heme-containing sensor domain and a functional domain. The binding of gas molecules to heme induces an alteration in the sensor domain, which changes the activity of the functional domain. The FixL protein found in rhizobia contains a heme-based O2 sensor PAS domain and an ATP-dependent histidine kinase domain.2,5,6 A high-oxygen atmosphere fully inhibits the kinase activity of FixL, whereas in the absence of O2 its histidine kinase is active and transfers a phosphoryl group to FixJ that initiates nitrogen fixation gene expression.7 Although FixL also can bind other diatomic ligands, such as CO and NO, these molecules produce incomplete inhibition of the kinase.7,8 Therefore, a ligand discrimination mechanism is required for FixL to distinguish between gases. Crystal structures reported for the sensor domain of FixL from Bradyrhizobium japonicum (BjFixL) and Sinorhizobium meliloti (SmFixL) in ferric, ferrous, CN−-bound, O2-bound, CO-bound, and NO-bound forms provide the structural basis for ligand discrimination and recognition of FixL.8−13 The overall structures for the sensor domain are quite similar between BjFixL and SmFixL. The key residues involved in the regulation of FixL proteins are highly conserved. In FixL, the histidine residue that coordinates to the heme iron is located in the α-helix (Fα), and two antiparallel β-strands (Gβ and Hβ) are © 2013 American Chemical Society

Figure 1. Crystal structures of O2-bound (blue) and deoxy (green) forms of BjFixL retrieved from the Protein Data Bank. F α-helix (Fα), G β-strand (Gβ), FG loop, Fα-10 Tyr, and Gβ-2 Arg are depicted. Special Issue: Michael D. Fayer Festschrift Received: July 8, 2013 Revised: August 13, 2013 Published: August 13, 2013 15786

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containing ampicillin (final concentration: 100 μg/mL). E. coli JM109 cells harboring the plasmid were grown to an OD600 of 0.7 in a 37 °C incubator, followed by addition of 50 μM isopropyl 1-thio-β-d-galactopyranoside. After 10 h of induction at 25 °C, cells were harvested by centrifugation at 4 °C. The His-tagged recombinant SmFixLH2 was purified using a TARON His-tag affinity column (CLONTECH). The purified SmFixLH2 was dialyzed against 20 mM Tris−HCl buffer at pH 8.0 containing 5% glycerol. The purity of the recombinant SmFixLH2 was confirmed by SDS-PAGE. The protein concentration for the UVRR measurements was 80 μM in 50 mM sodium phosphate at pH 7.0. The deoxy, CO, and O2 forms of the truncated sensor domain of SmFixL were prepared according to protocols described previously.19 Resonance Raman Measurements. Static and nanosecond time-resolved UVRR spectra were obtained as described previously.22,23 Briefly, probe pulses at 225 nm were the fourth harmonics of the output of an Nd:YLF-pumped Ti:sapphire laser (Photonics Industries, TU-L), and pump pulses at 532 nm were generated with a diode-pumped Nd:YAG laser (Megaopto, LR-SHG). Pulse widths of the pump and probe pulses were ∼20 ns. The probe pulse also was used to measure static UVRR spectra. The probe power was set to 0.5 μJ/pulse. The power of the pump pulse was adjusted to 630 μJ/pulse. Timing between the pump and probe pulses was adjusted with a computer-controlled pulse generator (Stanford Research Systems, DG 535). Jitters in the delay time were within ±10 ns. Scattered light was detected with a liquid-nitrogen-cooled CCD camera (Roper Scientific, Spec-10:400B/LN) attached to a custom-made prism prefilter (Bunko Keiki) equipped with a single spectrograph (Horiba, iHR550). Raman shifts were calibrated with Raman bands of cyclohexane. Sample solutions for time-resolved UVRR measurements were contained in an airtight 10 mmϕ NMR tube, spun at 2000 rpm at room temperature.

bond with the heme-bound O2 in the O2-bound form, while the Gβ-2 Arg moves toward the heme propionate 7 and forms a hydrogen bond in the deoxy form. In contrast, even though the size and shape of CO is very similar to those of O2, the structure of CO-bound FixL was nearly identical to that of the unligated form. The partial negative charge on bound O2 induces an inward movement of Gβ-2 Arg, which transmits a signal that cannot be induced by the binding of CO.8 Another proposed O2 sensing mechanism of FixL is that the binding of O2 to the heme iron converts the domed structure into a planar structure of heme, because the unligated form has a high spin state while the O2 form of heme exhibits a low spin state.9,11,15 The structural changes of heme could weaken the salt bridge between heme propionate 7 and Gβ-2 Arg, resulting in movement of the FG loop. The replacement of Gβ-2 Arg with Ala dramatically reduced the affinity for O2 but not that for CO.8 However, the CN-bound Gβ-2 Arg to Ala mutant still can inhibit kinase activity without movement of the FG loop.8 These results suggest that Gβ-2 Arg cannot be the sole factor in kinase regulation of FixL.8,16−18 To fully understand the O2 sensing mechanism of FixL, characterization of the structural dynamics of the protein induced by O2 association and dissociation is necessary. A previous study reported structural changes in the heme and heme environment of FixL upon O2 and CO dissociation using time-resolved visible resonance Raman spectroscopy.19 The spectroscopic measurements suggested that the hydrogen bond formation between Gβ-2 Arg and heme propionate 7 occurred in ∼0.3 and ∼1 μs for the truncated sensor domain and fulllength SmFixL, respectively. These results for structural changes upon O2 association provide strong evidence to support the contribution of Gβ-2 Arg to the signal transduction mechanism in FixL. However, the crystal structure of the sensor domain of FixL suggested that the distinct structural changes observed upon O2 association are not only in the FG loop and Gβ but also in the Fα region. Detailed structural information is needed to determine the complete mechanism for FixL activation. In the present study, the ultraviolet resonance Raman (UVRR) data for the O2- and CO-bound forms of the truncated sensor domain of SmFixL were compared. The intensity of the Y8a band arising from Fα-10 Tyr (corresponding to Tyr201 in SmFixL and Tyr207 in BjFixL) decreased upon O2 binding but not upon CO binding. These observations indicate that the spectral change reflects specific structural changes upon O2 binding for the sensor domain of FixL. Moreover, the time-resolved UVRR measurements revealed that temporal changes in the Y8a band arising from Fα-10 Tyr upon O2 dissociation occurred in ca. 0.1 μs. The reposition of Fα-10 Tyr upon O2 dissociation appears to occur with the structural rearrangement of Gβ-2 Arg, which is much faster than structural changes in the FG loop that links the F α-helix and the G β-strand. These results suggest that structural changes in the F α-helix and the G β-strand could help “unlock” the FG loop. Since the interaction between the FG loop and the histidine kinase domain plays an important role for signal transduction in FixL, these findings provide important information on the signal transduction mechanism in FixL.

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RESULTS Static Resonance Raman Measurements of FixL. Static UVRR spectra of the deoxy (unligated), O2-bound, and CObound form of the truncated sensor domain of WT SmFixL excited at 225 nm are shown in Figure 2. In this figure, the difference spectra were calculated using the ClO4− band (933 cm−1) as an internal intensity standard. Raman bands arising from the Trp, Tyr, and Phe residues were enhanced selectively upon excitation at 225 nm. These spectra were dominated by bands from four Tyr (Tyr172, Tyr190, Tyr197, and Tyr201) and 10 Phe (Phe55, Phe57, Phe75, Phe104, Phe162, Phe170, Phe236, Phe242, Phe243, and Phe246) residues, labeled Y and F, respectively. The sensor domain of SmFixL contained no Trp residue. Band positions of the deoxy, O2-bound, and CO-bound states were not significantly different. However, the intensity of the Y8a band arising from Tyr residue(s) decreased upon O2 binding but did not change upon CO binding, indicating that the spectral change reflected specific structural changes on O2 binding for the sensor domain of FixL. The sensor domain of SmFixL contains four Tyr residues at positions 172, 190, 197, and 201. Positions 190, 197, and 201 in the F α-helix are located close to the heme, while position 172 is relatively distant from the heme. Therefore, Y190F, Y197F, and Y201F mutant proteins were prepared to identify the key residues of O2 responsible for the structural changes in FixL. Figure 3 shows a magnified view of the resonance Raman spectra of the Y8a region. In the Y201F mutant protein, the

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EXPERIMENTAL SECTION Protein Expression and Purification. The truncated sensor domain of FixL from Sinorhizobium meliloti (SmFixLH2) was expressed in E. coli and purified as described previously.19−21 Briefly, cells were grown in TB medium 15787

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Figure 2. UVRR spectra of (a) O2-bound, (b) CO-bound, and (c) deoxy forms of the truncated sensor domain WT SmFixL. Spectra d and e are the difference spectra of c − a and c − b, respectively.

negative peak of the Y8a band was not present in the difference spectrum of the deoxy and O2-bound forms. The negative peak of the Y8a band remained in spectra of the Y197F and Y197F mutant proteins. These results indicate that the spectral changes upon O2 association in WT FixL arise from structural perturbation at position 201. In contrast, binding of CO to FixL proteins did not produce any spectral changes in this region. Therefore, the intensity changes of the Y8a band arising from Fα-10 Tyr (corresponding to Tyr201 in SmFixL and Tyr207 in BjFixL) reflect specific structural changes upon association of O2. Confirming whether the specific spectral change upon O2 association in FixL is related to the kinase activity is important. To examine the relation between kinase activity and specific spectral changes induced by O2 association, UVRR spectra were obtained for the Gβ-2 Arg mutant protein. A previous study reported that Gβ-2 Arg plays a crucial role in the stabilization of heme-bound O 2 . 8 Static UVRR spectra of the deoxy (unligated), O2-bound, and CO-bound forms of the truncated sensor domain of Gβ-2 Arg mutant, R214A (corresponding to R220A in BjFixL), excited at 225 nm are shown in Figure 4. No prominent peak was found in the difference spectra between the deoxy, CO-bound, and O2-bound forms for the Gβ-2 Arg mutant, suggesting that the structural changes around the Tyr residue(s) were small. Intensity changes of the Y8a band upon O2 association in the truncated sensor domain WT FixL would reflect structural arrangements associated with regulation of the kinase activity. Time-Resolved Resonance Raman Measurements of FixL. Figure 5 shows nanosecond time-resolved UVRR spectra of the O2-bound form of the truncated sensor domain of WT SmFixL upon ligand photodissociation for various delay times of the probe pulse with respect to the pump pulse. These difference spectra were calculated using the ClO4− band (933 cm−1) as an internal intensity standard. Differences were observed for the Y8a band in the 10 ns to 100 μs delay spectra. The differences became more pronounced as delay time increased, indicating structural changes around the Tyr residue(s) upon O2 dissociation. Since the static UVRR measurements for the truncated sensor domain of SmFixL showed that intensity changes of the Y8a band upon O2 binding

Figure 3. The Y8a bands in the UVRR spectra of (A) WT, (B) Y190F, (C) Y197F, and (D) Y201F truncated sensor domain SmFixL.

arise from structural rearrangement of Fα-10 Tyr residue, the difference in the time-resolved UVRR spectra depicts the structural dynamics around Fα-10 Tyr upon O2 dissociation. To examine temporal evolution of the intensity of the Y8a band following O2 dissociation, the relative intensities of the Y8a band were calculated and plotted against delay time (Figure 6). The inset is a magnified view of the initial changes in intensity of the Y8a band. The temporal changes of the Y8a band were fitted using a single-exponential function. The time constant of the truncated sensor domain of SmFixL upon O2 dissociation was approximately 0.1 μs.

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DISCUSSION A critical issue in understanding O2-specific regulation of kinase activity in FixL involves structural changes in the protein moiety upon O2 association and dissociation to and from the heme, respectively. Comparison of the crystal structures of the O2-bound and deoxy forms of BjFixL showed rotation of the F α-10 Tyr ring upon O2 binding but not upon CO binding.9,11,14 The mutational study on SmFixL combined 15788

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Figure 6. Time dependence of intensity changes for the Y8a bands of truncated sensor domain WT SmFixL. Solid lines indicate the best fit obtained using an exponential decay. The inset shows the magnified 0 to 1.2 μs region.

O2-bound, CO-bound, and deoxy forms of BjFixL.9,11,14 Although the crystal structures of the O2- and CO-bound forms of SmFixL are unavailable, O2 association would cause similar effects on structural changes in the Tyr residue in SmFixL. Static UVRR spectra for SmFixL suggested that the O2dependent activation mechanism of SmFixL is very similar to that of BjFixL. The Y8a mode is attributed to the in-plane stretching vibration on the phenyl ring.24 The Raman intensity of this mode is resonantly enhanced by the Franck−Condon A-term mechanism via La absorption (peak wavelength 222 nm). The maximum of its Raman excitation profile is ca. 225 nm.25 Under the probe condition used in the present study, the intensity loss of the Y8a band could arise from either a blue or red shift of the Raman excitation profile. The La absorption band systematically blue-shifts when the tyrosine residue is in a more protic environment.26 The Fα-10 Tyr forms a hydrogen bond to Hβ-8 Glu (Glu240 in BjFixL and Glu234 in SmFixL). Therefore, the intensity change of the Y8a band indicates a difference in the hydrogen-bond strength of the Fα-10 Tyr between the oxy and deoxy forms of SmFixL. Structural changes occurring in the heme pocket of the truncated sensor domain of SmFixL upon O2 dissociation have been reported using the time-resolved visible resonance Raman spectroscopy.19 Intensity changes of ν(Fe-His), γ7, and ν8 bands were observed with a time constant of approximately 0.3 μs. The former two bands are sensitive to the extent of heme doming,27,28 and the latter one is correlated with disorder in the orientation of the propionate.29 Especially, the ν(Fe-His) mode is a good indicator of protein structural changes induced by ligand photodissociation,19,30−32 because the heme is covalently linked to the protein solely through the proximal Fα-3 His. These spectral changes were attributed to formation of the salt bridge between Gβ-2 Arg and heme propionate 7 which intensifies heme doming and causes rearrangement of the F α-helix. Thus, it was concluded that, after photodissociation of O2 from the sensor domain of SmFixL, Gβ-2 Arg switches its interaction partner from the ligated-O2 to heme propionate 7 in approximately 0.3 μs.19 In the present data, intensity changes in the Y8a bands upon O2 dissociation suggested that the structural changes around Fα-10 Tyr occur at ∼0.1 μs. The reposition of Gβ-2 Arg appears to occur along with structural rearrangement of the F α-helix. However, structural changes in the FG loop that links the F α-helix and G β-strand were observed at ∼1 μs,19 based on the heme propionate bending band, of which the intensity changed upon

Figure 4. UVRR spectra of (a) O2-bound, (b) CO-bound, and (c) deoxy forms of the truncated sensor domain R214A mutant SmFixL. Spectra d and e are the difference spectra of c − a and c − b, respectively.

Figure 5. Nanosecond time-resolved UVRR spectra at the indicated delay times following photolysis of the O2-bound form of truncated sensor domain WT SmFixL. The UVRR spectrum of the deoxy form of truncated sensor domain WT SmFixL is shown at the top for comparison.

with UVRR spectroscopy showed that the Y8a band arising from Fα-10 Tyr is a characteristic marker for specific structural changes associated with kinase activation. Since intensity changes in the Y8a bands were not observed for the Fα-10 Tyr mutant, the structural changes for other Tyr residues would be relatively small compared to that for Fα-10 Tyr. Indeed, configurations of the Tyr residues (except for Fα-10 Tyr) were not significantly different between the crystal structures of the 15789

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mutation of residues in the FG loop.33 This change of the FG loop is much slower than the movement of Gβ-2 Arg and Fα-10 Tyr. Therefore, the structural changes around Fα-10 Tyr would not be transmitted via the FG loop. The crystal structures of BjFixL showed that the hydrogen bond between Fα-9 Arg and heme propionate 6 is formed in the O2-bound form but not in the deoxy form.34 The disruption of this hydrogen bond would induce structural changes in the F α-helix, resulting in the rotation of the Fα-10 Tyr ring. These results lead us to propose a mechanism of signal transduction that the initial changes occur in the F α-helix and the G β-strand within a microsecond, which can lead to alterations in the FG loop, as shown in Figure 7.

ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research on the Priority Area “Molecular Science for Supra Functional Systems” (Grant No. 19056013) to Y.M. from the Ministry of Education, Science, Sports, and Culture of Japan.

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REFERENCES

(1) Aono, S. Novel Bacterial Gas Sensor Proteins with Transition Metal-Containing Prosthetic Groups as Active Sites. Antioxid. Redox Signaling 2012, 16, 678−686. (2) Gilles-Gonzalez, M.-A.; Gonzalez, G. Heme-Based Sensors: Defining Characteristics, Recent Developments, and Regulatory Hypotheses. J. Inorg. Biochem. 2005, 99, 1−22. (3) Girvan, H.; Munro, A. Heme Sensor Proteins. J. Biol. Chem. 2013, 288, 13194−13203. (4) Jain, R.; Chan, M. Mechanisms of Ligand Discrimination by Heme Proteins. J. Biol. Inorg. Chem. 2003, 8, 1−11. (5) Liebl, U.; Lambry, J.-C.; Vos, M. Primary Processes in HemeBased Sensor Proteins. Biochim. Biophys. Acta 2013, 1834, 1684−1692. (6) Stock, J.; Ninfa, A.; Stock, A. Protein Phosphorylation and Regulation of Adaptive Responses in Bacteria. Microbiol. Rev. 1989, 53, 450−490. (7) Tuckerman, J.; Gonzalez, G.; Dioum, E.; Gilles-Gonzalez, M.-A. Ligand and Oxidation-State Specific Regulation of the Heme-Based Oxygen Sensor FixL from Sinorhizobium meliloti. Biochemistry 2002, 41, 6170−6177. (8) Dunham, C.; Dioum, E.; Tuckerman, J.; Gonzalez, G.; Scott, W.; Gilles-Gonzalez, M.-A. A Distal Arginine in Oxygen-Sensing HemePAS Domains Is Essential to Ligand Binding, Signal Transduction, and Structure. Biochemistry 2003, 42, 7701−7708. (9) Gong, W.; Hao, B.; Mansy, S.; Gonzalez, G.; Gilles-Gonzalez, M.; Chan, M. Structure of a Biological Oxygen Sensor: a New Mechanism for Heme-Driven Signal Transduction. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15177−15182. (10) Ayers, R.; Moffat, K. Changes in Quaternary Structure in the Signaling Mechanisms of PAS Domains. Biochemistry 2008, 47, 12078−12086. (11) Hao, B.; Isaza, C.; Arndt, J.; Soltis, M.; Chan, M. StructureBased Mechanism of O2 Sensing and Ligand Discrimination by the FixL Heme Domain of Bradyrhizobium japonicum. Biochemistry 2002, 41, 12952−12958. (12) Key, J.; Moffat, K. Crystal Structures of Deoxy and CO-Bound bjFixLH Reveal Details of Ligand Recognition and Signaling. Biochemistry 2005, 44, 4627−4635. (13) Miyatake, H.; Mukai, M.; Park, S.; Adachi, S.; Tamura, K.; Nakamura, H.; Nakamura, K.; Tsuchiya, T.; Iizuka, T.; Shiro, Y. Sensory Mechanism of Oxygen Sensor FixL from Rhizobium meliloti: Crystallographic, Mutagenesis and Resonance Raman Spectroscopic Studies. J. Mol. Biol. 2000, 301, 415−431. (14) Gong, W.; Hao, B.; Chan, M. New Mechanistic Insights from Structural Studies of the Oxygen-Sensing Domain of Bradyrhizobium japonicum FixL. Biochemistry 2000, 39, 3955−3962. (15) Miyatake, H.; Mukai, M.; Adachi, S.; Nakamura, H.; Tamura, K.; Iizuka, T.; Shiro, Y.; Strange, R.; Hasnain, S. Iron Coordination Structures of Oxygen Sensor FixL Characterized by Fe K-Edge Extended X-ray Absorption Fine Structure and Resonance Raman Spectroscopy. J. Biol. Chem. 1999, 274, 23176−23184. (16) Balland, V.; Bouzhir-Sima, L.; Anxolabéhère-Mallart, E.; Boussac, A.; Vos, M.; Liebl, U.; Mattioli, T. Functional Implications of the Propionate 7-Arginine 220 Interaction in the FixLH Oxygen Sensor from Bradyrhizobium japonicum. Biochemistry 2006, 45, 2072− 2084. (17) Balland, V.; Bouzhir-Sima, L.; Kiger, L.; Marden, M.; Vos, M.; Liebl, U.; Mattioli, T. Role of Arginine 220 in the Oxygen Sensor FixL from Bradyrhizobium japonicum. J. Biol. Chem. 2005, 280, 15279− 15288. (18) Jasaitis, A.; Hola, K.; Bouzhir-Sima, L.; Lambry, J.-C.; Balland, V.; Vos, M.; Liebl, U. Role of Distal Arginine in Early Sensing

Figure 7. Proposed mechanism for signal transduction of FixL following oxygen dissociation. Following the oxygen dissociation, the initial changes occur in the F α-helix and the G β-strand within a microsecond. These changes lead to alterations in the FG loop, which is relevant to signal transduction to the catalytic domain.

The static UVRR for SmFixL revealed that the intensity change in the Y8a band reflects specific O2 movement of the Fα-10 Tyr, while an intensity change in the difference spectrum was not observed for the Gβ-2 Arg mutant protein. Although the Fα-10 Tyr residue exists on the side of the heme opposite to the Gβ-2 Arg, substitution of Gβ-2 Arg to Ala suppresses the structural changes around Fα-10 Tyr. Interestingly, the CObound BjFixL did not form a hydrogen bond between Fα-9 Arg and heme propionate 6 or between the Gβ-2 Arg and ligated CO.11 Hydrogen bond formation between the heme-bound O2 and Gβ-2 Arg was involved in the interaction between the F αhelix and the heme propionate, resulting in structural alteration in the Fα-10 Tyr. Note that Gβ-2 Arg is a critical residue for the regulation of catalytic activity. The hydrogen-bonding switch of Gβ-2 Arg plays an important role in the signal transduction mechanism of FixL. Therefore, the intensity change in the Y8a band in the UVRR spectra is likely to be relevant to the regulation of FixL.

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CONCLUSIONS The UVRR spectral changes specific to O2 binding in SmFixL were successfully identified. The spectral changes indicated that structural changes upon O2 dissociation occurred in both the F α-helix and G β-strand within a few hundred nanoseconds. The structural changes in the F α-helix and G β-strand could promote “unlocking” of the FG loop.

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Corresponding Author

*Phone: +81-6-6850-5776. Fax: +81-6-6850-5776. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 15790

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Intermediates in the Heme Domain of the Oxygen Sensor FixL. Biochemistry 2006, 45, 6018−6026. (19) Hiruma, Y.; Kikuchi, A.; Tanaka, A.; Shiro, Y.; Mizutani, Y. Resonance Raman Observation of the Structural Dynamics of FixL on Signal Transduction and Ligand Discrimination. Biochemistry 2007, 46, 6086−6096. (20) Nakamura, H.; Saito, K.; Ito, E.; Tamura, K.; Tsuchiya, T.; Nishigaki, K.; Shiro, Y.; Iizuka, T. Identification of the Hydrophobic Amino Acid Residues Required for Heme Assembly in the Rhizobial Oxygen Sensor Protein FixL. Biochem. Biophys. Res. Commun. 1998, 247, 427−431. (21) Saito, K.; Ito, E.; Hosono, K.; Nakamura, K.; Imai, K.; Iizuka, T.; Shiro, Y.; Nakamura, H. The Uncoupling of Oxygen Sensing, Phosphorylation Signalling and Transcriptional Activation in Oxygen Sensor FixL and FixJ Mutants. Mol. Microbiol. 2003, 48, 373−383. (22) Yamada, K.; Ishikawa, H.; Mizutani, Y. Protein Dynamics of Isolated Chains of Recombinant Human Hemoglobin Elucidated by Time-Resolved Resonance Raman Spectroscopy. J. Phys. Chem. B 2012, 116, 1992−1998. (23) Yoshida, Y.; Ishikawa, H.; Aono, S.; Mizutani, Y. Structural Dynamics of Proximal Heme Pocket in HemAT-Bs Associated with Oxygen Dissociation. Biochim. Biophys. Acta 2012, 1824, 866−872. (24) Harada, I.; Takeuchi, H. Raman and Ultraviolet Resonance Raman Spectra of Proteins and Related Compounds, In Spectroscopy of Biological Systems; John Wiley & Sons: Chichester, U.K., 1986. (25) Luding, M.; Asher, S. A. Ultraviolet ResonanceRaman Excitation Profiles of Tyrosine: Dependence of Raman Cross Sections on Excited-State Intermediates. J. Am. Chem. Soc. 1988, 110, 1005−1011. (26) Chi, Z.; Asher, S. A. UV Raman Determination of the Environment and Solvent Exposure of Tyr and Trp Residues. J. Phys. Chem. B 1988, 102, 9595−9602. (27) Bangcharoenpaurpong, O.; Schomacker, K. T.; Champion, P. M. Resonance Raman Investigation of Myoglobin and Hemoglobin. J. Am. Chem. Soc. 1984, 106, 5688−5698. (28) Hu, S.; Smith, K. M.; Spiro, T. G. Assignment of Protoheme Resonance Raman Spectrum by Heme Labeling in Myoglobin. J. Am. Chem. Soc. 1996, 118, 12638−12646. (29) Peterson, E. S.; Friedman, J. M.; Chien, E. Y. T.; Sligar, S. G. Functional Implications of the Proximal Hydrogen-Bonding Network in Myoglobin: A Resonance Raman and Kinetic Study of Leu89, Ser92, His97, and F-Helix Swap Mutants. Biochemistry 1998, 37, 12301−12319. (30) Kitagawa, T.; Nagai, K.; Tsubaki, M. Assignment of the Fe-Nϵ (His F8) Stretching Band in the Resonance Raman Spectra of Deoxy Myoglobin. FEBS Lett. 1979, 104, 376−378. (31) Sato, A.; Gao, Y.; Kitagawa, T.; Mizutani, Y. Primary Protein Response after Ligand Photodissociation in Carbonmonoxy Myoglobin. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9627−9632. (32) Murakawa, Y.; Nagai, M.; Mizutani, Y. Differences between Protein Dynamics of Hemoglobin upon Dissociation of Oxygen and Carbon Monoxide. J. Am. Chem. Soc. 2012, 134, 1434−1437. (33) Mukai, M.; Nakamura, K.; Nakamura, H.; Iizuka, T.; Shiro, Y. Roles of Ile209 and Ile210 on the Heme Pocket Structure and Regulation of Histidine Kinase Activity of Oxygen Sensor FixL from Rhizobium meliloti. Biochemistry 2000, 39, 13810−13816. (34) Gilles-Gonzalez, M.-A.; Caceres, A.; Sousa, E.; Tomchick, D.; Brautigam, C.; Gonzalez, C.; Machius, M. A Proximal Arginine R206 Participates in Switching of the Bradyrhizobium japonicum FixL Oxygen Sensor. J. Mol. Biol. 2006, 360, 80−89.

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